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Environmental Sciences of the University of Helsinki, in Lecture Hall 2 at Haartman ..... 5.2 SIGMA-1 RECEPTOR AGONIST PRE084 IS PROTECTIVE AGAINST ...
Endoplasmic Reticulum Stress, Regulation of Autophagy and Ubiquitin-proteasome System in Cellular Models of Huntington’s Disease Alise Hyrskyluoto

Division of Biochemistry and Developmental Biology Institute of Biomedicine Faculty of Medicine University of Helsinki Division of Biochemistry and Biotechnology Department of Biosciences Faculty of Biological and Environmental Sciences University of Helsinki Minerva Foundation Institute for Medical Research Finnish Graduate School of Neuroscience / Doctoral Program Brain and Mind

Academic dissertation To be presented for public examination with the permission of the Faculty of Biological and Environmental Sciences of the University of Helsinki, in Lecture Hall 2 at Haartman Institute, Helsinki, on September 12th 2014 at 12 o´clock noon.

Hansaprint, Helsinki 2014

Thesis supervised by:

Professor Dan Lindholm, MD, PhD Institute of Biomedicine University of Helsinki and Minerva Foundation Institute for Medical Research Biomedicum Helsinki Docent Laura Korhonen, MD, PhD Institute of Biomedicine University of Helsinki and Division of Child Psychiatry Helsinki University Central Hospital and Minerva Foundation Institute for Medical Research Biomedicum Helsinki

Thesis committee members:

Professor Kid Törnquist, PhD Minerva Foundation Institute for Medical Research Biomedicum Helsinki Docent Urmas Arumäe, PhD Institute of Biotechnology University of Helsinki

Thesis reviewed by:

Docent Eeva-Liisa Eskelinen, PhD Department of Biosciences University of Helsinki Docent Urmas Arumäe, PhD Institute of Biotechnology University of Helsinki

Opponent:

Professor Raimo Tuominen, MD, PhD Division of Pharmacology and Toxicology Faculty of Pharmacy University of Helsinki

Custodian:

Professor Kari Keinänen, PhD Department of Biosciences University of Helsinki

ISBN 978-951-51-0071-9 (paperback) ISBN 978-951-51-0072-6 (PDF, http://ethesis.helsinki.fi) ISSN 2342-3161 (paperback), ISSN 2342-317X (PDF)

“Nothing in life is to be feared. It is only to be understood.” -

Marie Curie

TABLE OF CONTENTS LIST OF ORIGINAL PUBLICATIONS ABSTRACT ABBREVATIONS 1 INTRODUCTION ..........................................................................................................................1 2 REVIEW OF THE LITERATURE ..............................................................................................2 2.1 HUNTINGTON’S DISEASE ......................................................................................................2 2.1.1 HUNTINGTIN ............................................................................................................................4 2.1.1.1 Structure ..........................................................................................................................5 2.1.1.2 Proteolytic cleavage of HTT ............................................................................................6 2.1.1.3 Transcriptional regulation and interacting proteins .......................................................6 2.1.1.4 Post-translational modifications ......................................................................................7 2.1.2 MUTANT HUNTINGTIN..............................................................................................................8 2.1.2.1 Aggregation .....................................................................................................................9 2.1.3 NEUROPATHOLOGY ...............................................................................................................10 2.1.4 MODEL SYSTEMS OF HUNTINGTON’S DISEASE .......................................................................11 2.1.4.1 Cell models ....................................................................................................................11 2.1.4.2 Animal models ...............................................................................................................12 2.1.5 TREATMENT AND THERAPY....................................................................................................13 2.2. ENDOPLASMIC RETICULUM STRESS .............................................................................16 2.2.1 UNFOLDED PROTEIN RESPONSE ..............................................................................................18 2.2.1.1 IRE1 ...............................................................................................................................18 2.2.1.2 PERK .............................................................................................................................19 2.2.1.3 ATF6 ..............................................................................................................................19 2.2.2 ENDOPLASMIC RETICULUM STRESS IN HD .............................................................................19 2.3 CELL DEATH ...........................................................................................................................21 2.3.1 CELL DEATH INDUCED BY ER STRESS ....................................................................................22 2.4 OXIDATIVE STRESS IN HD ..................................................................................................24 2.4.2 OXIDATIVE STRESS AND CELL DEATH ....................................................................................25 2.4.3 SIGMA-1 RECEPTOR ...............................................................................................................27 2.5 AUTOPHAGY ...........................................................................................................................28 2.5.1 AUTOPHAGY PATHWAY .........................................................................................................30 2.5.2 REGULATION OF AUTOPHAGY ................................................................................................31 2.5.2.1 mTOR-dependent signaling pathways ...........................................................................31 2.5.2.2 mTOR-independent signaling pathways ........................................................................33 2.5.2.3 GADD34 in autophagy ..................................................................................................34 2.5.2.4 Ubiquitination and selective autophagy ........................................................................35

2.5.3 AUTOPHAGY IN HD ...............................................................................................................36 2.6 PROTEASOMAL DEGRADATION .......................................................................................38 2.6.1 UBIQUITIN-PROTEASOME SYSTEM..........................................................................................38 2.6.2 PROTEASOMAL DEGRADATION IN HD ....................................................................................39 2.6.4 THE DEUBIQUITINATING ENZYMES.........................................................................................40 2.6.4.1 Ubiquitin specific protease 14 (Usp14) .........................................................................41 3 AIMS OF THE STUDY ...............................................................................................................43 4 MATERIALS AND METHODS .................................................................................................44 4.1 CELL CULTURES (I-III)..............................................................................................................44 4.1.1 PC6.3 cell cultures (I-III) .................................................................................................44 4.1.2 Hela cell cultures (III) ......................................................................................................44 4.2 ANIMALS (III) ...........................................................................................................................45 4.3 TRANSFECTIONS AND STIMULATIONS (I-III) .............................................................................45 4.4 IMMUNOBLOTTING (I-III)..........................................................................................................45 4.5 IMMUNOCYTOCHEMISTRY.........................................................................................................46 4.6 IMMUNOPRECIPITATION ............................................................................................................46 4.7 SUBCELLULAR FRACTIONATION (III) ........................................................................................48 4.8 POLYMERASE CHAIN REACTION (PCR) (I) ................................................................................48 4.8.1 Reverse transcription and quantitative PCR (II) ..............................................................48 4.9 AUTOPHAGY DETECTION (I) ......................................................................................................49 4.10 SOLUBILITY ASSAY (II-III)......................................................................................................50 4.11 LUCIFERASE REPORTER ASSAY (II) .........................................................................................50 4.12 CASPASE ASSAY ......................................................................................................................51 4.13 CELL DEGENERATION ASSAYS (I-III) ......................................................................................51 4.14 MEASUREMENTS OF INTRACELLULAR ROS ............................................................................51 4.15 MEASUREMENTS OF INTRACELLULAR CALCIUM .....................................................................52 4.16 AEQUORIN-BASED MEASUREMENTS OF MITOCHONDRIAL CALCIUM ........................................52 4.17 DETERMINATION OF CELL VIABILITY WITH MTT ASSAY (I-II) ................................................53 4.18 STATISTICAL ANALYSES .........................................................................................................53 5 RESULTS AND DISCUSSION ...................................................................................................54 5.1 GADD34 PLAYS AN IMPORTANT ROLE IN CELL PROTECTION IN MHTT EXPRESSING CELLS BY INCREASING AUTOPHAGY AND CELL SURVIVAL. (I) ........................................................................54 5.1.1 Mutant HTT expression induces autophagy and inhibits the mTOR pathway in neuronal PC6.3 cells .................................................................................................................................54 5.1.2 GADD34 interacts with tuberous sclerosis complex (TSC) proteins in mHTT expressing cells and mediates mTOR inhibition ..........................................................................................55 5.1.3 Autophagy is inhibited at 48h in mHTT expressing cells - relationship to changes in GADD34 levels and AMP-activated protein kinase (AMPK) activity .......................................56 5.1.4 Overexpression of GADD34 enhance autophagy and protects cells against mHTTinduced cell degeneration ..........................................................................................................56

5.2 SIGMA-1 RECEPTOR AGONIST PRE084 IS PROTECTIVE AGAINST MUTANT HUNTINGTIN-INDUCED CELL DEGENERATION: INVOLVEMENT OF CALPASTATIN AND THE NF-ΚB PATHWAY (II) ................58 5.2.1 The Sig1-R agonist PRE084 counteracts mHTT induced cell death and caspase activation ...................................................................................................................................................59 5.2.2 Stimulation with PRE084 increases cellular antioxidants and reduces ROS in mHTT expressing cells ..........................................................................................................................60 5.2.3 PRE084 increases NF-B-p65 levels and activate NF-B-p65 signaling in neuronal PC6.3 cells .................................................................................................................................60 PRE084 and overexpression Sig1-R elevate calpastatin in mHTT expressing cells ..................61 5.3 UBIQUITIN SPECIFIC PROTEASE-14 (USP14) PROTECTS AGAINST MUTANT HUNTINGTIN-INDUCED CELL DEGENERATION AND REDUCES CELLULAR AGGREGATES (III) ................................................62 5.3.1 Usp14 levels are unchanged but Usp14 is redistributed in mutant huntingtin expressing cells ............................................................................................................................................62 5.3.2 Overexpression of Usp14 reduces cellular aggregates in mHTT expressing cells mainly via the ubiquitin proteasome system ..........................................................................................63 5.3.3 IRE1α, involved in ER stress response, is activated in mHTT expressing cells as well as in the striatum of mHTT transgenic (BAC-HD) mouse ..............................................................64 5.3.4 Overexpression of Usp14 protected against cell degeneration and caspase-3 activation induced by mHTT .......................................................................................................................65 6 CONCLUSIONS AND FUTURE PROSPECTS ........................................................................66 7 ACKNOWLEDGEMENTS .........................................................................................................69 8 REFERENCES .............................................................................................................................71

LIST OF ORIGINAL PUBLICATIONS This thesis is based on the following articles, which are referred in the text by their roman numbers:

I

Hyrskyluoto A, Reijonen S, Kivinen J, Lindholm D*, Korhonen L*. GADD34 mediates cytoprotective autophagy in mutant huntingtin expressing cells via the mTOR pathway. Exp Cell Res. 2012;318(1):33-42

II

Hyrskyluoto A, Pulli I, Törnqvist K, Ho T, Korhonen L*, Lindholm D*. The sigma-1 receptor agonist PRE084 is cytoprotective against mutant huntigtin cell degeneration: involvement of calpastatin and the NF-κB pathway. Cell Death Dis. 2013 May 23;4:e646. doi: 10.1038/cddis.2013.170.

III

Hyrskyluoto A, Bruelle C, Lundh S, Kivinen J, Do H, Rappou E, Reijonen S, Waltimo T, Petersén Å, Lindholm D*, Korhonen L*. Ubiquitin specific protease-14 reduces cellular aggregates and protects against mutant huntingtin-induced cell degeneration: involvement of the proteasome and ER stress-activated kinase IRE1α. Hum Mol Genet. 2014 Jun 20; pii: ddu317.

* Equal contribution

The articles are printed with the permission of copyright holders.

Author’s contribution to the publications: I Contributed to the design of experiments and writing of the manuscript. Performed most of the experimental work and data analysis. II Contributed to the design of experiments and writing of the manuscript. Performed data analysis and the experimental work, except Ca2+ concentration measurements and qPCR. III Contributed to the design of experiments and writing of the manuscript. Performed data analysis and the experimental work, except the experiments using tissue samples from BACHD mice and immunoprecipitation experiments.

ABSTRACT Huntington’s disease (HD) is a fatal neurodegenerative disease with progressive motor dysfunction, cognitive decline and psychiatric disturbances. HD is caused by a CAG repeat expansion in the huntingtin (IT15) gene, which encodes the huntingtin protein. Mutations in huntingtin cause accumulation of protein aggregates with subsequent cell death and loss of neurons in the striatum and cortex. The exact molecular mechanisms by which mutant huntingtin (mHTT) induces cell death are not completely understood. Huntingtin protein participates in many cellular functions such as protein trafficking, transcriptional regulation and apoptosis. Mutant protein is cleaved to form N-terminal fragments containing the first 100-150 residues including the polyglutamine repeats, which are thought to be the toxic species found in aggregates. Previous studies have shown that endoplasmic reticulum (ER) stress is involved in the early pathogenesis of HD. However, the precise mechanisms by which mHTT proteins cause ER stress are still unclear. The aim of this thesis was to elucidate the early pathological changes in HD. The specific goal was to study in more detail how ER stress, alterations in autophagy and ubiquitin proteasome system and oxidative stress trigger the disease and by which mechanisms. This thesis also aimed to identify novel therapeutic targets for early pathogenic changes in HD. The results showed that growth arrest and DNA damage inducible gene 34 (GADD34) plays an important role in cell protection and mediates cytoprotective autophagy via the mammalian target of rapamycin (mTOR) pathway in mHTT expressing cells. Modulation of GADD34 may thus prove useful in counteracting cell degeneration accompanying HD. Our results also showed that the sigma-1 receptor (Sig1R) agonist PRE084 increased levels of cellular antioxidants by affecting the NF-κB pathway that is reduced by expression of mHTT proteins. The Sig1R agonist increased cell survival and counteracted the deleterious effects caused by N-terminal mHTT proteins. Compounds that influence the Sig1R may have beneficial effects in models of HD, which warrants further studies. This thesis also shows that overexpression of ubiquitin-specific protease-14 (Usp14) reduces cellular aggregates in mHTT expressing cells mainly via the ubiquitin proteasome system. Overexpression of Usp14 was able to inhibit phosphorylation of inositol requiring enzyme 1 (IRE1) in mHTT expressing cells and to protect against cell degeneration and caspase-3 activation. These results show ER stress induced IRE1 activation is part of mHTT toxicity, which is inhibited by Usp14.

ABBREVATIONS AD AIF ALS AMP AMPK ASK1 ATF ATG ATP BAC BDNF Bip CHOP DUB FL flHTT E1 E2 E3 eEF2 eIF2α ER ERAD GADD34 HD HEAT IRE1α JNK kDa LC3 mHTT mTOR MSN NF-κB PC6.3 PD PGC-1α PI3K PERK polyQ polyP

Alzheimer’s disease Apoptosis inducing factor Amyloid lateral sclerosis Adenosine monophosphate Adenosine monophosphate-activated protein kinase Apoptosis signal-regulating kinase 1 Activating transcription factor Autophagy-related gene Adenosine triphosphate Bacterial artificial chromosome Brain-derived neurotrophic factor Binding immunoglobulin protein CCAAT/enhancer-binding protein homologous protein Deubiquitinating enzyme Full-length Full-length huntingtin Ubiquitin-activating enzymes Ubiquitin-conjugating enzymes Ubiquitin-protein ligases Eukaryotic translation elongation factor 2 Eukaryotic translation initiation factor 2 subunit alpha Endoplasmic reticulum ER associated degradation Growth arrest and DNA damage-inducible protein 34 Huntington’s disease Huntingtin, elongation factor 3, the PR65/A subunit of protein phosphatase 2A and target of rapamycin 1 Inositol requiring enzyme 1 alpha JUN N-terminal kinase Kilodalton Light chain 3 protein Mutant huntingtin Mammalian target of rapamycin Medium-sized spiny neurons Nuclear factor-kappa beta Pheochromocytoma cell line subline 6.3 Parkinson’s disease Peroxisome proliferator-activated receptor- coactivator-1 alpha Phosphatidylinositol 3-kinase Protein kinase (PRK)-like endoplasmic reticulum kinase / pancreatic endoplasmic reticulum kinase Polyglutamine Polyproline

PRE084 ROS siRNA TSC Ub UPR UPS Usp14 WB wt YAC 3-MA

2-(4-morpholino)ethyl1-phenylcyclohexane-1-carboxylate hydrochloride Reactive oxygen species Small interfering RNA Tuberous sclerosis complex Ubiquitin Unfolded protein response Ubiquitin proteasomal system Ubiquitin specific protease 14 Western Blotting Wild type Yeast artificial chromosome 3-methyladenine

1 INTRODUCTION Huntington’s disease (HD) is the most common dominantly inherited neurodegenerative disorder, affecting 1 in 10,000 people of Western world. It leads to progressive motor dysfunction, cognitive decline, psychiatric disturbances and marked brain atrophy with loss of neurons, especially in caudate-putamen and the frontal lobes (van Dellen et al., 2005). The disease is caused by a CAG (encoding glutamine) repeat expansion in the first exon of the IT15 gene, resulting in the expression of mutant huntingtin (HTT) with greater than ~36 glutamines (Gil and Rego, 2008). Huntingtin protein (350kDa) is expressed in most tissues, including the nervous system (DiFiglia et al., 1995; The Huntington's Disease Collaborative Research Group, 1993). In neurons, HTT expression is localized to neuronal cell bodies, axons and dendrites with subcellular localization to vesicles and presynaptic terminal fibers and nerve-endings (DiFiglia et al., 1995). Mutation in HTT causes accumulation of intracellular and intranuclear protein aggregates. Mutant protein is cleaved to form N-terminal fragments containing the first 100-150 residues including the polyglutamine repeats, which are thought to be the toxic species found in aggregates (Ross and Tabrizi, 2011). Huntingtin protein participates in many cellular functions such as protein trafficking, transcriptional regulation and apoptosis (Ross and Tabrizi, 2011). HTT is a subject to many post-translational modifications, including proteolysis, phosphorylation and modification by ubiquitin and ubiquitinlike proteins (Lu et al., 2013). HD is a complex disease and the mechanisms by which mHTT causes neuronal dysfunction and degeneration are not yet fully understood. Thus, it has been difficult to develop therapies that effectively target HD’s toxic effects. Huntingtin can interact with over 150 protein partners and, when mutated, interferes directly and indirectly with dozens of cellular pathways. Impaired ubiquitin-proteasome

activity,

defective

autophagy-lysosomal

function,

transcriptional

dysregulation, oxidative stress, apoptosis, mitochondrial and metabolic dysfunction, and abnormal protein-protein interaction have been shown to play important roles in the pathogenesis of HD (Anderson, 2011; Gil and Rego, 2008).

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2 REVIEW OF THE LITERATURE 2.1 Huntington’s disease Huntington’s disease (HD) is an inherited neurodegenerative disorder, also known as Huntington chorea. Its clinical features and pattern of familial transmission was first described by a young physician George Huntington in 1872 (Huntington, 1872; Huntington, 2003). But it was not until the end of next century, in 1993, that the actual HD gene mutation and cause of the disease was discovered by a multicenter consortium, organized by the Hereditary Disease Foundation (The Huntington's Disease Collaborative Research Group, 1993). Huntington’s disease is caused by a cytosine-adenine-guanine (CAG) nucleotide repeat expansion in Huntingtin (HTT, also known as the interesting transcript IT15) gene in chromosome 4. Mutation in HTT gene leads to an expanded polyglutamine (polyQ) stretch in the huntingtin (HTT) protein (The Huntington's Disease Collaborative Research Group, 1993). HD is inherited in an autosomal dominant manner, thus a child has a 50% chance of inheriting HD from a diseased parent (Myers et al., 1993). Individuals with Huntington's disease can become symptomatic at any time between the ages of 1 and 80 years, but in most cases the onset of the disease occurs in midlife, between the ages of 35 and 50 years. Patients with juvenile HD, defined as having disease onset before age 20, typically have a very large number of CAG repeats, usually greater than 55 (Foroud et al., 1999). The disease progresses over time and is invariably fatal 15–20 years after the onset of the first symptoms (Ross and Tabrizi, 2011; Walker, 2007). In addition to HD, there are eight other polyglutamine diseases: dentatorubral-pallidoluysian atrophy (DRPLA), spinal and bulbar muscular atrophy (SBMA) and the spinocerebellar ataxias (SCAs) 1, 2, 3, 6, 7 and 17. (Table 1.) These diseases have a striking genotype–phenotype correlation. Although the expected functions of the causative genes and the surrounding amino acids are quite different from each other, most of the diseases result from more than 40 polyQ repeats and share several common features, including neuronal dysfunction and loss in the central nervous system. Clinical features include ataxia and other movement disorders, loss of cognitive ability, and psychiatric disabilities (Marsh et al., 2009; Takahashi et al., 2010). Prevalence of HD is four to ten cases per 100 000 individuals in the western world (Ross and Tabrizi, 2011). Exceptions can be seen in areas where the population can be traced back to a few founders, such as Tasmania (Pridmore, 1990) and the area around Lake Maracaibo in Venezuela 2

Also 10-25% of HD patients exhibit diabetes mellitus (Farrer, 1985). As motor and cognitive deficits become severe, patients eventually die, usually from complications of falls, inanition, dysphagia, or aspiration. Typical latency from diagnosis to death is 20 years (Walker, 2007).

2.1.1 Huntingtin Huntingtin (HTT) is a large (~350kDa) multidomain protein expressed in all human and mammalian cells, with the highest concentrations in the brain and testes. Moderate amounts of HTT are present in the liver, heart, and lungs. HTT is ubiquitously distributed in neurons, particularly in cell bodies and dendrites (DiFiglia et al., 1995). Within the cell, cytoplasmic HTT is associated with mitochondria, the Golgi apparatus, the endoplasmic reticulum, synaptic vesicles and several components of the cytoskeleton (Gil and Rego, 2008). To a lesser extent HTT is also present in the nucleus (Kegel et al., 2002). The role of the wild-type (wt) protein is still poorly understood but different studies have shown that it is engaged in many intracellular functions such as: protein trafficking, vesicle transport and anchoring to the cytoskeleton, clathrin-mediated endocytosis, postsynaptic signaling, transcriptional regulation and anti-apoptotic function (Gil and Rego, 2008). HTT is also suggested to be essential for normal embryonic development; knockout mutations that disrupt the promoter, exon 4 or exon 5 of the mouse HD gene homolog (Hdh) leads to complete inactivation of the gene and finally to embryonic lethality (Duyao et al., 1995; Nasir et al., 1995; Zeitlin et al., 1995). Later it was shown that mHTT (YAC46 and YAC72) was able to compensate for the absence of wtHTT and rescue the embryonic lethality of mice homozygous for a targeted disruption of the endogenous Hdh gene (Leavitt et al., 2001). Moreover HD mutations seem not to abrogate the developmental functions of huntingtin, as HD patients appear to develop normally and the symptoms start to manifest at the later stage of life (Gil and Rego, 2008). Huntingtin is thought to have pro-survival and anti-apoptotic role in the cell. HTT protects immortalized striatal-derived cells from apoptotic stimuli by acting down-stream of mitochondrial cytochrome C release and preventing the formation of the apoptosome complex and caspase 9 and 3 activation (Rigamonti et al., 2000; Rigamonti et al., 2001).

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2.1.1.1 Structure Huntingtin does not show structural homology with any other know protein (The Huntington's Disease Collaborative Research Group, 1993). HTT is composed of 3114 amino acids depending on the amount of polyQ repeats. (Figure 1.) Characterization of full-length HTT by biophysical methods suggests that the protein is an elongated superhelical solenoid with a diameter of ∼200 Å (1 Å=0.1 nm) (Li et al., 2006). Downstream of the polyQ residues is a polymorphic polyproline (polyP) stretch. The polyP and Proline rich domains of mHTT are implicated in a number of aberrant protein interactions (Southwell et al., 2008). Multiple HEAT domains are the structural domains in HTT (Cattaneo et al., 2001). A HEAT (Htt, Elongation factor 3, the PR65/A subunit of protein phosphatase 2A, and the lipid kinase TOR) repeat is a ∼50 amino acid long motif consisting of two anti-parallel α-helices forming a helical hairpin, which assembles into a superhelix with a continuous hydrophobic core (Southwell et al., 2008; Southwell et al., 2008). HEAT repeat proteins generally mediate important protein-protein interactions involved in cytoplasmic and nuclear transport, microtubule dynamics, and chromosome segregation (Li et al., 2006). It has been predicted that HTT contains 28–36 HEAT repeats that span the entire protein (Takano and Gusella, 2002). The first 17 amino acids of HTT are part of a membrane-targeting domain. This N-terminal stretch of huntingtin has been recognized to play an important role together with the first three HEAT repeats in targeting huntingtin to various intracellular membrane-bound organelles, including the plasma membrane, mitochondria, endosomes, autophagic vesicles, the Golgi apparatus and the ER (Atwal and Truant, 2008; Kegel et al., 2005; Rockabrand et al., 2007).

Figure 1. Diagram representing huntingtin protein. Amino acid numbering is for the human sequence (accession number NP 002102). A stretch of glutamine residues near the N terminus is expanded (polyQ) in individuals affected with Huntington's disease. Unaffected individuals typically have fewer than 38 CAG repeats. Downstream of polyQ repeats are polyP stretch and ten conserved HEAT repeats. HTT is cleaved at various sites by different proteases. PolyP, polyproline; polyQ, polyglutamine; wt, wild-type

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2.1.1.2 Proteolytic cleavage of HTT Earlier studies have shown that inclusions in brains of HD patients and in HD mouse model are primarily composed of short truncated derivatives of mHTT. This raised a possibility that proteolytic cleavage of HTT plays an important role in HD pathogenesis (DiFiglia et al., 1997; Wheeler et al., 2000). Nowadays it is known that HTT protein is cleaved by several different proteases; it has caspase, Ca2+ -dependent cysteine protease calpain and aspartyl protease cleavage sites as well as less well-defined cleavage fragments. Proteolysis has been shown to increase in the presence of longer polyQ tract (Gafni and Ellerby, 2002; Sun et al., 2002). Caspase cleavage of HTT and its nuclear accumulation represent early neuropathological changes in HD patients. Caspase 3 cleaves HTT at residues 513 and 552 and produces N-terminal fragments of HTT including the polyglutamine tract. The cleavage site at residue 552 is also susceptible to caspase 2 (Hermel et al., 2004; Wellington et al., 1998). It has been found that caspases 2, 6, and 7 recruit HTT in an apoptosome-like complex. Caspases 2 and 7 bound full-length HTT while caspase 6 bound the N-terminal caspase cleavage product (Hermel et al., 2004). Caspase 6 cleavage site resides at site 586. Caspase 6 is reported to have a critical role in early pathogenesis of HD and by inhibiting cleavage of caspase 6 substrates, it is possible to influence caspase activation patterns in vivo. Mutant HTT fragments are required to initiate a toxic amplification cycle that contributes to neuronal dysfunction in HD (Graham et al., 2010). Calpains can also cleave huntingtin. Calpain cleavage site at residue 536 leads to the formation of a 72 kDa N-terminal fragment of huntingtin as an intermediate product. This fragment may be cleaved further to generate a 47 kDa product, which is small enough in size to shuttle in and out of the nucleus (Gafni and Ellerby, 2002). HTT has also been reported to be a substrate for aspartic proteases. Lunkes and colleagues showed that clearance of HTT is ensured by a multistep proteolysis involving aspartic proteases and the proteasome, which generate N-terminal mHTT fragments with high aggregation potential. This proteolysis generates smaller fragments of HTT than caspases and calpains and may be a crucial factor for the formation of intranuclear inclusions (Lunkes et al., 2002). 2.1.1.3 Transcriptional regulation and interacting proteins One of the pathogenic processes that have been suggested as the basis for neurodegeneration in HD involves interaction of huntingtin with other proteins to produce a change of function and alterations of gene transcription. Mutant HTT can interact with different nuclear proteins and transcription

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factors by recruiting them into the aggregates and thus inhibiting their transcriptional activity (Gil and Rego, 2008). Several transcription factors have been shown to interact with mHTT. These include the TBP-associated factor (TAF), specific protein-1 (sp1), CREB [cycline-adenosine monophosphate (cAMP) response element (CRE) binding protein]-binding protein and the proapoptotic transcription factor p53 (Dunah et al., 2002; Li et al., 2002; Schaffar et al., 2004; Steffan et al., 2000). More recently, expression of HTT has been shown to down-regulate transcription of peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α), a key regulator of mitochondrial biogenesis and respiration. (McGill and Beal, 2006) Decreased expression of PGC-1α has been considered because mHTT interferes with CREB/TAF4-mediated expression of PGC-1α (Cui et al., 2006; Jin and Johnson, 2010). Also the transcription of brain-derived neurotrophic factor (BDNF) was reduced in patients with HD (Zuccato and Cattaneo, 2009). Different studies have shown that wtHTT interacts with numerous proteins. A study by Kaltenbach and colleagues identified 243 high-confidence huntingtin-associated proteins. Relevant examples include huntingtin- associated protein 1 (HAP1), a novel protein with at least two isoforms (HAP1A and HAP1-B), which is expressed in several tissues including the brain and which interacts with the p150 subunit of dynactin, thus being involved in intracellular transport; huntingtin-interacting protein 1 (HIP1), a protein that binds to α–adaptin and clathrin, and is thus implicated in cytoskeleton assembly and in endocytosis; and huntingtin-interacting protein 2 (HIP2), a ubiquitinconjugating enzyme that catalyzes the covalent attachment of ubiquitin units to intracellular proteins, tagging them for degradation by the proteasome (Kaltenbach et al., 2007). The presence of an expanded polyQ tail can disrupt and/or modify the interactions between HTT and the interacting proteins and affect the normal functions of these proteins. 2.1.1.4 Post-translational modifications Post-translational modifications affect protein-protein interactions of HTT and influence its subcellular localization. The activity of both wild type and mHTT can be post-translationally modified by phosphorylation, ubiquitination, acetylation, palmitoylation and sumoylation (Gil and Rego, 2008). HTT has several phosphorylation sites and most of these modifications seem to be non-toxic. Phosphorylation at serines 13 and 16 has been shown to prevent mHTT induced disease pathogenesis in HD mouse models. Phosphorylation of HTT by Akt/protein kinase B at serine 421 reduces toxic effects of mHTT in vitro (Gu et al., 2009; Humbert et al., 2002; Rangone et al., 2004). 7

Serine 434 phosphorylation by the cyclin-dependent kinase 5 reduces caspase-mediated HTT cleavage and attenuates aggregate formation and toxicity in vitro (Luo et al., 2005). Covalent attachment of SUMO-1 (small ubiquitin-like modifier) to lysine residues is called SUMOylation whereas covalent attachment of a single ubiquitin protein or chains of ubiquitin to lysine residues is called ubiquitination. (Ehrnhoefer et al., 2011) In neurons, SUMOylation is involved in the transcriptional regulation of neuronal development and function, mitochondrial dynamics, the modulation of kinase pathways, synaptic function, and axonal trafficking (Wilkinson et al., 2010). Subramaniam et al. showed that Rhes (Ras homolog enriched in striatum) induces SUMOylation of mHTT, which leads to cytotoxicity and mHTT disaggregation (Subramaniam et al., 2009). Ubiquitination plays a role at the synapse in the targeted protein degradation during synaptogenesis, synapse elimination and synaptic plasticity (Haas and Broadie, 2008). The ubiquitin proteasome system will be discussed later in more detail. Acetylation may also control the pathogenicity of HTT. One study showed that acetylation acts as a mechanism for removing accumulated HTT protein in HD (Jeong et al., 2009). Jeong et al. showed that acetylated mHTT was trafficked to autophagosomes for degradation which reversed its toxic effects. Palmitoylation (attachment of a palmitoyl group to proteins) plays an important role in neuronal development and synaptic plasticity and can anchor proteins to the plasma membrane and other membranes in the cell (Fukata and Fukata, 2010). HTT is palmitoylated at cysteine 214 by the palmitoyl transferases HIP14 and HIP14L and a mutation rendering the protein palmitoylation resistant leads to increased inclusion body formation and nuclear localization as well as enhanced toxicity (Yanai et al., 2006).

2.1.2 Mutant huntingtin In HD patients, wt and mHTT have a similar distribution and expression in the body. Although the mutant protein is ubiquitously expressed throughout the organism, cell degeneration occurs mainly in the brain (Ross and Tabrizi, 2011; Walker, 2007). HD pathogenesis is thought to result from a combination of gain-of-function of the mHTT together with the loss-of-function of wtHTT physiological function (Cattaneo et al., 2005; Tobin and Signer, 2000). Mutant HTT, in either its soluble or insoluble aggregate form, has been shown to disrupt several intracellular pathways, which ultimately lead to cell loss in HD. This is discussed in the following sections.

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2.1.2.1 Aggregation The accumulation of nuclear and cytoplasmic protein aggregates or inclusion bodies containing mHTT N-terminal polyQ fragments is a pathological hallmark of HD. The protein aggregates have been reported in the brains of human patients, in transgenic mouse models and in transfected cell models of HD (Nucifora et al., 2001). The mechanisms by which polyglutamine aggregation leads to selective neuronal dysfunction and neurodegeneration in HD has not yet been elucidated, but several key processes have been identified. Aggregates have been considered as the cause of neuronal death based on the facts that there is a direct correlation between the frequency of inclusions and severity of the disease (Becher et al., 1998; Sieradzan et al., 1999). Prolonged mHTT production and formation of aggregates are believed to eventually overcome the ability of cells to degrade them via either autophagy or proteasomes, leading to an increased load of unmanageable aggregates (Walker, 2007). Mutant HTT is more likely to be cleaved by proteases than wtHTT and its truncation facilitates aggregation. It has been shown that aggregates of truncated HTT are toxic and likely to translocate to the nucleus (Peters et al., 1999; Wellington et al., 2000). The mechanism of HTT aggregation is complex. The normal tertiary protein conformation is destabilized by the presence of the expanded polyQ tract, which results in the formation of a toxic conformation, which includes a compact β-conformation with short β strands interspersed with β turns so that the strands are held together in antiparallel conformation by hydrogen bonds. Initial step in the aggregation seems to be accelerated by hydrophobic interactions within an amphipathic α-helical structure of 17 amino acids at the N-terminus (Tam et al., 2009; Thakur et al., 2009). Mutant HTT can have many different conformations (Kim et al., 2009; Legleiter et al., 2009; Legleiter et al., 2010). Soluble, intermediate, mHTT species are thought to be more toxic to neurons than the large visible intracellular aggregates (Ross and Tabrizi, 2011). Although mHTT aggregates are a pathological feature of HD, there has been a great controversy about the nature of the toxic species of mHTT in the pathogenesis of the disease. Aggregates have been considered as the cause of neuronal death based on the facts that there is a direct correlation between the frequency of inclusions and severity of the disease (Becher et al., 1998; Sieradzan et al., 1999). Also the formation of aggregates is shown to increase apoptosis in the cell models of HD (Wellington et al., 1998). However there are some studies suggesting that aggregates might be a cellular protective process to sequester soluble forms of toxic mHTT species. One study showed that formation of the inclusion bodies predicted 9

improved cell survival and leaded to decreased levels of mHTT elsewhere in neurons, thus suggesting that formation of aggregates could function as a coping response to toxic mHTT (Arrasate et al., 2004). Consistent with this view, in a YAC transgenic mouse model of HD, neuronal death was observed in the absence of inclusions (Leavitt et al., 2006).

2.1.3 Neuropathology HD neuropathology is characterized by the selective loss of predominantly medium-sized spiny neurons (MSNs) in the striatum and to a lesser extent the pyramidal neurons in the deep layers of the cortex (Ross and Tabrizi, 2011). In striatum, up to 95% of GABAergic medium spiny projection neurons are lost, which projects to the globus pallidus and the substantia nigra (Vonsattel, 2008). The most commonly used neuropathological classification of HD was done in 1985 by Vonsattel and colleagues. They used postmortem brain specimens from HD patients and made a grading system based on the severity of neurodegeneration. This system has five grades (0-4) designated in ascending order of severity. Grade 0 has no discernible neuropathological abnormalities. The earliest changes were seen in the medial paraventricular portions of the caudate nucleus (CN), in the tail of the CN, and in the dorsal part of the putamen. Counts of neurons in the CN reveal that 50% are lost in grade 1 and that 95% are lost in grade 4; astrocytes are greatly increased in grades 2-4 (Vonsattel et al., 1985). In stages 1 and 2 nonstriatal structures show generally a slight atrophy, whereas in grades 3 and 4 the cerebral cortex, globus pallidus, thalamus, subthalamic nucleus, substantia nigra, white matter and cerebellum can be affected (Vonsattel and DiFiglia, 1998). Also hypothalamus can be atrophied in HD patients (Kassubek et al., 2004). The overall brain weight can decrease ultimately by up to 40% in HD patients. The loss of brain weight is known to precede the loss of body weight and the onset of neurological symptoms (Vonsattel and DiFiglia, 1998). Advances in neuroimaging have given a better understanding of HD pathology. Structural neuroimaging has elucidated the correlation between morphological brain changes in striatum and cortex, and the development of cognitive deficits in attention, working memory and executive functions in HD (Montoya et al., 2006). Mutant HTT is ubiquitously expressed in the brain and it is still unknown why HD neuropathology is largely restricted to the striatum. Loss of BDNF (brain-derived neurotrophic factor) support from cortical-striatal projections and excitotoxicity has been proposed to account for striatal selectivity (Cowan and Raymond, 2006; Wang et al., 2008). Also interaction between HTT and the Rhes (ras 10

homolog enriched in striatum) protein could account for striatal selectivity. The small guanine nucleotide–binding protein Rhes, localized very selectively to the striatum, induces SUMOylation of mHTT, which leads to cytotoxicity (Subramaniam et al., 2009). SUMOylation of mHTT was shown to increase its soluble form and to elicit cytotoxicity and neurotoxicity in a Drosophila model of HD (Steffan et al., 2004). Deletion of Rhes reduces striatal degeneration and motor dysfunction in a toxin model of HD and Rhes deleted mice have delayed onset of symptomatology (Baiamonte et al., 2013; Mealer et al., 2013). A recent study showed that Rhes has a role in autophagy; deletion of endogenous Rhes decreased autophagy, while Rhes overexpression activated autophagy. These effects are independent of mTOR and opposite in the direction predicted by the known activation of mTOR by Rhes. Rhes robustly binds the autophagy regulator Beclin-1, decreasing its inhibitory interaction with Bcl-2 independent of JNK-1 signaling. The regulation of autophagy by Rhes may account for the delayed onset of HD neuropathology (Mealer et al., 2013).

2.1.4 Model systems of Huntington’s disease Our knowledge about Huntington’s disease biology mainly arises from studies of model systems, ranging from cellular models to invertebrates to mammals. All of these models have limitations but also strengths. Although not perfect, these models are essential for modeling the human disease, which is not feasible with postmortem human HD brains (Ross and Tabrizi, 2011). Different model systems are necessary in understanding the pathogenesis of HD and developing early diagnostic and therapeutic applications. 2.1.4.1 Cell models Cell lines are useful for biochemical studies and can be used for transient, stable or inducible expression strategies. Disadvantage with different cell lines is that they might not recapitulate the cell biology of neurons, yet primary neurons can be used to model many features of neurons in vivo (Ross and Tabrizi, 2011). The HD Consortium reported the generation and characterization of 14 induced pluripotent stem cell (iPSC) lines from HD patients and controls. Through an international consortium effort involving eight research groups, it was shown that HD iPSC lines had clear, reproducible CAG repeat- expansion-associated phenotypes upon differentiation. The utility of this model system includes elucidation of HD cellular pathogenesis, development of HD specific biomarkers, and ultimately screening for small molecule or other therapeutic interventions (HD iPSC Consortium, 11

2012). One recent study showed that iPSCs derived from HD patient fibroblasts can be corrected by the replacement of the expanded CAG repeat with a normal repeat using homologous recombination. The corrected cells retained pluripotent characteristics and were able to be differentiated into striatal neurons. Correction of the HD-iPSCs normalized pathogenic HD signaling pathways and reversed disease phenotypes such as susceptibility to cell death and altered mitochondrial bioenergetics in neural stem cells. (An et al., 2012) 2.1.4.2 Animal models Animal models of Huntington’s disease are useful in the study of the early disease stages for which patient material is rarely available. First animal models for HD were developed already in the 1970s on the basis of selective vulnerability of striatal neurons to exitotoxic aminoacids (Coyle and Schwarcz, 1976). Nowadays there are several commercially available genetically engineered animals carrying part or the full length of the mutated gene causing Huntington disease (HD). Transgenic animal models for HD were first created in mice and subsequently in invertebrates (Bates et al., 1997; Mangiarini et al., 1996; Marsh et al., 2003). Invertebrate models of HD include Drosophila and Caenorhabditis elegans. These models are useful for rapid tests of therapeutic interventions. However the extent to which they resemble human HD biology is uncertain (Marsh et al., 2003; Ross and Tabrizi, 2011). Intranuclear inclusions of mHTT were first discovered in the R6/2 mouse model, which expresses the first exon of the human HD gene carrying 141–157 CAG repeat expansions. Mouse models expressing N-terminal fragments of HTT (for example the R6/2 model and the N171-82Q model) seem to have the most robust and rapidly progressive phenotypes, including incoordination, cognitive and other behavioral abnormalities and weight loss progressing to early death. Thus these models have frequently been used in therapeutic trials (Heng et al., 2008; Ross and Tabrizi, 2011). Mice expressing flHTT (BAC and YAC mouse models) generally present more subtle phenotypes than mice expressing N-terminal fragments, but have more selective neurodegeneration. The BAC, YAC and knock-in models are useful for studies where the entire HTT protein is needed, such as studies of cleavage of flHTT and studies of stages before behavioral and pathological phenotypes (Ross and Tabrizi, 2011). In addition to mouse models, a transgenic rat model carrying N-terminal HTT (51Q) is available (von Horsten et al., 2003). This model displays adult-onset neurological phenotypes with reduced anxiety, cognitive impairments, and slowly progressive motor dysfunction as well as neuronal nuclear inclusions in the brain and aggregates in the striatum. HD transgenic rats are well suited for complex behavioral studies and the evaluation of in vivo progression markers 12

using high-resolution PET and MRI (von Horsten et al., 2003). In conclusion, a variety of different rodent models of HD are available and different features of these models confer different advantages and disadvantages, depending on specific experimental aims (Heng et al., 2008). HD non-human primate model was established in a rhesus macaque using a lentiviral vector to deliver green fluorescent protein-tagged HTT exon 1 with 84 CAG repeats into unfertilized monkey egg cells. These transgenic monkeys have hallmark features of HD, including nuclear inclusions and aggregates, in the brain, and showed important clinical features of HD including dystonia and chorea. Thus the transgenic HD monkeys may provide the opportunity for a wide range of behavioral and cognitive assessments that are identical to or close to the assessments used for human patients (Yang et al., 2008). The sheep, Ovis aries L., is another mammalian model for studying HD. This ovine model was generated through a ligation of a FL human HTT cDNA containing 73 polyglutamine repeats under the control of the human promoter into the genome using pronuclear microinjection. This novel transgenic animal could represent a practical model for drug/clinical trials and surgical interventions especially aimed at delaying or preventing HD initiation (Jacobsen et al., 2010). Although useful in research, transgenic animal models are not always applicable to human HD because of species differences and variations in HTT gene length, promoters, and mechanisms of expression. Animal models are usable in drug screenings as an animal with a lifespan of days or months elucidates a fast screening of different compounds. (Bates et al., 1997; Walker, 2007).

2.1.5 Treatment and therapy Although the genetic basis of the disease has been identified, our understanding of the HD pathogenesis is still incomplete, and there is no cure or treatment to delay the onset or slow the progression of the disease. Reducing the amount of mHTT is believed to be an effective strategy against HD. However there are only few validated targets that regulate mHTT protein levels. Many clinical trials in HD have been done in the past ten years, but no drugs have been proven to be efficacious. Clinical trials are really challenging, because of the slow progress of HD and its clinical heterogeneity (Mason and Barker, 2009; Ross and Tabrizi, 2011). Most studies have been openlabel, using a small number of patients and tended to concentrate on the motor features of the

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disease, primarily the chorea (Mason and Barker, 2009; Ross and Tabrizi, 2011). The best therapeutic option for HD treatment is to start in the asymptomatic phase of the disorder. Currently there are only symptomatic drugs available for treating the illness. (Table 2.) Dopamine, glutamate, and γ-aminobutyric acid are thought to be the most affected neurotransmitters in HD and are currently the focus of pharmacotherapy. Pharmacological interventions typically address the hyperkinetic movement disorders that may be associated with HD, such as chorea and dystonia (Frank, 2013). To date, tetrabenazine (TBZ), a dopamine depleting agent, is the only pharmacotherapy shown to have a clinically meaningful, statistically significant effect on chorea (Pidgeon and Rickards, 2013). 2.1.5.1 Potential new treatments There are a number of ongoing or recently completed clinical studies in HD (Frank, 2013). However, recently completed studies have shown no or little benefit for cognitive or motor symptoms (Huntington Study Group TREND-HD Investigators, 2008; Kieburtz et al., 2010). Hence there is a need for treatment options that could change the course of the disease. Some ongoing studies are evaluating supplements that may affect metabolism or mitochondrial function implicated in HD to potentially change the course of disease. The largest include the 5-year study of coenzyme Q10 (2CARE) and the 3-year study of creatine (CREST-E). The green tea extract polyphenon (2)epigallocatechin-3-gallate is under study for its effect on cognition in patients with HD (Frank, 2013; Yang et al., 2009). Another potential intervention under study is the use of RNA interference to reduce the expression of mHTT. RNA interference may be delivered using a viral vector or through direct infusion into the basal ganglia, and both systems are under study, although not yet in humans (Drouet et al., 2009; Frank, 2013). Cell-replacement therapy for HD has given positive outcomes in clinical trials. In this therapy lost striatal neurons are replaced by fetal medium spiny neuron transplants or diverse stem cell transplants. These experiments were done using human fetal transplants grafted into animal hosts. There are still many issues to be addressed concerning reproducibility, stability, safety and quality control, as well as the more complex issues involving directed differentiation into the precise neuronal phenotypes required to replace the function of lost striatal neurons connected within complex neuronal circuits of the host basal ganglia (Kelly et al., 2009).

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Studies with cellular and animal models of HD have shown that rapamycin can protect against neurodegeneration induced by aggregated mHTT, by enhancing their clearance via autophagy. Rapamycin is a US Food and Drug Administration (FDA)-approved antibiotic and immunosuppressant drug. It induces autophagy by inhibiting the activity of mammalian target of rapamycin (mTOR) and attenuates mHTT toxicity in various HD models (Bove et al., 2011). Several mTOR-independent inducers of autophagy had similar beneficial effects than rapamycin, including lithium, trehalose and a series of autophagy-inducing compounds identified from either a small-molecule yeast screen or a library of FDA-approved drugs, (Bove et al., 2011; Sarkar et al., 2005; Sarkar et al., 2007; Sarkar and Rubinsztein, 2008). Effects of rapamycin in mHTT clearance are mostly observed at early stages of protein aggregation in the presence of micro aggregates and soluble HTT. This indicates that treatment with rapamycin should be aimed at early stages of the disease, when large, highly stable aggregates have not yet been formed. Pre-symptomatic treatment with autophagy inducers could potentially be feasible in HD, and eventually delay, or even prevent, disease onset (Bove et al., 2011). Autophagy in the context of HD will be discussed in more detailed in later chapters.

2.2 Endoplasmic reticulum stress Secreted and membrane proteins are synthetized in the ribosomes bound to ER. After that they fold and mature in the ER before their delivery to other compartments in the endomembrane system, display on the cell surface or release to the extracellular space. The rates of protein synthesis, folding and trafficking are controlled to ensure that only properly folded proteins exit the ER (Tabas and Ron, 2011; Wang and Kaufman, 2012). Disturbance in the function of this organelle leads to the accumulation of unfolded or misfolded proteins inside the ER, a cellular condition referred as ER stress (Hetz et al., 2013). Factors that can contribute to the development of ER stress include; increased protein synthesis or protein misfolding rates that exceed the capacity of protein chaperones, alterations in calcium stores in the ER lumen, oxidative stress and disturbances to the redox balance in the ER lumen (Tabas and Ron, 2011). Misfolded proteins are either retained within the ER or degraded by the proteasome-dependent ER-associated protein degradation (ERAD) pathway or by autophagy (Wang and Kaufman, 2012). ER stress can induce autophagy for removal of unfolded proteins independently of the ubiquitin/proteasome system, which may be particularly important when severe protein misfolding results in insoluble protein aggregates that cannot be eliminated by the proteasome (Kim et al., 2008).

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ER stress is sensed by three upstream signaling proteins that trigger different cascades. The activity of these three pathways collectively constitutes an ER-specific unfolded protein response (UPR). Activation of the UPR strives to restore protein-folding homeostasis, but when the cell damage is sufficiently severe, UPR signaling results in cell death by apoptosis (Hetz et al., 2013; Tabas and Ron, 2011). Dysfunction of ER quality control system causes conformational diseases (caused by adoption of non-native protein conformations that lead to aggregation) such as diabetes mellitus, ischemic diseases and neurodegenerative disorders, like Huntington’s disease. Thus, the understanding of the molecular mechanisms of the ER stress may provide insight on the potential therapeutic target for diseases (Hetz et al., 2013).

Figure 2. The Unfolded protein response. Three sensors control UPR-dependent responses; IRE1α, PERK and ATF6. Each of these ER transmembrane proteins uses a different mechanism of signal transduction: ATF6 signals via regulated proteolysis, PERK via translational control and IRE1 via nonconventional mRNA splicing. A) IRE1α RNase activity processes the mRNA encoding XBP1. This leads to the expression of an active transcription factor (XBP1) that upregulates a subset of organelle biogenesis. IRE1α also degrades select mRNAs through a process called regulated IRE1-dependent decay pathway. In addition, IRE1α activates the JNK-ASK1pathway through the binding to adaptor proteins. B) Activation of PERK attenuates general protein synthesis through phosphorylation of eIF2α, which allows the selective translation of the ATF4 mRNA encoding a transcription factor that induces the expression of genes involved in antioxidant responses, amino acid metabolism, autophagy and apoptosis. ATF4 controls the expression of the pro-apoptotic components GADD34 and CHOP. GADD34 dephosphorylates eIF2α. C) ER stress induces translocation of ATF6 to Golgi apparatus where it is processed by a site 1 protease (S1P) and site 2 protease (S2P) releasing its cytosolic domain (ATF6f) which controls the upregulation of select UPR target genes. Modified from (Hetz et al., 2013; Walter and Ron, 2011).

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2.2.1 Unfolded protein response In mammals there are three UPR sensors: inositol-requiring enzyme 1 (IRE1), protein kinase RNAlike ER kinase (PERK) and activating transcription factor 6 (ATF6) (Figure 2.) (Hetz et al., 2013). Each UPR sensor binds to the ER luminal chaperone Bip. When misfolded proteins accumulate in the ER, they bind to and sequester Bip (immunoglobulin binding protein, also called Grp78) and activate the ER stress sensors (Bertolotti et al., 2000). However, additional mechanisms that initiate and modulate the activity of different UPR sensors have been reported (Gardner and Walter, 2011; Promlek et al., 2011). While IRE1, PERK and ATF6 activation proceeds independently in ERstressed cells, the three arms of the UPR communicate with each other extensively. For example CHOP is a transcriptional target not only for ATF4 (PERK pathway) but also XBP-1 (IRE1 pathway) and ATF6 (Tabas and Ron, 2011). 2.2.1.1 IRE1 The IRE1 branch is the most conserved pathway of the UPR and present from lower eukaryotes to human. In mammals IRE1 has two homologues, IRE1α and IRE1β. IRE1α is expressed in all cells and tissues, whereas IRE1β is expressed only in the gastrointestinal and respiratory tracts. The function of IRE1β is still unknown and UPR signaling is mainly mediated through IRE1α (Wang and Kaufman, 2012). Under ER stress, IRE1 is activated which involves its transautophosphorylation, oligomerization and dimerization. These conformational changes lead to activation of its cytoplasmic kinase and ribonuclease domains. Activation of IRE1 is probably initiated both by ER stress induced changes in protein interactions, such as dissociation of Bip and by direct binding to unfolded proteins (Ron and Walter, 2007). Active cytoplasmic nuclease domain of IRE1 cleaves the mRNA encoding a UPR-specific transcription factor, called XBP1 (X-box binding protein 1). The resulting spliced mRNA is translated to the active form of the transcription factor XBP1, which targets a wide variety of genes encoding proteins involved in ER membrane biogenesis, protein folding, ERAD, and protein secretion from the cell (Wang and Kaufman, 2012). Under certain conditions, the IRE1 nuclease can degrade and block the translation of several mRNA species. The possible physiologic and pathologic functions of this action are still poorly understood (Hollien and Weissman, 2006; Hollien et al., 2009). In addition IRE1 can cleave microRNAs, which affect the regulation of apoptosis (Upton et al., 2012). IRE1 promotes not only the UPR but also the apoptotic signaling. IRE1α interacts with TNF (the adaptor tumor necrosis factor) receptor-associated factor 2 (TRAF2) to instigate the downstream

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activation of ASK1 (apoptosis signal-regulating kinase 1) and JNK (JUN N-terminal kinase) (Han et al., 2009; Hetz et al., 2013). 2.2.1.2 PERK Activation of PERK involves trans-autophosphorylation and dimerization, similar to IRE1 activation. Under ER stress active PERK phosphorylates eukaryotic translation initiator factor 2α (eIF2α), which leads to the inhibition of protein synthesis. This has a pro-survival role in the cell, as it reduces the number of proteins entering the ER (Harding et al., 2000). Phosphorylation of eIF2α can also change the efficiency of AUG initiation codon utilization and allow the translation of mRNAs containing short open reading frames in their 5’-untranslated regions, such as activating transcription factor 4 (ATF4) (Lange et al., 2008). ATF induces the expression of genes involved in ER function but also in apoptosis, including the transcription factor C/EBP-homologous protein (CHOP) and growth arrest and DNA damage-inducible protein 34 (GADD34) (Hetz et al., 2013). A recent study showed that transcriptional induction through ATF4 and CHOP increased protein synthesis leading to oxidative stress and cell death. They showed that increased protein synthesis generates ROS, which are a necessary signal to induce apoptosis in response to ER stress. Their findings suggested that limiting protein synthesis would be therapeutic for diseases caused by protein misfolding in the ER (Han et al., 2013). Phosphorylation of eIF2α is a key mechanism to slow protein translation and prevent oxidative stress and apoptosis under prolonged ER stress. GADD34 promotes the dephosphorylation of eIF2α and thus restores protein translation and represents a pro-apoptotic mechanism (Han et al., 2013). 2.2.1.3 ATF6 ATF6 is an ER-associated type 2 transmembrane basic leucine zipper transcription factor. Under ER stress ATF6 dissociates from Bip and translocates to the Golgi apparatus for cleavage by serine site1 protease (S1P) and site 2 protease (S2P), which releases the transcription-activating form of ATF6, ATF6f (Schindler and Schekman, 2009). Among other target genes, ATF6f regulates the expression of genes of the ERAD pathway and genes encoding ER-resident proteins involved in protein folding (Lee et al., 2002).

2.2.2 Endoplasmic reticulum stress in HD The role of ER stress in the nervous system is not fully understood but activation of the UPR is observed in several neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, and Huntington’s disease (Lindholm et al., 2006). 19

Several studies suggest a possible involvement of ER stress in the pathogenesis of HD, however the actual contribution of the UPR to the disease process in vivo is still poorly defined. Carnemolla and colleagues showed in a mouse model of HD that ER stress is an early event, which precedes the formation of intranuclear and/or cytoplasmic mHTT aggregates. They also confirmed ER stress in the postmortem brains of HD patients, by observing the transcriptional regulation of UPR genes, like CHOP and Bip (Carnemolla et al., 2009). Many studies have also shown that ER stress contributes to neurodegeneration in cellular models of HD (Kouroku et al., 2002; Urano et al., 2000; Vidal et al., 2011). In neuronal PC6.3 cells mHTT expression was shown to induce ER stress with elevation of Bip, cleavage of caspase-12 and the activation of different ER signaling pathways including the CHOP and the JNK pathways (Reijonen et al., 2008). HTT functions as an ER-associated protein. It has a domain within the first 18 amino acids that has an amphipathic α-helical structure able to reversibly associate with ER. HTT can translocate to the nucleus and back out in response to ER stress (Atwal and Truant, 2008). ER stress response to temperature shift or induction of the UPR seems to release HTT from the ER in a striatal-derived mouse cell line (Atwal and Truant, 2008). Although HTT interacts with the surface of the ER, it has not been described inside the ER lumen. There is evidence of dysfunction in many cellular pathways in different HD models, including ERAD/protein quality control mechanisms, ER/Golgi trafficking, endocytosis, vesicular trafficking, ER calcium homeostasis and autophagy/lysosomalmediated protein degradation. These defects are predicted to impact the protein folding status in the ER and to generate ER stress (Vidal et al., 2011). Therapeutic strategies to alleviate ER stress may be beneficial for HD patients. In a cellular HD model, inhibition of ER stress by the compound salubrinal, a specific inhibitor of eIF2α phosphatase enzymes, reduced ER stress and counteracted cell degeneration. Salubrinal also reduced mHTT aggregates, suggesting that inhibition of ER stress may directly or indirectly regulate the accumulation of protein aggregates in the cell and in the membrane (Reijonen et al., 2008). A recent study indicated a protective effect of XBP1 deficiency in neurodegeneration using knockout mice and suggested a potential use of gene therapy strategies to manipulate the UPR in the context of HD (Zuleta et al., 2012).

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2.3 Cell Death Cell death is classified into two categories according to their morphological structures: apoptosis and necrosis. Whereas apoptosis is considered as a controlled, active form of cell death, necrosis is an uncontrolled and passive breakdown of cellular contents (Kroemer et al., 2005). However certain cell types have been shown to undergo “programmed necrosis” or “necroptosis” which is a programmed necrosis that involve specific molecular machinery (Bizik et al., 2004). Necroptosis participates in the pathogenesis of diseases, including ischaemic injury, neurodegeneration (Vandenabeele et al., 2010). The initiation of “necroptosis” involves many proteins, including Tumor necrosis factor (TNF) and caspase inhibitors. Excitotoxicity, oxidative stress and mitochondrial dysfunction, all contribute to the execution of necroptosis and are also implicated in Huntington's disease (Lin and Beal, 2006; Vandenabeele et al., 2010). Autophagy, discussed later, has been morphologically defined as a type of cell death, but its role in executing cell death is controversial (Kroemer et al., 2005).

Figure 3. Extrinsic and intrinsic apoptotic pathways. The extrinsic pathway involves extracellular signals through cellular death receptors that cause intracellular interactions that lead to cell death. Cell death is also caused intrinsically. Any type of stress in the endoplasmic reticulum (ER), such as an accumulation of unfolded and unstable proteins or a disruption in the homeostasis of the ER can cause the activation of caspase 12, which then cleaves caspase 9 initiating the death cascade. Cytochrome C release is initiated via the intrinsic pathway in which intracellular death signals are translocated to the mitochondria stimulating the release of apoptogenic factors. Upon release from the mitochondria, cytochrome C interacts with cytosolic protein factors, Apaf1 and procaspase 9, and the complex begins the cascade which is completed by the executioner caspases, such as caspases 3, 6, and 7 in the cascade result in cell death. Calpain is activated by intracellular Ca2+ overload. Once activated, calpain hydrolyses its substrates in the cytosol, nucleus and membrane, resulting in apoptosis. Modified from (Pattison et al., 2006).

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Apoptosis is a genetically regulated form of cell death with distinct biochemical and morphological features. Deregulation of apoptosis causes several human disorders including cancer, autoimmune and neurodegenerative diseases (Agostini et al., 2011). Apoptosis was first defined by Kerr and colleagues in 1972. They stated that the structural changes during apoptosis occur in two discrete stages. The first comprises nuclear and cytoplasmic condensation and breaking up of the cell into a number of membrane-bound, ultra structurally well-preserved fragments. In the second stage, these apoptotic bodies are shed from epithelial-lined surfaces or are taken up by other cells, where they undergo a series of changes resembling autolysis within phagosomes, and are rapidly degraded by lysosomal enzymes derived from the ingesting cells (Kerr et al., 1972). Other features of apoptosis are a reduction in the membrane potential of the mitochondria, intracellular acidification, generation of free radicals, and externalization of phosphatidylserine residues on the plasma membrane (Agostini et al., 2011). The major executioners in the apoptotic program are proteases known as caspases (cysteinedependent aspartate-specific proteases). Caspases are cysteine proteases that exist as latent precursors, which, when activated, initiate the death program by destroying key components of the cellular infrastructure and activating factors that mediate damage to the cells. Procaspases are composed of p10 and p20 subunits and an N-terminal recruitment domain. Active caspases are heterotetramers consisting of two p10 and two p20 subunits derived from two procaspase molecules. A critical aspect of caspase-mediated cell death lies in the events regulating the activation of initiator caspases. Two major pathways exist by which initiator caspases lead to cell death - one is extrinsic, the other intrinsic (stress or mitochondria-mediated) pathway (Figure 3.). The extrinsic pathway involves extracellular signals through cellular death receptors that cause intracellular interactions that lead to cell death. In HD caspases are also transcriptionally up-regulated (Pattison et al., 2006). Other proteases called calpains are also involved in the apoptotic signaling process. Calpains are calcium-activated cysteine proteases that are involved in neuronal cell death and activated by increased intracellular calcium from the ER or mitochondria or an influx of extracellular calcium. Calpains are known to cleave many important regulators of apoptosis and to cross-talk with caspase cascades (Harwood et al., 2005; Kidd et al., 2000). Activated calpains have been detected in striatum of HD knock-in mouse models and postmortem HD human brain (Cowan and Raymond, 2006; Gafni and Ellerby, 2002; Gafni et al., 2004).2.3.1 Cell death induced by ER stress

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Cell death is induced under prolonged ER stress, typically by apoptosis. However, ER-stressinduced cell death can proceed even without caspase activity, and the cell death mechanisms triggered as a result of ER stress are diverse, involving both caspase-dependent apoptosis and caspase-independent necrosis (Kim et al., 2008). ER stress-induced cell death is a complex and highly regulated process carefully controlled by ER localized stress receptors. Based on recent studies IRE1α and PERK signals are important in cell death commitment (Logue et al., 2013). Prolonged activation of IRE1 and CHOP can trigger apoptosis. At high level of stress IRE1 contributes to the degradation of membrane-associated mRNAs through a process called regulated IRE1 dependent decay (RIDD) which may be a mechanism of apoptosis and other possible link between IRE1 and ER stress induced apoptosis is the interaction of IRE1 with different proteins involved in apoptosis signaling. For example IRE1 may be involved in the ER stress-induced apoptosis by interacting with the B-cell leukaemia/lymphoma 2 (BCl-2) family of proteins, a diverse group of proteins that regulate cell death. The pro-apoptotic proteins BCl-2-associated X protein (BAX) and BCl-2 antagonist/killer (BAK) are reported to physically interact with and activate IRE1α. BAX and BAK are pro-apoptotic and they initiate intrinsic apoptosis through the release of cytochrome C and assembly of the apoptosome (Figure 3.). The IRE1α–ASK1–JNK signaling pathway is also connected to a cell death mechanism, whereby JNK-mediated phosphorylation has been reported to activate the pro-apoptotic BCl-2 family member BIM, while inhibiting the antiapoptotic protein BCl-2. IRE1 activation also triggers the recruitment of caspase 12, which different studies have claimed functionally important in ER-stress-induced apoptosis (Kim et al., 2008; Lei and Davis, 2003; Szegezdi et al., 2003; Tabas and Ron, 2011). Loss of PERK-mediated eIF2α phosphorylation markedly sensitizes cells to death from ER stress. However, not all of the effectors of this arm of the UPR contribute to protection; a notable exception is the transcription factor CHOP, which is itself transcriptionally induced by eIF2α phosphorylation. CHOP operates downstream of all ER-stress pathways. The IRE1–ASK1–p38 MAPK pathway enhances CHOP activity at a post-transcriptional level. Overexpression of the CHOP protein is shown to induce apoptosis through a mechanism that could be inhibited by BCl-2 (McCullough et al., 2001; Wang and Ron, 1996). Another mechanism implicated in CHOP-induced apoptosis is oxidative stress. Prolonged ER stress can both hyperoxidize the ER lumen, which may result in H2O2 leakage into the cytoplasm, and directly induce cytotoxic reactive oxygen species (ROS) in the cytoplasm. Oxidation of the ER lumen is induced by the CHOP transcriptional target ER oxidase 1α (ERO1α). The CHOP-ERO1α pathway can induce pro-apoptotic oxidative stress (Tabas and Ron, 2011). 23

2.4 Oxidative stress in HD It has been speculated that there is energetic impairment in HD because the weight loss of the patients despite sustained caloric intake. Further, PET scans revealed marked reductions in glucose metabolism in the striatum of HD patients in early stages prior to pronounced striatal atrophy. These studies revealed that energy dysfunction precedes the onset of clinical symptoms of the disorder, suggesting that an energy failure may play a primary role in the pathogenesis of HD (Browne, 2008; Kuwert et al., 1990; Quintanilla and Johnson, 2009). Several different studies have demonstrated that alterations in mitochondrial function play a key role in the pathogenesis of HD. The net results of these events are compromised energy metabolism and increased oxidative damage, which eventually contribute to neuronal dysfunction and death (Andersen, 2004). Studies in HD patients and HD post-mortem tissue have shown decrease/dysfunction in the activities of different mitochondrial complexes (Gil and Rego, 2008). Mutant HTT was also shown to disrupt mitochondrial-dependent Ca2+ handling (Oliveira et al., 2006). In addition mHTT can affect mitochondrial function by inhibiting the expression of PGC-1α, which is related to impaired ATP production and a reduction in intact mitochondria (Weydt et al., 2006). PGC-1α is a transcriptional co-regulator of mitochondrial biogenesis and antioxidant enzymes. The suppression of PGC1-α by mHTT is also cause of mitochondrial-dependent generation of reactive oxygen species (ROS).

Figure 4. Effect of increased ROS production in neurodegeneration. mHTT causes damage to mitochondria, which then produce harmful ROS, which in turn damage more mitochondria. Modified from (Andersen, 2004).

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2.4.1 Reactive oxygen species and antioxidants ROS are highly toxic to cells. ROS include superoxide, hydroxyl and peroxyl free radicals, as well as nitrogen intermediates (NO and peroxynitrile). Oxidative stress can lead to a toxic increase in ROS production causing damage to cell membranes and aborting normal cellular functions. (Figure 4.) ROS can inflict oxidative molecular damage to lipids, proteins and nucleic acids. Mitochondria are the major source of ROS. Apart from mitochondria, the ER produces ROS during normal cell metabolism and protein folding. Because of the high metabolic rate and relatively reduced capacity for cellular regeneration, the brain is particularly susceptible to oxidative stress and ROS (Andersen, 2004; Johri and Beal, 2012). Oxidative alterations to proteins may cause increased protein misfolding and impaired degradation, leading to toxic accumulation of insoluble mHTT aggregates in the brain and ultimately to neurodegeneration. Increased oxidative damage and an increased production of ROS have been reported in cellular an animal models of HD (Perez-Severiano et al., 2002; Reijonen et al., 2010). The burden of ROS production is largely counteracted by an antioxidant defense system including the enzymatic scavengers superoxide dismutase (SOD), thioredoxins 1 and 2 (Trx1, Trx2), catalase and glutathione peroxidase. SOD speeds the conversion of superoxide to hydrogen peroxide, whereas catalase and glutathione peroxidase convert hydrogen peroxide to water and thioredoxins catalyze reduction of disulfides. The balance between ROS production and antioxidant defenses determines the degree of oxidative stress (Finkel and Holbrook, 2000). Antioxidants have been shown to be effective in slowing disease progression in transgenic mouse models of HD, and have shown promise in human clinical trials. Strategies to transcriptionally increase the expression of antioxidant enzymes by increasing the expression of PGC-1α have also been proposed as a therapeutical alternative to slow or halt the progression of HD (Johri and Beal, 2012).

2.4.2 Oxidative stress and cell death Apart from being a major contributor to oxidative stress, mitochondria are also central players in the process of caspase activation and apoptosis. HTT is known to associate with the outer mitochondrial membrane and a truncated mHTT fragment can directly induce the opening of the mitochondrial permeability transition pore (MPTP) in isolated mouse liver mitochondria, which is accompanied by a significant release of cytochrome C (Choo et al., 2004). Cytochrome C leads to caspase activation which in turn can cleave mHTT and promote its translocation into the nucleus, where it abnormally interacts with several transcription factors. Transcription factor p53 regulates the expression of 25

various mitochondrial proteins such as Bax and interacts with mHTT in HD lymphoblasts and in neuronal cells expressing mHTT (Bae et al., 2005; Steffan et al., 2000). In neuronal cultures, mHTT induces an upregulation of the nuclear levels of p53 and enhance its activity. Interestingly, p53 levels are also increased in HD transgenic mice and in HD patients. Thus, it is likely that mHTTinduced increase in p53 activity induces further mitochondrial abnormalities that contribute to HD (Bae et al., 2005). Proteins that prevent or promote apoptosis can affect intracellular Ca2+ dynamics and homeostasis through binding and modulation of the intracellular Ca2+ release and Ca2+ uptake mechanisms. Oxidative stress becomes an additional factor that affects ER and mitochondrial function and thus their role in Ca2+ signaling (Decuypere et al., 2011). The expression of Bcl-2, the major antiapoptotic member of the Bcl-2 family, is under complex control of several factors, including ROS. Bcl-2 prevents apoptosis caused by a variety of cellular stresses such as oxidative stress. It localizes at mitochondrial outer membranes, as well as to the ER. One of the primary actions of Bcl2 is to block homodimerization of proapoptotic Bax at mitochondria. Bcl-2 also regulates the Ca2+ mobilization at the ER (Meunier and Hayashi, 2010). ROS is known to decrease the expression of bcl-2 mRNA by promoting CRE-binding protein (CRBP) or NF-κB to CRE and κB sequences on the bcl-2 promoter (Pugazhenthi et al., 2003). Downregulation of NF-κB signaling was linked to decreased antioxidant levels, increased oxidative stress, and enhanced cell death in neuronal cells expressing N-terminal mutants and the disease-inducing variant of full-length HTT (Reijonen et al., 2010). Several studies have reported impairment of the intracellular Ca2+ modulation in HD. Mutant HTT has been associated with the altered expression of some genes involved in Ca 2+ homeostasis both in human patients and in HD animal models. Direct binding of mHTT to proteins involved in Ca2+ handling has also been reported. However, it is still unclear whether the transcriptional effects in HD neurons are a cell adaptation response to the variations of intracellular Ca2+, which could be due to the direct interaction of mHTT with Ca2+ binding/ Ca2+ transport proteins, or whether the changes of Ca2+ levels are instead a secondary consequence of primary effects of mHTT on the expression of regulatory proteins (Giacomello et al., 2013).

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2.4.3 Sigma-1 receptor Communication between the ER and mitochondria is important for cellular survival. The ER supplies Ca2+ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP3Rs) at close contacts between the two organelles called mitochondrion-associated ER membrane (MAM). Mitochondrial Ca2+ contributes to many physiological events, including bioenergetics, neuroplasticity, and cell death (Hayashi and Su, 2007). The sigma receptors are ER proteins classified into at least two subtypes; sigma-1 and sigma-2 (Hayashi and Su, 2004). The sigma-1 receptor (Sig1R) has been cloned (Hanner et al., 1996) but the sequence of the sigma-2 receptor remains unknown. Hayashi and Su (Hayashi and Su, 2007) have shown that Sig-1R is a chaperone protein located at the MAM and forms a Ca2+-sensitive chaperone-complex with Bip prolonging Ca2+ signaling from ER into mitochondria by stabilizing IP3Rs. In nervous systems, Sig-1R also regulates neuritogenesis, K+ channels, memory, and drug addiction (Hayashi and Su, 2004; Hayashi and Su, 2007). Apart from the nervous system, Sig-1Rs are expressed in peripheral organs, including liver and pancreas, and in cancer cells (Hanner et al., 1996; Vilner et al., 1995). Sig1R is upregulated by ER stress and overexpression of Sig1R is known to regulate UPR signaling (Hayashi and Su, 2007). It is also known that Sig1Rs can translocate from MAM to other parts of the ER, which could be provoked by ER stress. Knockdown of Sig1R was shown to promote apoptosis induced by ER stress and by oxidative stress (Hayashi and Su, 2007; Meunier and Hayashi, 2010). Previous studies have demonstrated potent anti-apoptotic actions of Sig-1Rs in neurodegeneration caused by β-amyloid and ischemia (Marrazzo et al., 2005; Vagnerova et al., 2006). Nevertheless, the basic molecular action of Sig-1Rs remains unknown and very little is known about its function in HD. Although the mechanisms involved in neuroprotective properties of Sig-1Rs are poorly known, stimulation of Sig-1Rs was reported to protect against oxidative stress. For example, siRNA-mediated Sig-1R knockdown decreased free radical scavenging activity in neuronal cell lysates (Tsai et al., 2009). Another study demonstrated that chloroquine, a widely used anti-malarial and anti-rheumatoid agent, at concentrations below its ability to inhibit autophagy and induce cell death, was able to rescue cells from glutamate-induced oxidative stress and cell death via Sig-1R (Hirata et al., 2011). Sig-1Rs are also suggested to play a critical role in the regulation of ROS levels. Sig-1R has also been shown to promote cell survival by transcriptionally regulating bcl-2 expression via the ROS-inducible transcription factor NF-κB pathway (Meunier and Hayashi, 2010). 27

Although more studies are needed, Sig1-Rs may have therapeutic potential in treating oxidative stress-related neurodegenerative diseases. The level of Sig1Rs was shown to decrease in different neurodegenerative diseases. A postmortem study reported that Sig1Rs were reduced in the hippocampus in Alzheimer’s disease (Mishina et al., 2008). Sig-1R protein levels were also decreased in the early Parkinson’s patients (Mishina et al., 2005). Another study reported a novel mutation in Sig1R in the patients of juvenile Amyotrophic Lateral Sclerosis (ALS) (Al-Saif et al., 2011). Sigma-1R is also considered to be involved in aging, schizophrenia and depression (Mishina et al., 2008). Several selective Sig1R ligands have been synthesized, since the Sig1R binding sites were identified in 1982 (Su, 1982). Some of them have been shown to promote cellular survival by preventing oxidative stress caused by ischemia (Schetz et al., 2007), and by protecting from β-amyloid toxicity (Meunier et al., 2006). The compound PRE084 (2-(4-morpholino)ethyl1-phenylcyclohexane-1carboxylate hydrochloride) is a phencyclinide derivative and a highly selective ligand for the sigma1 receptor (Su et al., 1991). PRE084 has been shown to promote neuronal survival (Guzman-Lenis et al., 2009; Penas et al., 2011). A recent study reported that PRE084 also improves motor function and motoneuron survival in ALS mice (Mancuso et al., 2012). In a recent study immunohistochemical investigations were performed to clarify the localization of Sig-1R in the brains of patients with neurodegenerative disorders. The study showed that Sig-1R is consistently co-localized with neuronal nuclear inclusions in five polyglutamine diseases. The results indicated that Sig-1R shuttles between the nucleus and the cytoplasm, but during ER stress it relocates into the nucleus. Under normal conditions, HTT also shuttles between the ER and the nucleus, and regulates autophagy triggered by ER stress. However, mHTT loses its ability to return to the ER and starts to aggregate in the nucleus (Miki et al., 2013).

2.5 Autophagy Autophagy is a process by which organelles and proteins are delivered in autophagosomal vesicles to the lysosomes for degradation. Autophagy is categorized to three different types; macroautophagy, microautophagy and chaperone-mediated autophagy (CMA) (Chen and Klionsky, 2011). In macroautophagy, here referred to simply as autophagy, cytoplasmic components are engulfed by a double-membrane structure, the phagophore, which expand and fuses to form a double-membrane vesicle called the autophagosome. Autophagosomes fuse with lysosomes and 28

form autolysosomes, in which the content is degraded by hydrolases (Figure 5.) (Cheung and Ip, 2011). The source of the isolation membrane is still unclear. It is generally believed to involve de novo membrane synthesis, but the ER, mitochondria, Golgi apparatus and plasma membrane have all been suggested as membrane sources for autophagosome formation (Hailey et al., 2010; Hayashi-Nishino et al., 2009; Ravikumar et al., 2010; van der Vaart and Reggiori, 2010; Yla-Anttila et al., 2009). Both microautophagy and CMA involve direct transport of cargo into the lysosomes. Microautophagy occurs through bulk sequestration of cytoplasmic content by lysosomes. CMA, on the other hand, requires direct transport of unfolded proteins into the lysosomes in a chaperoneaided manner (Cheung and Ip, 2011).

Figure 5. Macroautophagy is characterized by the formation of a cytosolic double-membrane vesicle, the autophagosome. During macroautophagy, cytoplasmic proteins, organelles or other materials are surrounded by phagophores, which expand and close to form autophagosomes. These autophagosomes fuse with lysosomes to form autolysosomes, in which the cytoplasmic cargos (mutant aggregated proteins or cellular organisms) are degraded by resident hydrolases. LC3 localize to the phagophore throughout its elongation process. Upon completion of autophagosome formation, LC3-I dissociates from the membrane, whereas LC3-II remains on it. Autophagy can be inhibited by drugs such as 3-MA at the formation of autophagic vacuole stage (Sarkar and Rubinsztein, 2008). (Image modified from the original image of Melissa Cory)

Although autophagy was generally considered to be nonspecific, however recent studies have revealed many examples of selective autophagy, including mitophagy (for mitochondria), ribophagy (for ribosomes), pexophagy (for peroxisomes) and reticulophagy (for the ER). There is a growing body of evidence, suggesting that the specificity factor for selective autophagy is determined by ubiquitination and binding of this ubiquitin signal by autophagic adaptor proteins (Shaid et al., 2013). The primary role of autophagy is to protect cells under stress conditions, such as starvation. During periods of starvation, autophagy degrades cytoplasmic materials to produce amino acids and fatty acids that can be used to synthesize new proteins or are oxidized by mitochondria to produce ATP 29

for cell survival. Autophagy is also involved in cell growth, development and death. The levels of autophagy must be properly regulated, as indicated by the fact that dysregulated autophagy has been linked to many human pathophysiologies, such as cancer, myopathies and neurodegeneration (Chen and Klionsky, 2011). Neurons are particularly vulnerable to disruptions of autophagy, especially as the brain ages. Because neurons have unusually large expanses of dendritic and axonal cytoplasm, they face particular hurdles in preventing dysfunctional organelles and cellular waste from accumulating over a lifetime without the aid of cell division, which mitotic cells can rely on to dilute these waste burdens. (Nixon, 2013) Hence autophagy dysfunction leads to the accumulation of autophagic vacuoles and/or toxic protein aggregates, which can interfere with normal cellular functions. Initial studies have revealed the accumulation of autophagic vacuoles in the brains of Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis and HD patients (Ogata et al., 2006; Rubinsztein, 2006).

2.5.1 Autophagy pathway The core molecular machinery that controls the different stages of autophagy is composed of proteins that are encoded by autophagy-related genes (ATGs). More than 30 of these genes were originally characterized in yeast, and many orthologs have subsequently been identified and confirmed as autophagy regulators in higher eukaryotes. Autophagy consists of four stages: initiation, elongation, maturation and fusion. The phagophore accumulates ATG proteins, which enables the membrane to elongate and form a double-membrane-bound structure called the autophagosome (Harris and Rubinsztein, 2011). To date, LC3-II is the best-identified mammalian protein species that specifically associates with autophagosome membranes (Kabeya et al., 2000). LC3 is a microtubule-associated protein light chain 3 (LC3), one of the mammalian Atg8 homologues, and exists in two forms, LC3-I (18 kDa) and its lipidation form LC3-II (16 kDa), which are localized in the cytosol and in autophagosomal membranes, respectively. Another autophagy-related gene, Beclin 1, is the mammalian orthologue of yeast Atg6 and belongs to the class III phosphatidylinositol 3-kinase (PI3K) complex, which participates in autophagosome formation (Rami, 2009). Autophagy can be inhibited by different drugs. 3-methyladenine (3-MA) is a popular inhibitor of the autophagic pathway. 3-MA inhibits the activity of PI3K which blocks the formation autophagosomes (Liu et al., 2011).

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2.5.2 Regulation of autophagy Autophagy is regulated by a complex network that consists of different signaling pathways. Simplified summary of the regulation of autophagy is illustrated in Figure 6. One important regulator of autophagy and ATGs is the mammalian target of rapamycin (mTOR) kinase. The mechanism by which mTOR regulates autophagy are not fully known, and this kinase controls several cellular processes besides autophagy. There are also other, mTOR-independent pathways that regulate autophagy (Williams et al., 2008).

Figure 6. Simplified scheme of autophagy regulation. In the presence of amino acids, growth factors and energy, the mTOR complex 1 (mTORC1) represses autophagy by inhibiting the kinase activity of ULK1. In contrast, in the absence of amino acids and growth factors, or in response to an increase in the AMP/ATP ratio, via activation of AMPK, mTORC1 is inhibited and autophagy is initiated by the ULK1 complex. Atg proteins mediate the different steps of autophagy and LC3-II binds to autophagosome membrane to facilitate membrane elongation. Rapamycin and and the TSC1/2 complex inhibit mTOR. TSC1/2 mediates the inhibition by suppressing the mTOR regulator Rheb. Upstream of TSC1/2, AMPK activates AKT and inhibits TSC1/2 activity via phosphorylation. Beclin 1 induces the autophagic vesicle formation in a complex with other cofactors, like PI3K. Anti-apoptotic Bcl-2 and 3-MA inhibit Beclin 1 and PI3K activity. Autophagy inhibition by Ca2+ -dependent calpains is counteracted by calpain inhibitor calpastatin. Activators and inhibitors of autophagy are shown in blue and purple, respectively.

2.5.2.1 mTOR-dependent signaling pathways The serine–threonine kinase mTOR is a master negative regulator of autophagy that acts by blocking the activity of the ULK1 complex that acts during initiation of autophagosome formation. The activity of mTOR depends on a variety of inputs from upstream signals that include the energy and nutrient status of the cell, as well as the presence of amino acids and growth factors. mTOR is inhibited when nutrients are scarce, growth factor signaling is reduced, and ATP concentrations fall. 31

Thus, under conditions of nutrient starvation, mTOR is inhibited and the repressive effect of mTOR on autophagosome formation is relieved, leading to enhanced autophagosome biogenesis. mTOR activity can also be inhibited by drugs (such as rapamycin) that promote autophagy (Harris and Rubinsztein, 2011). The mTOR pathway involves two functional complexes: mTORC1 (Figure 7.) consisting of mTOR, raptor (regulatory associated protein of mTOR), PRAS40, mLST8 and Deptor; and mTORC2 comprising mTOR, rictor, mLST8, Deptor, mSIN, and Protor1. The unique compositions of mTORC1 and mTORC2 determine the selectivity of their binding partners. Up to date we know more about mTORC1 rather than mTORC2 probably due to the lack of available and wide-spreaded inhibitors of mTORC2 activity. (Tchevkina and Komelkov Andrey, 2012)

A major signalling cascade regulating mTOR activity is the class I PI3K pathway. The binding of growth factors or insulin to cell surface receptors activates the class 1a PI3K. Activated PI3K catalyzes the production of phosphatidylinositol-3,4,5-triphosphate (PIP3) at the plasma membrane, which increases the membrane recruitment of Akt/PKB and its activator PDK1, leading to the activation of Akt. The phosphorylation-dependent Akt activation results in the phosphorylation of a host of other proteins, including the tuberous sclerosis complex 1/2 (TSC1/TSC2). The TSC1/TSC2 complex integrates upstream signals from various kinases, including AKT and ERK1/2 [16]. Phosphorylation of TSC2 by these kinases leads to the disruption of the heterodimer with TSC1, resulting in loss of TSC1/TSC2 activity. TSC1/TSC2 complex forms a functional complex that has GTPase-activating protein (GAP) activity toward Ras homolog enriched in brain (Rheb) to inhibit mTOR. Hence, the loss of TSC1/TSC2 activity results in mTOR activation and autophagy inhibition (Garcia-Arencibia et al., 2010; Kang et al., 2011). Cells with mutation in either TSC1 or TSC2 are hypersensitive to ER stress and undergo apoptosis (Kang et al., 2011).

Figure 7. mTORC1 and TSC complexes. mTORC1 consists of mTOR, Raptor, PRAS40, mLST8 and Deptor. mLST8 binds to the mTOR kinase domain, where it seems to be crucial for their assembly. Deptor acts as an inhibitor. Phosphorylation inhibits the TSC1/2 complex, thereby relieving the TSC1/2-mediated repression of Rheb and allowing activation of TORC1. Modified from (Tchevkina and Komelkov Andrey, 2012)

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mTOR can also act as a sensor of changes in cellular energy states via AMP-activated protein kinase (AMPK). AMPK is activated in response to low cellular energy (high AMP/ATP ratio). Activated AMPK downregulates energetically demanding processes like protein synthesis and stimulates ATP-generating processes such as fatty acid oxidation.

Activated AMPK directly

phosphorylates TSC2 and thereby enhances its GAP activity, leading to the inhibition of mTOR signaling. AKT activates mTOR not only by direct phosphorylation of TSC2, but also by regulation of cellular energy by maintaining a high ATP level that causes a decrease in the AMP/ATP ratio that in turn inhibits AMPK-mediated phosphorylation and activation of TSC2 (Garcia-Arencibia et al., 2010; Wullschleger et al., 2006). 2.5.2.2 mTOR-independent signaling pathways Pathways that act independently of mTOR can also regulate autophagy. For instance, in nutrientrich conditions, Beclin-1 is bound to the anti-apoptotic protein Bcl-2. On nutrient starvation (4h of starvation in Hank's balanced salt solution), the stress-activated enzyme JNK1 phosphorylates Bcl-2, which induces the dissociation of Bcl-2 from Beclin-1. Once dissociated, Beclin-1 can interact with other members of the autophagic machinery and stimulate the induction of autophagy. In nutrientrich conditions, Beclin-1 is bound to the antiapoptotic protein Bcl-2 (Harris and Rubinsztein, 2011; Wei et al., 2008). Another mTOR-independent pathway is cyclical and can be mediated by the cAMP-Epac-PLC-εIP3 pathway and the Ca2+-calpain-G-stimulatory protein α (Gsα) pathway. PLC-ε-mediated generation of IP3 induces the release of Ca2+ from stores in the ER. High levels of intracytosolic Ca2+ inhibit autophagy by activating a family of Ca2+ cysteine proteases, calpains. After cleavage by calpains, Gsα becomes activated which, in turn, causes the production of more inhibitory cAMP. A decrease in IP3 is likely to result in reduced activity of IP3 receptors on the ER. Decreased levels of IP3 signaling leads to reduced Ca2+ release from the ER and lower rates of mitochondrial Ca 2+ uptake, causing a small but effective reduction in mitochondrial activity and ATP levels, which results in AMPK activation. Activated AMPK directly phosphorylates ULK1, which leads to the induction of autophagy (Harris and Rubinsztein, 2011; Vicencio et al., 2009; Williams et al., 2008). Autophagy is also modulated by ROS. Starvation triggers accumulation of ROS, most probably H2O2, which is necessary for autophagosome formation and the resulting pathway of degradation. The oxidative signal is partially class I PI3K dependent. Treatment with antioxidants ameliorates the ability of starvation to induce autophagy. One way in which ROS may be acting to regulate autophagy is by the modulation of the action of Atg4 on Atg8/LC3. The Atg4-mediated delipidation 33

of LC3-II on the cytosolic surface of autolysosomes allows it to be recycled. Atg4 is inactive and unable to cleave Atg8 from membranes when in its oxidized state. It is therefore possible that under oxidative conditions Atg4 is oxidized and inactive, which allows Atg8 to lipidate and thus initiates autophagy, while reduced Atg4 is active favoring Atg8 delipidation (Garcia-Arencibia et al., 2010; Scherz-Shouval et al., 2007). A recent study reported a function of Rhes, a striatal-specific protein implicated in the selective pathology of HD, in autophagy. In neuronal PC12 cells, deletion of endogenous Rhes decreased autophagy, whereas Rhes overexpression activated autophagy. These effects were shown to be independent of mTOR. Rhes bound the autophagy regulator Beclin-1 and decreased its inhibitory interaction with Bcl-2 independent of JNK-1 signaling. Mutant HTT blocked Rhes-induced autophagy activation (Mealer et al., 2013). 2.5.2.3 GADD34 in autophagy GADD34 (Growth arrest and DNA damage-inducible gene 34) is a stress‐induced gene encoding a regulatory subunit of a protein phosphatase 1 (PP1) ‐containing complex that can dephosphorylate eIF2α in vitro and in vivo. GADD34 protein is induced by cell damage. (Novoa et al., 2003) GADD34 is a major regulator of translation during conditions of cell stress such as heat shock, virus infection, nutrient deprivation and exposure to agents that cause improper folding of proteins in the ER. Cells respond to stress by turning off protein synthesis through phosphorylation of eIF-2α, which also induces autophagy (Talloczy et al., 2002). Phosphorylated eIF-2α induces the expression of GADD34, which forms a functional complex with PP1 to dephosphorylate eIF-2α, which leads to the restoration of protein synthesis (Watanabe et al., 2007). Studies of GADD34 gene deficient mice have shown that GADD34 promotes cell survival and the recovery from protein synthesis inhibition induced by ER stress (Kojima et al., 2003). Watanabe and colleagues proposed a novel pathway mediated by GADD34 that inhibits mTOR signaling and maintains translation during conditions of cellular stress. They showed that GADD34 forms a stable complex with TSC1/2 and causes TSC2 dephosphorylation, which inhibits signaling by mTOR (Watanabe et al., 2007). Uddin and colleagues showed that GADD34 is induced by starvation and that it plays a critical role in the induction of autophagy mediated by inhibition of the mTOR signaling pathway (Uddin et al., 2011). GADD34 may find clinical potential as a drug target for the treatment of autophagy dysregulation associated diseases.

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2.5.2.4 Ubiquitination and selective autophagy Ubiquitination is the hallmark of protein degradation by the 26S proteasome. Ubiquitin is a small 76 amino acid polypeptide that was originally characterized as a covalently attached signal for ATPdependent proteasomal degradation of substrate proteins (Hershko and Ciechanover, 1998). Ubiquitin, a protein implicated in the proteasome degradation pathways, is also involved in selective autophagy. (Figure 8.) Identification of autophagy receptors, like p62/SQSTM1 and NBR1, which simultaneously

bind

both

ubiquitin

and

autophagy-specific

ubiquitin-like

modifiers

LC3/GABARAP, has provided a molecular link between ubiquitination and autophagy (Kirkin et al., 2009).

Figure 8. The Ubiquitin code links between proteasomal and lysosomal degradation. As illustrated, a misfolded protein can be degraded by proteasome or lysosomal system. Ubiquitinated misfolded protein is targeted for the proteasome. If the UPS is overloaded, protein aggregation occurs, which are then targeted for autophagic clearance. Thereby, ubiquitin chains on misfolded proteins can undergo remodelling by combined activity of deubiquitinating enzyme (DUB) and E3-ligases. Newly formed ubiquitin chains are then recognized by the UBD of p62, NBR1 or other autophagy receptors to form inclusion bodies or by the corresponding UBD of HDAC6, which direct protein aggregates to the aggresome. Aggresomes can be degraded via the proteasome or via autophagy pathway. If degradation occurs via autophagy, targeting of the protein aggregates are determined by the LIR motif of p62 and NBR1. LC3, light chain 3 protein; LIR, LC3-interacting region; Ub, ubiquitin; NBR1, neighbor of breast cancer 1; p62/SQSTM1, sequestosome 1; UBD, ubiquitin binding domain; UPS, ubiquitin–proteasomal system; HDAC6, histone deacetylase 6. Modified from (Shaid et al., 2013).

Autophagy is considered selective when a precise cargo is specifically targeted into autophagosomes. This process involves ubiquitination (Shaid et al., 2013). The molecular characterization of ubiquitin binding proteins such as p62 and NBR1 (neighbour of breast cancer 1) has demonstrated that, analogous to the proteasome where ubiquitinated cargos are delivered by 35

ubiquitin receptors, an ubiquitin-dependent sensor system is responsible for substrate specificity. Before autophagic clearance, these receptors need to gather the ubiquitinated cargo to the nascent autophagosome, which carries LC3 or GABARAP (gamma-aminobutyric acid receptor-associated protein) proteins on its surface. Thus, autophagy receptors binding to both ubiquitin and LC3 or GABARAP proteins are able to control protein degradation by selective autophagy (Bjorkoy et al., 2005; Weidberg et al., 2011). Filimonenko and colleagues showed that Alfy, a phosphatidylinositol 3-phosphate-binding protein, is required for the macroautophagic elimination of aggregated proteins, but not for macroautophagic elimination of bulk cytosol in response to starvation. Alfy was shown to function as a scaffold that gathers together the E3-like ligase (promoting protein-lipid conjugation), Atg5-Atg12-Atg16L, and LC3 to the target substrate to permit the degradation of mHTT proteins. Alfy overexpression decreased aggregated mHTT levels and protected cells from expanded polyQ toxicity in a primary neuronal HD model (Filimonenko et al., 2010)

2.5.3 Autophagy in HD Autophagy was first demonstrated to have a protective role in neurodegeneration in a Drosophila model of HD. This study showed that activation of autophagy by the mTOR inhibitor rapamycin attenuated HTT toxicity (Ravikumar et al., 2004). In a cell model of HD, treatment with rapamycin reduced both the levels of soluble mHTT and the formation of intracellular aggregates of this protein, which protected cells against its toxic effects (Ravikumar et al., 2006). The mood stabilizer lithium induces autophagy. Lithium, a mood stabilizer drug, was shown to induce autophagy and enhance clearance of mHTT independently of mTOR through the inhibition of IP3 (Sarkar et al., 2005). Autophagy inhibitors are known to increase the accumulation of transfected HTT in different types of cultured cells. For example, inhibition of autophagy in PC12 cells by 3-MA can raise the level of mHTT (Li et al., 2010). In mHTT expressing cells, the aggregates sequester mTOR, reducing the level of the soluble mTOR protein. This results in decreased mTOR activity, and a consequent increase in autophagy. This phenomenon seems to be confined to cells with aggregates, and is not seen in cells expressing soluble mutant protein. The ability of cells to induce autophagy when they contain polyQ aggregates may account for the observations that cells containing aggregates are less prone to cell death than cells expressing non-aggregated mutant polyglutamine proteins. This may be due to the more rapid 36

clearance of the soluble protein in cells containing mHTT aggregates, leading to lower levels of diffuse HTT, which seems to correlate with lower toxicity and the protective effects of autophagy (Arrasate et al., 2004; Rubinsztein, 2006). Another study revealed aberrant cargo recognition in cellular and mouse model of HD. Detailed analysis indicated that despite normal autophagic flux, the autophagic vacuoles contain minimal cargo, thus resulting in a general decrease in protein turnover in different HD models (Martinez-Vicente et al., 2010). Beclin 1 has been shown to mediate the catabolism of mHTT. A study pointed out that the agedependent decline of beclin 1 expression and possible associated reduction of PI3K (III) is critical for the decline of autophagic function, which may lead to the accumulation of mHTT and the agedelayed disease onset of HD. Furthermore, the study suggested that pharmacological means to increase of Beclin 1 function could be beneficial in delaying the onset as well as slowing the progression of HD (Shibata et al., 2006). Despite the observed neuroprotective effect of autophagy activation in different HD models, it still remains largely enigmatic how autophagy is impaired in HD (Cheung and Ip, 2011). The relationship between mTOR and autophagy in the clearance of mHTT is also complex. Inhibition of mTOR by drugs like rapamycin and lithium, which induce autophagy, are clearly beneficial in HD models. However, the clearance of mHTT by autophagy can occur in the presence of elevated mTOR activity but depends on Beclin-1, and separate studies demonstrated that mHTT accumulation is regulated by Beclin-1 (Yamamoto et al., 2006). Futher mentioned above, mHTT aggregates sequester mTOR, leading to decreased kinase activity and autophagy activation, presumably as a compensatory mechanism to cope with mHTT toxicity (Ravikumar et al., 2004). The gradual accumulation of mHTT affects the cellular processes involved in protein degradation, and as HD patients get older, these processes start to fail. This could also be the reason for the delayed onset of HD. Early in life, proteasomal degradation is robust and capable of preventing cytotoxicity. With advancing age, proteasomal function becomes compromised, forcing cells to rely more heavily on the autophagic pathway. Autophagic capacity also decreases with aging, which potentially leads to both normal aging and the pathologic aging seen in HD. Accordingly, in most tissues, the cytotoxic influences of mHTT are initially minimized by the proteasome before autophagy is required (Li and Li, 2011; Mealer et al., 2013). In the next chapter proteasomal degradation is discussed in more detail.

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2.6 Proteasomal degradation With autophagy–lysosome pathway, the ubiquitin–proteasome system is one of the two main routes of protein and organelle clearance in eukaryotic cells. Protein degradation plays a central role in many cellular functions. The ubiquitin-proteasome system functions in cellular quality control by degrading misfolded, unassembled, or damaged proteins that could otherwise form potentially toxic aggregates (Li and Li, 2011).

2.6.1 Ubiquitin-proteasome system The ubiquitin–proteasome system (UPS) is critical for cell survival and a variety of cellular functions. Altered UPS function can lead to a wide range of disturbances, such as cell degeneration. Dysfunction of the UPS has been implicated in the pathogenesis of many neurological diseases, including Alzheimer's disease, spinocerebellar ataxia, and several motor neuron diseases (Chen et al., 2009). Two sequential reactions are involved in protein clearance by the UPS: one is a tagging reaction and the other is a subsequent degradation of the tagged proteins in the proteasome (Figure 9.) (Li and Li, 2011). Proteasomes are barrel-shaped multi-protein complexes that predominantly degrade shortlived nuclear and cytosolic proteins. The 26S proteasome is a multicatalytic protease localized both in the nucleus and the cytoplasm. It is composed of three major subunits: one 20S catalytic core and two 19S regulatory caps. In the inner part of the 20S complex, there are three types of catalytic subunits that execute the corresponding catalytic activities of the proteasome (trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolyzing activity) (Li and Li, 2011). Most proteins are targeted for proteasomal degradation after being covalently modified with ubiquitin. This conjugation typically involves three types of enzymes: E1 (ubiquitin-activating enzyme) hydrolyses ATP and forms a thioester-linked conjugate between itself and ubiquitin; E2 (ubiquitin-conjugating enzyme) receives ubiquitin from E1 and forms a similar thioester intermediate with ubiquitin; and E3 (ubiquitin ligase) binds both E2 and the substrate, and transfers the ubiquitin to the substrate. Frequently, the ubiquitin itself forms a substrate for further rounds of ubiquitination, resulting in the formation of a polyubiquitin chain. Chains of four or more ubiquitin molecules appear to form a recognition signal that allows substrates to be shuttled to the proteasome (Rubinsztein, 2006).

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Figure 9. The Ubiquitin-proteasome system. First, a ubiquitin monomer is activated by forming an intermolecular thioester with the ubiquitin-activating enzyme (E1) in an ATP-requiring reaction. Next, activated ubiquitin is transferred to the active site of an ubiquitin-conjugating enzyme (E2). Finally, ubiquitin is linked in the substrate protein, a step that is catalyzed by an ubiquitin–protein ligase (E3). The E3 ligase confers specificity to the process by selectively binding to a protein target. Activated ubiquitin molecules are then sequentially added to the first ubiquitin proteins, forming a polyubiquitin chain. Proteins tagged with chains of four or more ubiquitins are then recognized by the 26S proteasome for degradation. After proteasome degradation, ubiquitin monomers are released or actively removed by the ubiquitin carboxyl-terminal hydrolases. Pi, inorganic phosphate; PPi inorganic diphosphate; Ub, ubiquitin.

2.6.2 Proteasomal degradation in HD Multi-ubiquitylated proteins are usually efficiently degraded by proteasomes; hence the presence of elevated ubiquitin conjugates associated with protein aggregates in diseased neurons in nearly all neurodegenerative diseases has long suggested a linkage between UPS dysfunction and pathogenesis (Li and Li, 2011). In various cellular and animal models of HD, as well as in the postmortem brains of HD patients nuclear polyQ inclusions are positively labeled by antibodies against ubiquitin (DiFiglia et al., 1997; Gutekunst et al., 1999). Various studies indicate that the UPS is involved in processing both wild type and mHTT (Li et al., 2010; Roscic et al., 2011). Aggregates of mHTT are mainly present in the nucleus in human HD postmortem brains. As autophagy is a cytoplasmic degradation pathway, it is not sufficiently effective in clearing nuclear mHTT aggregates. To target nuclear aggregates, the UPS gets into the picture, as proteasomes are present in both the cytoplasm and nucleus (Schipper-Krom et al., 2012). It has been reported that protein aggregation can directly impair the function of the UPS. Transient expression of two unrelated aggregation-prone proteins, a HTT fragment containing a pathogenic polyQ repeat and a folding mutant of cystic fibrosis transmembrane conductance regulator, caused nearly complete inhibition of the UPS (Bence et al., 2001). Several studies have shown that inhibition of proteasome activity leads to accumulation of mHTT aggregates (Bence et al., 2001; 39

Wang et al., 2008). PolyQ-expanded proteins are found to impair UPS function in various cell models or in vitro systems, but in vivo studies of UPS function in polyQ disease mouse models have not yielded consistent results (Bence et al., 2001; Bennett et al., 2005; Bennett et al., 2007; Bett et al., 2006). Interestingly, misfolded polyQ proteins or filamentous mHTT can directly affect the function of proteasomes before the formation of inclusions in vitro. Thus, an important issue is whether UPS impairment also occurs in vivo (Bennett et al., 2005). Seo and colleagues examined postmortem brain tissues of HD patients and observed a decrease in proteasomal activity in the striatum and cortex. However, this decrease could be due to atrophy or degeneration already present in the HD patient brains (Seo et al., 2004). Wang and colleagues measured synaptic UPS activity and found a decrease in UPS activity in the synapses of HD mice (Wang et al., 2008). It is possible that the effect of polyQ proteins on UPS activity is dependent on its accumulation and subcellular localization, which cannot be detected by examining whole cell homogenates. Because UPS activity varies in different types of cells and in subcellular regions, it is important to evaluate UPS activity in different subcellular compartments of neurons (Wang et al., 2008). A recent study supported the finding that mHTT clearance is mediated by both proteasomal and lysosomal degradation. The study showed that overexpression of negative regulator of ubiquitin-like protein 1 (NUB1) reduced mHTT amounts by enhancing polyubiquitination and proteasomal degradation of mHTT. NUB1 showed mHTT-specific neuroprotection in the mammalian neuronal models as well as in an in vivo transgenic fly model (Lu et al., 2013). Another study has demonstrated that overexpression of the C terminus of Hsc70-interacting protein (CHIP) increase the ubiquitination and proteasomal degradation of mHTT and suppresses aggregation (Jana et al., 2005).

2.6.4 The deubiquitinating enzymes To maintain ubiquitin homeostasis, ubiquitin must be recycled once a substrate has been committed to the degradation pathway. Hence, both the proteasome and lysosomal sorting machinery have deubiquitylating enzymes (DUBs) associated with them (Clague et al., 2012; Komander et al., 2009). The human genome encodes approximately 100 DUBs that are predicted to be active and which oppose the function of E3 ligases (Komander et al., 2009). The largest of the five families is the ubiquitin-specific protease (USP) family (~55 members). The substrate specificity of DUBs is determined by sub-cellular localization, specific binding interactions and the preference of the catalytic domain for particular types of ubiquitin chain linkages (Clague et al., 2012). DUBs are 40

classified into five families: the USP (the ubiquitin-specific processing protease), UCH (the ubiquitin C-terminal hydrolyase), OTU (the ovarian tumor), MJD (the Josephin domain) and JAMM (the Jab1/Mov34 metalloenzyme) families. First four families are cysteine proteases, while the fifth family is metalloproteases, and to date USP and UCH are the best characterized families (Tian et al., 2013). DUBs are thought to regulate three general processes: production of monomeric ubiquitin by cleaving ubiquitin from ubiquitin-fusion proteins; recycling ubiquitin from ubiquitin–protein conjugates at the proteasome; and editing ubiquitin chains to regulate ubiquitin chain length. A few DUBs have also been shown to associate with proteasomes. Both proteasome associated and nonassociated DUBs have been hypothesized to edit conjugated ubiquitin side chains and therefore, play an important role in regulating ubiquitin-signaling events such as protein stability and localization (Anderson et al., 2005). DUBs are crucial for various cellular functions and disturbances in these enzymes may lead to diseases in the nervous system (Singhal et al., 2008). Mutations in the DUB UCHL1 have been linked to an autosomal dominant form of PD. Oxidative modification and down-regulation of UCHL1 are associated with both PD and AD (Marshall et al., 2013). 2.6.4.1 Ubiquitin specific protease 14 (Usp14) Ubiquitin specific protease 14 (Usp14) is one of the DUBs. It functions at the proteasome to edit and/or remove ubiquitin chains (Borodovsky et al., 2001). Usp14 is expressed during embryogenesis and in adult tissues, including the nervous system (Chen et al., 2009; Crimmins et al., 2009). Usp14 has been shown to be important for maintaining ubiquitin levels in neurons (Crimmins et al., 2009). Through its association with the proteasome, Usp14 becomes catalytically active and deconjugates ubiquitin from substrates destined for proteasomal degradation (Chen et al., 2009; Crimmins et al., 2009). By facilitating ubiquitin recycling at the proteasome, Usp14 may act to stabilize ubiquitin pools at synaptic terminals (Chen et al., 2009). A mouse mutant with a spontaneous deletion of Usp14, the so called ataxia mouse (axJ), shows progressive neurological symptoms in the form of ataxia, tremor, hind limb paralysis and muscular weakness. These mice develop severe tremors by 2–3 weeks of age, followed by hindlimb paralysis, and death by 6–8 week (Anderson et al., 2005; D'Amato and Hicks, 1965; Wilson et al., 2002). While it is clear that the null mutant has a neurological phenotype, it remains unclear whether this is a non-specific (proteasomal) or more specific, non-proteasomal effect for Usp14 in neurons. 41

A recent study identified and characterized an N-ethyl-N-nitrosourea (ENU) induced mutation in Usp14 that leads to adult-onset neurological disease (nmf375 mice). The mutation causes aberrant splicing of Usp14 mRNA, resulting in a 95% reduction in USP14. The adult nmf375 mice exhibited deteriorating motor performance, depletion of monomeric ubiquitin and motor endplate disease, indicating that USP14 is required for stable maintenance of adult motor endplates and ubiquitin pools (Marshall et al., 2013). Usp14 might also have noncatalytic functions to modulate the degradation of proteins. Nagai and colleagues identified Usp14 as a binding partner of IRE1α. The interaction between IRE1α and Usp14 is inhibited by ER stress. Overexpression of Usp14 with the kinase negative form of IRE1α (IRE1αKN), which recruits Usp14 on the ER membrane, inhibits the degradation of unfolded ER proteins. Moreover, the inhibition of Usp14 expression by small interfering RNA (siRNA) resulted in enhanced of the degradation of unfolded ER proteins (Nagai et al., 2009).

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3 AIMS OF THE STUDY This study elucidated the early pathological changes in HD. The general aim was to study the role of ER stress, oxidative stress and autophagy in the cell models of Huntington’s disease. The specific aims of the study were: 1.

To study to role of GADD34 in the regulation of autophagy in a cell model of HD. (I)

2.

To study the role of Sigma-1 receptor and its agonist PRE084 in a cell model of HD. (II)

3.

Investigate the functions of Ubiquitin Specific Protease 14 (Usp14) in two different cell models of HD. (III)

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4 MATERIALS AND METHODS The detailed descriptions of the materials and methods are found in the original publications

4.1 Cell cultures (I-III) 4.1.1 PC6.3 cell cultures (I-III) PC6.3 cell line is a neuronal-like subline of PC12 pheochromocytoma cells from rat adrenal medulla that can be differentiated by NGF. These cells originate from the sympathoadrenal system and have several properties of the sympathetic neurons and are therefore considered as neuron-like cells. PC6.3 cells can be grown under conditions in which 90% of the cells undergo transcriptiondependent cell death following removal of NGF in either serum-free or serum-containing medium. PC6.3 cells are a good model system for characterizing cellular, biochemical and molecular events in programmed neuronal cell death (Pittman et al., 1993). In these studies PC6.3 cells were cultured without NGF. PC6.3 cells were cultured in cell culture dishes in RPMI-1600 (Lonza and Biochrom) medium supplemented with 5% fetal calf serum (Chemicon) and 10% horse serum (PAA), 7,5% NaHCO3, 100mM Na-glutamine (Gibco) and 100mM penicillin-streptomycin. 4.1.2 Hela cell cultures (III) Tetracycline (tet)-regulatable HeLa cell line stably express the first 17 amino acids of HTT followed by a polyQ expansion (65Q or 103Q) tagged with monomeric CFP (HttPolyQ-mCFP). As a control, we used cell lines that express 25 CAG repeats (25Q), which is under the aggregation threshold of 37 repeats, and thus remains soluble. The cell line was created by co-transfecting HeLa tet-htt (25Q, 65Q, or 103Q) exon1- mCFP and PTk-hygro (CLONTECH Laboratories, Inc.) and then selecting with hygromycin 800 μg/ml. Doxycycline (dox), belongs to tetracycline antibiotic class. Dox (100 ng/ml) was also included in the culture media during selection to maintain suppression of transgene expression. HeLa cells were cultured in cell culture dishes in DMEM (Lonza) medium supplemented with 10% fetal calf serum (Chemicon), 7.5% NaHCO3, 100mM Na-glutamine (Gibco) and 100mM penicillin-streptomycin (Filimonenko et al., 2010; Yamamoto et al., 2006).

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4.2 Animals (III) Animal experiments were done in collaboration with Dr. Petersén’s research group (Lund, Sweden). The BACHD model is a transgenic mouse expressing full-length mHTT that displays a metabolic phenotype and psychiatric-like behavior. The mouse was produced with the bacterial artificial chromosome-mediated transgenic approach and expresses full-length mHTT containing 97 polyglutamine repeats. (Lundh et al., 2012) BACHD mice were obtained from the Jackson Laboratories ((FVB/N strain; Bar Harbor, Maine). All animal experiments were approved by the local ethical committee and accomplished in accordance with the European Communities Council Directive (86/609/EEC). We analyzed the tissue samples from BACHD mice received from Lund.

4.3 Transfections and stimulations (I-III) PC6.3 cells were transfected with expression vectors encoding for different CAG-repeat (Q) lengths (18Q, 39Q, 53Q or 120Q) of huntingtin exon-1 fused to EGFP (Hasholt et al., 2003) or using fulllength huntingtin constructs with 17Q and 75Q repeats (a kind gift from Dr. Saudou). RFP-LC3 (Red fluorescent protein- light chain 3 protein), EGFP-LC3 (Enhanced green fluorescent proteinLC3), LAMP1-RFP (Lysosomal-associated membrane protein 1-RFP), Ubiquitin (RFP-Ub) and GADD34 expression plasmids were obtained from Addgene. Human wild type pRK-FLAG-USP14 and mutant pRK-FLAG-USP14 C114A plasmids were a kind gift from Dr. Yihong Ye (Wang et al., 2006). Transfectin reagent (Biorad) was used following the instructions provided by the vendor using 1 μg DNA per one well on 24-well plates, 3 μg DNA per one well on 6-well plates and 8 μg DNA per 10cm2 plates. Controls were transfected with EGFP expression plasmid (Clontech) and cells were incubated 24-48h prior to analysis. In some experiments, 1 mM or 5 mM 3-methyladenine (3-MA, Sigma), 20 µM MG-132 (Calbiochem), 200 nM Rapamycin (Calbiochem), 2 μM thapsigargin (Sigma) or 1 μg/ml tunicamycin (Sigma) was added. LC3 immunoblotting was done in the presence of lysosomal proteasome inhibitors: 5 μM E64d (Sigma) and 10 μM pepstatin A (Sigma). In some experiments 0.3 mM PRE084 (Tocris) and 3 mM progesterone (Sigma-Aldrich) were added 4 h after transfection.

4.4 Immunoblotting (I-III) For immunoblotting cells were rinsed twice with ice-cold PBS and lysed in RIPA buffer (150 mM NaCl, 1 % Triton-X-100, 0,5 % sodium deoxylate, 1 % SDS, 50mM Tris-HCl, pH 7.4) supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitors (Phosphostop, 45

Roche). Protein concentrations were determined (BioRad BC assay) and equal amounts of protein (40μg) were subjected to SDS-PAGE and blotted onto nitrocellulose filters (Amersham), which were incubated for 1h in 5% skimmed milk or BSA, in TBS-T (50mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween 20) and then with primary antibodies overnight at 4°C. After washing the filter was incubated with horseradish peroxidase-conjugated secondary antibodies (1:2500, Jackson ImmunoResearch Laboratories), followed by detection using enhanced chemiluminescence (Pierce). Immunoblots were quantified with ImageJ (NIH) quantification software. Primary antibodies used are shown in Table 3.

4.5 Immunocytochemistry (I-III) HeLa and PC6.3 cells plated on poly-lysine and laminin coated coverslips were fixed for 7-20 min using 4% paraformaldehyde. Cells were incubated for 1 h using phosphate-buffered saline (PBS) containing 0.1% Triton-X-100 and 5% bovine serum albumin (Sigma), followed by overnight incubation in +4°C with primary antibodies. (Table 3.) Cells were washed using PBS and incubated for 1 h using Alexa Fluor 488 and/or 594 (1:500, Invitrogen Molecular Probes) or Cy5 (Jackson laboratories) secondary antibodies. Cells were counterstained for 1 min using Hoechst 33342 blue (4 mg/ml; Sigma), mounted in gel mounting medium (DABCO) and analyzed using a Zeiss LSM confocal microscope (Zeiss) at the Helsinki Biomedicum Molecular Imaging unit or by fluorescent microscope (Leica DM4500B).

4.6 Immunoprecipitation (I, III) Cells were lysed in IP-lysis buffer (20mM HEPES-KOH, pH 7.5, 10mM KCl, 1.5mM MgCl2, 1mM EDTA, 1mM EGTA, 1mM DTT and 0.1mM PMSF) supplemented with protease inhibitor cocktail (Roche) and PhosphoStop (Roche). Protein concentrations were measured with Bio-Rad protein assay (BCA protein assay kit) and equal amounts of proteins (typically 200-300μg) were taken for immunoprecipitation. Pre-clearing was done by incubating the lysates with protein A- and Gagarose beads (Roche) followed by an overnight incubation in +4°C with either Usp14 (III) or GADD34 (I) antibodies. Following day 50 μl of protein G-agarose was added and incubated in a rotor at +4°C for 2 hours. Agarose beads were collected by centrifugation and washed three times with lysis buffer. The agarose pellet was dissolved to 50μl of 1xWB loading buffer and boiled for 10 min followed by centrifugation, at 14000 rpm for 5 min. Subsequently, immunoprecipitates were

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BRL). Incubation was carried out at 42°C for 55 min in 20mM Tris-HCl (pH 8.4), 5mM MgCl2, 500μM dNTPs, and 10mM DTT in a final volume of 20 μl. PCR reactions were started with a 5 min denaturation step at 95 °C followed by 30 cycles carried out at 95°C for 60 s, 63°C for 60 s and 72°C for 90 s. The following primers were used: GADD34: 5′-GTCCATTTCCTTGCTGTCTG-3′ (forward), 5′-AAGGCGTGCCCATGCTCTGG-3′ (reverse). ß-actin: 5′- CTTCAACACCCCAGCCATG-3′ (forward); 5′-GTGGTACGACCAGAGGCATAC- 3′ (reverse). 4.8.1 Reverse transcription and quantitative PCR (II) Total RNA was extracted from cells using the GenElute Mammalian total RNA kit (Sigma) and gene-specific reverse transcription was performed using Tetro reverse transcriptase (Bioline). cDNA synthesis was carried out in a 10-ml reaction volume containing Tetro reaction buffer, 0.5 mM each of the reverse primers (calpastatin, 5’-CCCCAGTAGACTTCTCTTTC-3’ ; Sig1R, 5’CTTCCTCTACATTC CTCTG-3’), 750 ng of the RNA template and 0.5 ml Tetro reverse transcriptase. The reaction was incubated at 75°C for 5 min to open RNA secondary structures, and samples were cooled to 42°C. Superscript III reverse transcriptase was then added and the samples were incubated at 42°C for 20 min followed by enzyme inactivation at 64°C for 20 min. The unbound reverse transcription primers were digested with 20 units Exonuclease I (New England Biolabs GmbH) at 37°C for 30 min, followed by denaturation at 80°C for 15 min. One microliter of product was then amplified with Light Cycler 480 SYBR Green I Master Mix (Roche Diagnostics GmbH, Mannheim, Germany) and using 0.5 mM of forward (fw) and reverse (rev) primers (calpastatin, fw: 5’-AGT AGTTCTGGACCCAATG-3’ , rev: 5’-CCCCAGTAGACTTCTCTTTC3’; Sig1R, fw: 5’-TGCCTTATCTCCATTCCA-3’, rev: 5’-CTCCTTCCTTCAGTCCTT-3’). qPCR amplification and relative quantification was performed on the LightCycler 480  II instrument (Roche) with 40 cycles (10 s denaturation at 95°C, 20 s annealing at 60°C and 20 s extension at 72°C). Melting curve acquisition and analysis was also carried out immediately after amplification to confirm the specificity of PCR.

4.9 Autophagy detection (I) PC6.3 cells were transfected with different huntingtin constructs together with RFP-LC3 and fixed after 24 h or 48 h using 4% paraformaldehyde. The number of cells with LC3 positive vesicles was analyzed. Cells with more than five dots were considered positive.

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Conversion of LC3 was also studied using immunoblotting and the appearance of the lower LC3-II band was used as an indication of autophagosome formation. Experiments were done with and without lysosomal proteasome inhibitors, E64d (Sigma) and pepstatin A (sigma). In the absence of lysosomal protease inhibitors we were able to measure the lysosome-dependent degradation. Autophagy flux was also detected by transfecting the cells with different huntingtin constructs together with GFP-LC3(B). When GFP-LC3 is delivered to a lysosome the LC3 part of the chimera is sensitive to degradation, whereas the GFP protein is relatively resistant to hydrolysis. Therefore, the appearance of free GFP on western blots can be used to monitor lysis of the inner autophagosome membrane and breakdown of the cargo (Klionsky et al., 2008).

4.10 Solubility assay (II-III) Cells were lysed in solubility buffer containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl2, 3 mM EGTA, 0.5% Triton X and protease inhibitors (Roche) and kept on ice for 5 min. Aliquotes (30 μl) of the extract were mixed with 10 μl 4× sodium dodecyl sulfate (SDS) loading buffer and sonicated (total cell lysate). The remaining cell lysate was centrifuged at 13 000 g for 10 min, and the supernatant was mixed with 4× SDS loading buffer (soluble fraction); the pellet was dissolved in 1× SDS loading buffer and sonicated (insoluble fraction). Equal amounts were then analyzed on SDS– PAGE gel and subsequent WB using a monoclonal GFP antibody (Roche) and horseradish peroxidase-coupled anti-mouse secondary antibodies (Jackson). To detect high molecular weight forms of the huntingtin fragment proteins, the stacking gel was also blotted.

4.11 Luciferase reporter assay (II) The Luciferase Assay System is a reporter quantitation assay that can be used to study gene expression. The luciferase assay kit is based upon the bioluminescent measurement of firefly luciferase. The intensity of light emission is linearly related to the amount of luciferase and is measured using a luminometer. PC6.3 cells in six-well plates were transfected with 0.5 mg of full-length huntingtin expression plasmids in conjunction with 0.5 mg of the NF-κB reporter plasmid, containing multiple NF-kB sites linked to firefly luciferase gene. A volume of 0.02 mg of Renilla luciferase (pRL-TK) was used to control for transfection efficiency. Cells were harvested 48h after transfection and lysed to Passive Lysis Buffer (Promega). For assaying the expression of Renilla and firefly luciferase we used the dual lusiferase substrate and the activities were measured by luminometer (GloMax 50

TD20/20 Luminometer Turner Designs). Results are shown as fold increase in luciferase normalized to the Renilla activity.

4.12 Caspase assay (II) PC6.3 cells were transfected with HTT 18Q- and 120Q-expressing plasmids as noted above and 0.3 mM PRE084 was added 4 h after transfection as indicated. Controls were transfected with EGFP expression plasmid. Cells were incubated for 2 days and caspase-3/7 activities were measured using a Caspase-Glo assay kit (Promega) as described by the vendor. Samples were incubated at room temperature for 90min and the luminescence was measured using a luminometer (GloMax TD20/20 Luminometer Turner Designs).

4.13 Cell degeneration assays (I-III) Hoechst 33342 (Sigma) was employed to stain dying cells showing condensed and fragmented DNA (Korhonen et al., 2005; Sokka et al., 2007). More than 300 fluorescent cells in each well were analyzed and experiments were repeated three times. Results are expressed as percentage of transfected cells.

4.14 Measurements of intracellular ROS (II) PC6.3 cells were cultured in 96-well plate and transfected with flHTT constructs with 17- and 75 polyglutamine repeats for 48 h, 0.3 mM PRE084 was added 4 h after transfection. Cells were treated with 10 mM 6-carboxy-20,70-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (H2DCFDA AM: Invitrogen, Carlsbad, CA, USA) and incubated at 37°C for 45 min. H2DCFDA is able to penetrate cells due to the acetoxymethyl ester, where it is hydrolyzed by intracellular esterases to form 20,70-dichloro-fluorescin (DCFH). Oxidation of DCFH by hydrogen peroxide and hydroxyl radicals yields a highly fluorescent product 20,70-dichlorofluorescein (DCF). The fluorescence intensity of DCF after excitation of the samples at a wavelength of 485 nm was measured at an emission wavelength of 535 nm using a fluorescence microplate reader. Results are shown as normalized to control (set to 100%).

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4.15 Measurements of intracellular calcium (II) PC6.3 cells were grown on poly-L-lysine-coated coverslips and transfected for 24h as described above. Cells were then washed three times with HBSS buffer (118mM NaCl, 4.6mM KCl, 1mM CaCl2, 10mM D-glucose, 20mM HEPES, pH 7.4), following a 30-min incubation with 2 mM Fura2 AM at room temperature. After washing twice using HBSS, the cells were transferred to a heated coverslip holder adjusted to 37 1C, and perfused with HBSS containing 100 nM bradykinin. An XBO 75W/2 xenon lamp served as the source for excitation light and the excitation filters were set at 340 or 380 nm, respectively. Emitted light was measured at 510 nm. Filters were controlled with Lambda 10-2 (Sutter Instruments). A SensiCam CCD camera was employed for image acquisition and the recorded images were processed using the Axon Imaging Workbench software (Axon Instruments). The F340/F380 fluorescence ratio served as the indication of intracellular calcium concentrations.

4.16 Aequorin-based measurements of mitochondrial calcium (II) Measurements of mitochondrial Ca2+ concentration ([Ca2+]mito) were carried out using recombinant aequorin targeted to mitochondrial matrix (mtAeq) and a luminometer system as described previously. (Brini et al., 1995; Brini, 2008) In brief, cells were grown to 70% confluency on poly-Llysine-coated 13-mm glass coverslips and transfected with the mtAeq (kind gift from Professor U Ruegg, Geneva, Switzerland) along with control (pcDNA), 17Q- or 75Q-huntingtin-expressing plasmids as described previously. At 24h after transfection, the cells were washed with HBSS buffer (118mM NaCl, 4.6mM KCL, 1mM CaCl2, 10mM D-glucose, 20mM HEPES, pH 7,4) and reconstituted with 5 mM native coelenterazine (Invitrogen) for 1h at room temperature. PC6.3 cells were then placed into a perfusion chamber and the luminescence was recorded. All measurements were conducted in HBSS at 37°C. Cells were stimulated with 100 nM bradykinin (Sigma-Aldrich) to induce the release of Ca2+ from the intracellular stores. Calibration of the measurements was done by permeabilizing the cells in HBSS containing 10 mM Ca2+ and 100 mM digitonin (SigmaAldrich), thus generating the maximal luminescence of the sample. Luminescence data was then converted to [Ca2+] according to Brini et al. (Brini et al., 1995).

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4.17 Determination of cell viability with MTT assay (I-II) Cell viability was determined by the MTT [3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (Calbiochem) assay (Sigma). PC6.3 cells were transfected with different plasmids for 2448h. Cells were incubated in the presence of MTT solution (Thiazolyl blue tetrazolium bromide, Sigma) for 2 h at 37°C. After incubation the medium (containing MTT) was removed. 40 mM HCl in isopropanol was added to solubilize the dye absorbed by the cells. The amount pf the dye was measured by absorbance at 560 nm with Labsystems Multiskan MS spectrophotometer. The absorbance was linear to the number of viable cells.

4.18 Statistical analyses (I-III) All experiments were repeated at least three times. Statistical comparison was performed using student’s t-test or one-way ANOVA and a bonferroni post hoc test. P value p < 0.05 was considered as statistically significant.

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5 RESULTS AND DISCUSSION 5.1 GADD34 plays an important role in cell protection in mHTT expressing cells by increasing autophagy and cell survival. (I) The pathophysiological changes in HD are complex and include alterations in gene transcription, cell signaling, energy metabolism, protein trafficking, transport and synthesis, as well as an impaired degradation of misfolded and aggregated proteins. The precise signals regulating these events are not fully understood but ER stress and changes in autophagy are proposed to be involved in the cell degeneration observed in HD (Duennwald and Lindquist, 2008; Levine and Kroemer, 2008). 5.1.1 Mutant HTT expression induces autophagy and inhibits the mTOR pathway in neuronal PC6.3 cells 24 h after transfection, expression of N-terminal mHTT fragment proteins, containing 120 polyglutamine repeats (120Q), increased the phosphorylation of eEF2 and eIF2 in neuronal PC6.3 cells, reflecting an inhibition of global protein synthesis (I/Fig.1A). The translational block is not absolute as the proteins ATF4 and CHOP, involved in cell protection and ER stress, were synthesized (I/Fig.1C). Phosphorylation of p70 S6K and Akt was decreased in mHTT expressing cells (I/Fig.1E-F). p70 S6K is a target for the mTOR kinase in cells and the decreased phosphorylation correlates with mTOR inhibition. Akt activates mTOR not only by direct phosphorylation of TSC2, but also by regulation of cellular energy. Akt maintains a high ATP level that causes a decrease in the AMP/ATP ratio that in turn inhibits AMPK-mediated phosphorylation and activation of TSC2 (Wullschleger et al., 2006). Our data showed that the number of LC3 positive autophagosomes was increased in mHTT (120Q) expressing cells compared with control and wt HTT (18Q) expressing cells showed by RFP-LC3 and staining for endogenous LC3 (I/Fig.2A and Fig.2D). Quantification showed that the number of autophagosome positive cells increased significantly in N-terminal mHTT fragment proteins expressing cells as well as in cells expressing the disease-causing FL mHTT (75Q) proteins (I/Fig.2B). Also immunoblotting for endogeneous LC3, with and without lysosomal inhibitors, showed a conversion of LC3 (LC3-I to LC3-II) in mHTT expressing cells (I/Fig.2C). These results indicated that mHTT protein expression induces autophagy 24 h after transfection. 54

5.1.2 GADD34 interacts with tuberous sclerosis complex (TSC) proteins in mHTT expressing cells and mediates mTOR inhibition Previous studies have shown that the inhibition of mTOR pathway by using rapamycin can reduce cell toxicity induced by mHTT (Ravikumar et al., 2004). However, little is known about the upstream mechanisms controlling mTOR in neurodegenerative diseases, including HD. GADD34 plays an important role in the control of protein synthesis after global translation block; it interacts with PP1 and dephosphorylates eIF2α, which stimulates protein synthesis during cell injury (Harding et al., 2000; Novoa et al., 2003). GADD34 inhibits protein synthesis via the TSC protein complex that is also involved in the control of neuronal responses to stress (Di Nardo et al., 2009; Watanabe et al., 2007). The loss of TSC1 or TSC2 triggers the UPR in the ER (Ozcan et al., 2008). Interestingly, we observed largely intact levels of TSC1 or TSC2 in mHTT expressing cells at 24h, when the ER stress pathways are already activated (Reijonen et al., 2008). This probably reflects the nature of the upstream signals that activate UPR and ER stress in different cell types. The TSC complex, consisting of the TSC1 and the TSC2 proteins, regulates mTOR and the complex has emerged as a critical integrator of growth factor, nutrient and stress signals to control protein synthesis, cell growth and other cellular processes (Huang and Manning, 2008). Phosphorylation of TSC2 by upstream kinases, such as Akt, inhibits the activity of this complex and reduces mTOR signaling. Dephosphorylation of TSC2 by various phosphatases, on the other hand, inhibits mTOR and activates autophagy. GADD34 is engaged in dephosphorylation reactions and was previously shown to interact with the TSC complex (Watanabe et al., 2007). GADD34 levels may increase during cell stress, but little is known about the precise temporal changes of GADD34 after various insults. Our data showed that the expression of GADD34 was not significantly changed 24 h after transfection with mHTT constructs as determined by semiquantitative PCR and immunoblotting, neither was the subcellular localization of GADD34 altered in mHTT expressing cells (I/Fig.3A-B). However, immunoprecipitation showed that GADD34 specifically interacted with TSC1 in mHTT (120Q) proteins expressing cells 24 h after transfection but not in cells expressing wt HTT and empty EGFP-vectors (I/Fig.3C). The binding of GADD34 to TSC1 may stabilize the TSC-complex, as shown previously for GADD34 in non-neuronal cells (Inoki et al., 2002). It remains to be studied whether GADD34 may specifically interact with mHTT and other polyQ proteins.

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By binding to TSC-complex GADD34 may inhibit mTOR signaling 24h after mHTT transfection (I/Fig.3D). In line with this, the phosphorylation of mTOR was decreased in mHTT (120Q) expressing cells 24 h after transfection but not 48 h transfection. (I/Fig.3D-E). 5.1.3 Autophagy is inhibited at 48h in mHTT expressing cells - relationship to changes in GADD34 levels and AMP-activated protein kinase (AMPK) activity In contrast to the situation 24 h after mHTT transfection, we observed that the protein levels of GADD34 were downregulated in mHTT (120Q) expressing cells 48 h after transfection (I/Fig.4AB). This effect seemed to be dependent on mHTT since treatment with two well-known ER stress inducers, tunicamycin and thapsigargin, did not have an effect on GADD34 protein levels (I/Fig.4C). The decrease in GADD34 at 48 h was accompanied by an increase in the phosphorylation of TSC2 that in turn leads to activation of mTOR (I/Fig.3D) and to the inhibition of cytoprotective autophagy (I/Fig.4J-K). In line with this, we observed that there was a restoration in the phosphorylation of p70S6K and Akt in mHTT (120Q) expressing cells 48 h after transfection (I/Fig.4F-H). The decrease in GADD34 48 h after transfection contrasts to the situation at 24h and is probably due to an inhibition of protein synthesis that ultimately reduces GADD34 levels in mHTT expressing cells. It has been known for some time that another important cellular energy sensor, AMPK, can play a role in autophagy induction by phosphorylating TSC2 to inactivate mTOR (Wong et al., 2013). This let us to study the activity of AMPK in mHTT expressing cells. Our data showed that AMPK was activated in mHTT expressing cells at 24 h as shown by phosphorylation of its substrate AcetylCoA carboxylase (ACC) (I/Fig.4I). This indicates that AMPK may contribute to cytoprotective autophagy at early time points in mHTT expressing cells. However at 48 h the AMPK activity decreased that may lead to an increase in mTOR and an inhibition of autophagy in these cells (I/Fig3D). To study this more directly, we stained for LC3 positive autophagosomes in mHTT expressing cells at 48 h (I/Fig.4J). Quantification showed that the number of autophagosome positive cells was low in both wt cells and in those expressing either the N-terminal mHTT fragment protein or the disease-causing flHTT protein (I/Fig.4K). This data is in contrast to results obtained at 24 h, indicating that the lack of autophagy at 48 h may contribute to the deleterious effects of the HTT protein at longer time points. 5.1.4 Overexpression of GADD34 enhance autophagy and protects cells against mHTT-induced cell degeneration Our results indicate that GADD34 may be an important regulator of mTOR and autophagy in mHTT expressing cells. To study this further, we overexpressed and downregulated GADD34 in 56

neuronal PC6.3 cells and analyzed the effects on the activity of the TSC/mTOR pathway and on autophagy. Our data showed that overexpression of GADD34 increased the dephosphorylation of TSC2 in mHTT expressing cells at 48 h (I/Fig.5B). GADD34 overexpression also activated autophagy in cells expressing the wt HTT as well as the disease-causing FL mHTT protein at 48 h, shown by immunoblot (I/Fig.5D). The elevated autophagy increased cell viability in mHTT expressing cells as shown by MTT assay (I/Fig.5E). Our data showed that mHTT (120Q) protein expression induced caspase-3 and PARP (a substrate for caspase-3) cleavage. GADD34 overexpression reduced the cleavage of Caspase-3 and PARP (I/Fig.5G,F). Silencing of endogenous GADD34 increased caspase-3 activation and cell viability (I/Fig5E-G,F). Also cell viability was abolished by GADD34 silencing (I/Fig.5E). This data linked the expression levels of GADD34 to cell death and to the susceptibility of neuronal PC6.3 cells to undergo cell degeneration after expression of mHTT proteins. To further elucidate the role of autophagy in cytoprotection, we showed that the autophagy inhibitor 3-MA, significantly decrease the viability in mHTT expressing cells (I/Fig.5). This indicates that an unperturbed autophagy mediates protection against cell degeneration induced by mHTT proteins in the neuronal PC6.3 cells. These results demonstrate that inhibition of autophagy cause cell degeneration in neuronal PC6.3 cells. Previous data also support the view that active autophagy may facilitate the removal of misfolded proteins, and other toxic products and damaged organelles during cell stress (Levine and Kroemer, 2008; Ogata et al., 2006).

Figure 10. Summarizing figure.

Autophagy has been linked to increased cell survival after ER stress and autophagy observed here probably serves as a protective measure against the deleterious effects of mHTT protein and thus is a cell protective signal (Ogata et al., 2006). This study showed that mHTT proteins regulate the 57

mTOR pathway in neuronal PC6.3 cells with a decrease in protein synthesis and time-dependent change in the process of autophagy. GADD34 binds TSC1 and dephosphorylates TSC2, which leads to the inhibition of mTOR activity and induced cytoprotective autophagy. However, autophagy was inhibited at later time points in mHTT expressing cells. Overexpression of GADD34 prolonged the inhibition of mTOR and stimulated autophagy and thereby protected cells from mHTT induced cell death. These data underscore the importance of autophagy in the pathogenesis of HD (Figure 10.).

5.2 Sigma-1 receptor agonist PRE084 is protective against mutant huntingtininduced cell degeneration: involvement of calpastatin and the NF-κB pathway (II) Alterations in mitochondria and increased oxidative stress are associated with the disease progression in HD (Federico et al., 2012). ER stress and oxidative damage are linked through the close communication between the ER and mitochondria (Hayashi and Su, 2007). Sig-1R is a chaperone protein in the ER that is involved in ER stress regulation, but little is known about its role in HD or the mechanisms for cell protection. In this work, we studied the role of Sig-1R and its agonist 2-(4-morpholinoethyl)-1-phenylcyclohexane-1-carboxylate hydrochloride (PRE084) in a cellular model of HD with overexpression of wt and mHTT in neuronal PC6.3 cells. Sig-1R has been linked to neurodegenerative disorders as shown by analyses of materials from patients afflicted by these diseases. A postmortem study reported that Sig-1Rs were reduced in the hippocampus in AD and Sig-1R levels were also decreased in specimens from patients with early Parkinson’s disease (Mishina et al., 2005; Mishina et al., 2008). The mechanisms involved and the functional significance of these changes, however, are not fully understood. Sig-1R protein levels were decreased in the total cell lysates after expression of mHTT proteins, but restored by the agonist PRE084 (II/Fig.1A-D). In cells, Sig-1R is largely localized in the ER in the MAM sub-compartment, which links it close to the mitochondria (Hayashi et al., 2009). We observed a partial co-localization of Sig-1R with aggregates in mHTT expressing cells, suggesting that the Sig-1R may be redistributed or delocalized in these cells (Figure 11). This has been shown also by others (Miki et al., 2013). The agonist PRE084 is thought to stabilize Sig-1R and may therefore hinder its relocalization to protein aggregates containing mHTT proteins. However more studies are required to reveal the precise mechanisms by which PRE084 affects the cellular functions of Sig-1R in neuroprotection. 58

Figure 11. Confocal microscopy. Immunofluorescence (green) of PC6.3 cells transfected for 24 h with HTT fragment proteins containing 18Q (upper panels) and 120Q repeats (middle panels). Immunostaining was done using specific antibody against Sig-1R (red fluorescence). Sig-1R is present in part in cytoplasmic aggregates in cells expressing120Q-HTT in the merged picture (yellow color). Lower panels, immunostaining using the EM48 antibody recognizing mHTT aggregates. Note a partial co-localization with Sig-1R. Scale bar 15 mm.

5.2.1 The Sig1-R agonist PRE084 counteracts mHTT induced cell death and caspase activation In this study we investigated whether the Sig-1R has a role in cell degeneration. Expression of Nterminal mHTT proteins in PC6.3 cells has been shown to lead to cell death characterized by activation of various caspases (Reijonen et al., 2008; Reijonen et al., 2010). Several selective Sig-1R ligands have been synthesized since the identification of the Sig-1Rbinding sites (Maurice and Su, 2009; Su, 1982). Pharmacological studies have shown that such ligands may have many physiological effects ranging from neuroprotection to neuropsychiatric and anti-depressant effects (Hayashi and Su, 2004; Kourrich et al., 2012; Walker et al., 1990). The PRE084 was shown to have beneficial effects in various models of brain diseases including neurodegenerative and acute brain disorders. PRE084 was shown to promote cell survival and reduce oxidative stress caused by ischemia and toxicity induced by β-amyloid peptide (Marrazzo et al., 2005; Schetz et al., 2007). PRE084 was also neuroprotective against nerve avulsion injury and it acted on motoneurons both in vivo and in vitro (Guzman-Lenis et al., 2009; Mancuso et al., 2012; Penas et al., 2011). Our results showed that PRE084 protected neuronal PC6.3 cells against mHTT induced cell degeneration (II/Fig.2A-G). PRE084 counteracted caspase-3 cleavage that was induced by mHTT and reduced the cleavage of PARP that is a target for the active caspase-3 (II/Fig.2C). Previous 59

studies have shown that procaspase-12, which resides in the ER is cleaved in cells expressing the 120Q-HTT fragment proteins. (Reijonen et al., 2008) Our results showed that PRE084 also prevented the cleavage of caspase-12 in mHTT cells, suggesting a cytoprotective effect of PRE084 related to reduced ER stress (II/Fig.2F-G). 5.2.2 Stimulation with PRE084 increases cellular antioxidants and reduces ROS in mHTT expressing cells Sig-1R is present in MAM and stabilizes the IP3R1 (Hayashi and Su, 2007). Furthermore, mHTT can interact with ER and modulate calcium signaling in HD (Bezprozvanny, 2011; Higo et al., 2010; Reijonen et al., 2010). Using the mitochondrial calcium reporter aequorin, we showed that neither PRE084 nor expression of mHTT proteins influenced [Ca2+] in mitochondria in the PC6.3 cells (II/Fig.3A-D). This is an important observation, as deregulation of mitochondrial [Ca2 +] may in turn lead to an increased ROS production by this organelle with deleterious functional consequences (Decuypere et al., 2011). PRE084 did not directly affect the mitochondrial [Ca2+] in mHTT expressing cells compared with controls (II/Fig.3A-D). However, PRE084 did have an effect on the mitochondria-localized antioxidants. We stimulated cells expressing 120-huntingtin fragment protein with PRE084. The immunoblotting results showed that PRE084 was able to increase the cellular levels of various antioxidants that were downregulated by the 120Q-huntingtin (II/Fig.4AE). Along this line, stimulation of PC6.3 cells with PRE084 reduced intracellular ROS levels that were elevated by the disease-causing 75QFL huntingtin protein (II/Fig.4F). Consequently PRE084 seems to be able to decrease the levels of ROS and oxidative stress in neuronal PC6.3 cells expressing mHTT proteins via the upregulation of cellular antioxidants. 5.2.3 PRE084 increases NF-B-p65 levels and activate NF-B-p65 signaling in neuronal PC6.3 cells Previous study showed that the antioxidants Sod2 and Trx2 in cells are regulated by NF-κB signaling (Kairisalo et al., 2007). To study the involvement of NF-κB signaling in the antioxidant effects of Sig-1R in PC6.3 cells, we determined the levels of NF-κB-p65 (p65, RelA) protein that is important for NF-κB signaling (Hayden and Ghosh, 2004). Treatment of the cells with PRE084 was able to restore the NF-κB-p65 levels that were downregulated by mHTT (II/Fig.5A-B). Using a NFκB luciferase reporter construct we showed that NF-κB activity was decreased in cells expressing mHTT (75Q), whereas the wt HTT (17Q) had no effect. Most importantly, treatment with PRE084 prevented the decrease in NF-κB activity in mHTT (75Q) expressing cells (II/Fig.5C). The activation of NF-kB by PRE084 may underlie the beneficial effects of this compound in oxidative stress. 60

PRE084 and overexpression Sig1-R elevate calpastatin in mHTT expressing cells Previous study has shown that calpastatin is decreased and the activity of calpain is elevated in cells expressing the 120Q-huntingtin-fragment protein. Calpastatin functions as an inhibitor of calpain (Reijonen et al., 2010). Calpains are proteolytic enzymes activated by increased cell calcium and are involved in the regulation of oxidative stress and other processes in the cell (Harwood et al., 2005). The results showed that stimulation of the PC6.3 cells with PRE084 increased the calpastatin levels that are reduced by overexpressing mHTT (120Q) (II/Fig.6A-B). The increase in calpastatin by PRE084 was accompanied by a reduced calpain activity as shown by decreased cleavage of the calpain substrate, α-spectrin (II/Fig.6C).

Figure 12. Summarizing figure.

To study whether PRE084 elevated calpastatin in control cells, we stimulated cells with the compound for up to 24h. Data showed that calpastatin levels were significantly induced by PRE084 at 24 h and that overexpression of Sig-1R had the same effect (II/Fig.6D-E). To reveal whether calpastatin mRNA levels were also increased by PRE084, we performed qPCR that showed no change in calpastatin expression following PRE084 treatment (II/Fig.6F). This suggests that the PRE084 may primarily influence the stability or the degradation of calpastain in protein level. This study identified a signaling pathway mediating neuroprotection in cells expressing mHTT involving the calpastatin/NF-κB signaling pathway. PRE084 counteracted the deleterious effects of mHTT in neuronal PC6.3 cells by increasing calpastatin and by activating NF-κB signaling which increased antioxidant levels and reduced ROS. The findings are summarized in Figure 12.

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5.3 Ubiquitin specific protease-14 (Usp14) protects against mutant huntingtininduced cell degeneration and reduces cellular aggregates (III) Autophagy and the UPS are the two main degradation pathways in the cell. It has been thought before that degradation of mHTT by the proteasome is unwieldy and that aggregates are subjected to degradation by autophagy. However it has been shown that inhibiting the UPS leads to a greater accumulation of mHTT than inhibiting autophagy (Li et al., 2010). This has led to a suggestion that the clearance of soluble N-terminal mHTT is more dependent on the function of the UPS. Also, since the proteasomes are present in the nucleus, the UPS function may be more important for clearing nuclear aggregates. Autophagy might function as a backup system to clear mHTT and is more efficient to remove the aggregated forms of mHTT (Li et al., 2010). Although it is clear that the UPS is important for clearing mHTT, it remains unclear whether HD affects the capacity of neuronal UPS to remove toxic and misfolded proteins (Li and Li, 2011). Studies of HD mouse models have provided evidence that the extent of protein misfolding and aggregation is correlated with the length of polyQ tract and that smaller N-terminal HTT fragments containing an expanded polyQ tract are more prone to misfolding and aggregation. They are also more toxic than FL mHTT, which is evident by the fact that cellular models expressing smaller HTT fragments show more aggregates than those expressing FL mHTT (Gutekunst et al., 1999; Hackam et al., 2000). The aggregates contain ubiquitin and part of the HD pathophysiology could result from an imbalance in cellular ubiquitin levels (de Pril et al., 2004). Deubiquitinating enzymes are important for replenishing the ubiquitin pool but little is known about their roles in HD. Overexpression of mHTT induces ER stress and activation of ER signaling pathways (Reijonen et al., 2008; Vidal et al., 2011). Mutant HTT also impairs the ERAD pathway (Duennwald and Lindquist, 2008; Yang et al., 2010). 5.3.1 Usp14 levels are unchanged but Usp14 is redistributed in mutant huntingtin expressing cells Usp14 is an important DUB in the nervous system and we reasoned that it could be involved in neurodegenerative diseases caused by disturbed protein metabolism. We studied the Usp14 levels in neuronal PC6.3 cells after expression of HTT fragment proteins linked to EGFP having different polyQ repeats (18Q, 39Q, 53Q, 120Q). There was no overt change in the total cellular levels of Usp14 as observed by immunoblotting and immunohistochemistry (III/Fig.1A-B).

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To study whether the distribution of Usp14 in the cells might change we performed cell fractionation studies using PC6.3 control cells, 18Q and 120Q huntingtin fragment proteins. Data showed that the amount of Usp14 associated with the ER membrane fraction substantially decreased in mHTT expressing cells whereas the level of Usp14 increased in the cytosolic fraction (III/Fig.1CD). This indicates that the subcellular localization of Usp14 is altered in mHTT cells. Apart from its association with the ER, USP14 is also localized to proteasomes and specifically binds the proteasome 19S subunit S2 as shown by an immunoprecipitation assay for Usp14 using control and mHTT stably expressing HeLa cells (III/Fig.1E). Data showed that the binding of Usp14 to 19S S2 were roughly the same in cells expressing wt and mHTT, suggesting that Usp14 binds the proteasome also in mHTT expressing cells. 5.3.2 Overexpression of Usp14 reduces cellular aggregates in mHTT expressing cells mainly via the ubiquitin proteasome system As observed before, expression of the 120Q HTT fragment protein produces aggregates both in the nucleus and cytoplasm in neuronal PC6.3 cells (Reijonen et al., 2008). Accumulation of mHTT aggregates in the cell is a balance between their synthesis and degradation, involving the autophagy and/or the UPS. We noticed that overexpression of Usp14 decreased the amount of aggregates in mHTT expressing cells. We counted the number of aggregates from confocal images of 18Q and 120Q-huntingtin expressing PC6.3 cells in the absence or presence of Usp14 co-expression and performed a solubility assay (III/Fig.2A-C). Usp14 overexpression reduced SDS-insoluble aggregates in 120Q-huntingtin expressing PC6.3 cells as shown by the western blot analysis of the SDS-soluble and insoluble samples. Usp14 seemed to reduce mHTT aggregates mainly via the UPS. Mutant HTT expressing cells were treated with 20 μM MG-132 (a proteasome inhibitor) for 8 h and 5 mM 3-methyladenine (an autophagy inhibitor) for 4 h. The inhibitors, per se, had no significant effect on the number of cells with aggregates. However, when the UPS was inhibited, Usp14 overexpression did not reduce the amount of aggregates as it did without inhibition. Also inhibiting the UPS lead to a greater accumulation of aggregates than inhibiting autophagy after Usp14 overexpression (III/Fig.2D). Using mutant, enzymatically inactive, Usp14 construct, we observed that the reduction of aggregates required the intact DUBs domain (III/Fig.2E). Although the autophagy inhibitor 3-MA increased the cell aggregates in mHTT expressing cells, the effect was not statictically significant (III/Fig.2D). These results indicate that the Usp14-mediated decrease of mHTT aggregates largely involves the UPS and less so the autophagy system. Mutant 63

HTT aggregates contained ubiquitin as shown by the confocal images of cells transfected with GFPubiquitin and flHTT (17Q and 75Q). Data showed that ubiquitin was present throughout the cell in wt HTT expressing cells (17Q) but accumulated in the aggregates in cells expressing mHTT (75Q) (III/Fig.2F). To examine the function of Usp14 further, we used stably mHTT expressing HeLa cells in our studies (Filimonenko et al., 2010). These tetracycline-regulatable HeLa cells stably express the first 17 amino acids of HTT followed by a polyQ expansion (65Q or 103Q) tagged to monomeric CFP (HttPolyQ-mCFP). Doxycyclin (dox) can be used to shut off production of new HTT protein in these cells. To analyze aggregate clearance, HeLa cells were treated with dox, which led to inclusion clearance over several days in a macroautophagy- dependent manner (Yamamoto et al., 2006). Our data showed that inhibition of Usp14 or its activity did not influence the dox-mediated clearance of mHTT further indicating that Usp14 mainly affects the UPS in the aggregate clearance (III/Fig.3A). We also studied the interaction of Usp14 with the mHTT aggregates by performing immunoprecipitation experiments. Data showed that Usp14 binds both wt and mHTT (III/Fig.3F) 5.3.3 IRE1α, involved in ER stress response, is activated in mHTT expressing cells as well as in the striatum of mHTT transgenic (BAC-HD) mouse We and others have shown that the expression of mHTT induces ER stress by activating different ER signaling pathways such as the IRE1 pathway (Reijonen et al., 2010). Interestingly, previous data suggest that Usp14 interacts with IRE1 and influences ERAD (Nagai et al., 2009). However, nothing has been known about the role of Usp14 in mHTT induced ER stress and cell death. We therefore studied whether the reduced ER membrane association of Usp14 in mHTT expressing cells (III/Fig.1C-D) is reflected by a decreased binding of Usp14 to IRE1α. Immunoprecipitation experiments, with tissue lysates from BAC-HD mice, showed that Usp14 binds IRE1α in cells expressing wtHTT, but less so in mHTT expressing cells (III/Fig.5A) Immunostaining further showed that Usp114 is partly co-localized with IRE1α in wt, but not mHTT, expressing cells (III/Fig.5B). To study whether the decreased binding of Usp14 influences the activation of IRE1α, we performed WB experiments. Overexpression of Usp14 was able to inhibit phosphorylation of IRE1α in mHTT overexpressing cells (III/Fig.5C-D).

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The change in Usp14 and IRE1α was also observed in brain samples obtained from BAC-HD mice. IRE1α was activated in the striatum but not in brain cortex of BAC-HD mice compared with respective controls (III/Fig.6). We also observed that the binding of Usp14 to IRE1α was less prominent in samples from BAC-HD striatum than in wt controls (III/Fig.6). 5.3.4 Overexpression of Usp14 protected against cell degeneration and caspase-3 activation induced by mHTT Knowing that Usp14 binds to IRE1 in unstressed cells and dissociates in stressed conditions, we wanted to study if overexpression of Usp14 could protect cells against mHTT triggered ER stress and cell death. For this purpose we co-transfected neuronal PC6.3 cells with huntingtin and Usp14 plasmids. Overexpression of Usp14 significantly decreased cell death and increased cell survival in mHTT expressing cells (III/Fig4.A-C) Overexpression of Usp14 inhibited the cleavage of caspase-3 and PARP. Usp14 also reduced the number of cells with chromatin condensation and fragmentation (III/Fig.4C).

Figure 13. Summarizing figure.

Usp14 might have a dual role in neuroprotection by trimming the ubiquitin chains in the protein aggregates and thus rendering them accessible for the proteasome and by reducing IRE1α phosphorylation and ER stress in cells expressing mHTT. Hence, Usp14 can be a potential target to consider in developing novel therapeutics for HD and other aggregate disorders. Our results showed that ER stress induced IRE1 activation is part of mHTT toxicity which is inhibited by Usp14. Usp14 effectively reduced cellular aggregates and counteracted cell degeneration indicating its important role in mHTT induced cell toxicity. The results are summarized in Figure 13.

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6 CONCLUSIONS AND FUTURE PROSPECTS Since the identification of the gene and mutation responsible for the disease two decades ago, the field of HD has been the focus of intense research (The Huntington's Disease Collaborative Research Group, 1993). Important advances have been made, but still the precise pathophysiological mechanisms of HD are poorly understood. There is no cure for HD and currently there are only symptomatic drugs available for treating the illness. Both a loss-of-function of wtHTT and a toxic gain-of-function of mHTT are thought to contribute to HD pathology, promoting the activation of proteases, protein misfolding, inhibition of protein degradation, transcription deregulation, autophagy dysfunction, metabolic and mitochondrial disruption, and oxidative stress, which over the years culminate in neurodegeneration (Gil and Rego, 2008) Some of the pathological changes mentioned above were investigated in this thesis work. The studies clarified how ER stress, alterations in autophagy and in ubiquitin proteasome system and oxidative stress are involved in the early pathological processes in HD. The experiments were mainly carried out using cellular models of HD, PC6.3 and HeLa expressing HTT constructs with different polyQ stretches. PC6.3 cells are known to be useful and valid models for characterizing cellular and molecular events in cell death. However, further studies, executed in in vivo animal models and eventually in HD patients, will be needed in future. Alterations in autophagy have been proposed to contribute to pathogenesis in HD, but the precise mechanism behind autophagy malfunction is poorly understood. This thesis work unraveled that GADD34 is involved in autophagy regulation in cellular model of HD (I). Our results demonstrated that the mTOR pathway is downregulated and autophagy is induced in neuronal PC6.3 cells at 24 h after expression of mHTT. TSC complex interacted with GADD34, which caused TSC2 dephosphorylation and induction of autophagy in mHTT expressing cells. However, GADD34 protein levels and autophagy decreased at later time points, 48 h after transfection with the concomitant increase in mTOR activity. By overexpressing GADD34 we were able to induce autophagy in mHTT expressing cells. GADD34 induced autophagy served as a protective signal against the deleterious effects of mHTT and thus was cytoprotective. In view of neuroprotective effects of GADD34, it may be a useful target to consider in defining novel therapeutic strategies for HD and other neurodegenerative diseases.

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It remains to be studied whether GADD34 has other protein targets, in addition to PP1 and TSC complex that are active during cell stress and neurodegeneration. Another interesting prospect to study in future is the role of mHTT induced ER stress in mTOR-independent autophagy. mTOR has many vital cellular functions like regulation of translation and cell growth, and therefore, it is highly desirable to discover mTOR-independent, autophagy-inducing pathways or drugs as therapeutic targets. The present work also identified a signaling pathway mediating neuroprotection in cells expressing mHTT, involving the Sig-1R, its agonist PRE084, and the calpastatin/NF-κB signaling pathway (II). The beneficial effect of the Sig-1R agonist PRE084 was observed both in cells expressing the Nterminal mHTT-fragment protein (120Q) and the FL mHTT protein (75Q). The mechanism by which PRE084 induced neuroprotection was ascribed to the restoration of the calpastatin and NFκB-p65 levels, which then upregulated various cellular antioxidants and decreased ROS levels with a positive effect on cell survival. Our data indicated that PRE084 and other compounds influencing Sig-1R may constitute promising targets for the development of hopefully more effective therapeutic strategies for HD. However the next step would be to study whether PRE084, or other Sig-1R agonists, have neuroprotective effects in other experimental models of HD. Several compounds with agonistic activities at Sig-1R are already approved for clinical use and seem to be well tolerated (Francardo et al., 2014). This thesis revealed new interesting data about the involvement of the deubiquitinating enzyme, Usp14, and proteasome function in HD (III). HTT aggregates contain ubiquitin and part of the HD pathophysiology has been thought to result from inhibition of proteasomal function, but very little is known about the functions of deubiquitinating enzymes in HD. Our results showed that Usp14 enhanced proteasome-mediated clearance of mHTT aggregates and acted as a link between mHTTinduced ER stress induction and the enhanced neuronal degeneration. We further showed that overexpression of Usp14 was able to counteract cell death in mHTT expressing cells. We also observed that IRE1α, acting as an ER stress sensor, was preferentially activated in the striatum of BACHD mice. Along with this, the interaction of Usp14 with IRE1α was reduced in the ER membrane in mHTT expressing cells and in the striatum of BACHD mice. The precise mechanisms regulating the Usp14-IRE1α interaction and its role in protein quality control in the ER remains to be studied in more detail in the future. The role of Usp14 in protein degradation is controversial. Some studies suggest that Usp14 plays an inhibitory role in protein degradation and some studies have shown that Usp14 induces protein 67

degradation. A study showed that overexpression of Usp14 inhibited the ER-associated degradation (ERAD) pathway, and Usp14 depletion by siRNA effectively activated ERAD (Nagai et al., 2009). A more recent study showed that Usp14 removes ubiquitin chains from I-κB, therefore inducing IκB degradation (Mialki et al., 2013). In our study, we considered that the cleavage of the ubiquitin chain by Usp14 could render the mHTT aggregates more accessible for degradation via the proteasomes. It remains to be studied whether the same strategy involving increased cleavage of ubiquitin chains from protein aggregates may be beneficial in other neurodegenerative diseases as well. In recent years, our understanding of the dynamic aspect of ubiquitin modifications has increased significantly. We have realized that DUBs have equal prominence than the ubiquitin ligases. In addition to UPS, DUBs also have roles in the endocytic and ERAD pathways. In future it is important to study their role in proteasomal degradation and autophagy in more detail. It would also be interesting to study how Usp14 affects ERAD and autophagy in HD. HD can be regarded as a model neurodegenerative disease as it is monogenic and similar to several other neurodegenerative disorders. Thus, research in HD may convey information about the early intervention strategies relevant also to other neurodegenerative diseases like Parkinson’s disease and Alzheimer’s disease. Current animal models replicate insufficiently the selective striatal cell death of human HD, so better disease models, both in vivo and cellular models, are needed. Induced pluripotent stem cells and other patient-derived cell models could be good alternatives for future modeling of the disease.

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7 ACKNOWLEDGEMENTS This work was carried out between 2009 and 2014 at the Institute of Biomedicine, University of Helsinki and at the Minerva Foundation Institute for Medical Research, and was financially supported by the Finnish Graduate School of Neuroscience / Doctoral Program Brain and Mind, the Academy of Finland and Minerva Foundation. I would like to warmly thank my supervisors Professor Dan Lindholm and Docent Laura Korhonen for giving me an opportunity to do my doctoral thesis in their research group. I am grateful to Dan for trusting in me and giving me guidance and support in becoming a scientist. I appreciate your knowledge in neuroscience and enthusiasm for my projects. Thank you Laura for your expertise and guidance, especially at the beginning of my PhD project. I would like to thank my opponent Professor Raimo Tuominen and my custos Professor Kari Keinänen. The follow-up group members, Professor Kid Törnquist and Docent Urmas Arumäe, have been supportive and instructive during the meetings and I want to thank them for the advice they have given me. Urmas also served as a reviewer for my thesis together with Docent Eeva-Liisa Eskelinen, and I am thankful for their helpful comments. I acknowledge the co-authors for their contribution; Dr. Sami Reijonen, M.Sc. Jenny Kivinen, M.Sc. Ilari Pulli, Professor Kid Törnqvist, M.D. Tho Ho, Dr. Céline Bruelle, M.Sc. Sofia Lundh, M.Sc. Hai Do, M.Sc. Elisabeth Rappou, B.Med. Tuure Waltimo and Dr Åsa Petersén. I wish to thank all of the past and present members of Dan’s lab and all the co-workers in Minerva for the friendly atmosphere, filling sushi lunches, nice breakfasts and inspiring discussions. I want to thank Johanna for her company in the meetings and for our conversations over lunch and in the office and Raili for her company in the FGSN meetings. I also wish to express my thanks to Noora, Sami, Céline, Hai, Olga, Tima and Tuija for all their help in the lab and great moments outside the lab. I also want to thank my summer students Elisabeth and Tuure for their contribution to my research. Kristiina Söderholm is thanked for her help and technical assistance during my thesis project. I appreciate our discussions about life and dogs. Carita Estlander-Kortman, Cia Olsson and Anu Taulio are acknowledged for their kind help concerning administrative issues. I also want to thank FGSN/B&M coordinator Dr. Katri Wegelius for her help during my PhD studies.

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Thanks to Jenni, for the fun times in FGSN/B&M events, lunch dates and for the company at the gym. I also want to thank Maria for the encouragement and friendship throughout my PhD project. I want to thank my dear friend Hanna-Maria and my other friends for all the fun moments we have had during these years. Special thanks to Jenny for her help in the lab, our “life coaching” sessions, memorable trips to Neuroscience meetings and all other adventures. I am so happy to have you as my friend! Thank you Ville for your support, encouragement and being my best friend. At the end, I especially want to thank my family. My mom and dad, thank you for your love, support and always having my back. My nephew, Joakim, for bringing so much joy to my life. And last but not least, my lovely sisters, Anu and Annina for being the best sisters anyone could imagine. You are my rocks.

Helsinki, July 2014

Alise

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