Synuclein oligomers oppose longterm ... - Wiley Online Library

JOURNAL OF NEUROCHEMISTRY

| 2012 | 120 | 440–452

doi: 10.1111/j.1471-4159.2011.07576.x

,1

*Departments of Neuroscience & Cell Biology, University of Texas Medical Branch, Galveston, Texas, USA Departments of Neurology, University of Texas Medical Branch, Galveston, Texas, USA

Abstract Intracellular deposition of fibrillar aggregates of a-synuclein (aSyn) characterizes neurodegenerative diseases such as Parkinson’s disease (PD) and dementia with Lewy bodies. However, recent evidence indicates that small aSyn oligomeric aggregates that precede fibril formation may be the most neurotoxic species and can be found extracellularly. This new evidence has changed the view of pathological aSyn aggregation from a self-contained cellular phenomenon to an extracellular event and prompted investigation of the putative effects of extracellular aSyn oligomers. In this study, we report that extracellular application of aSyn oligomers detrimentally impacts neuronal welfare and memory function. We found that oligomeric aSyn increased intracellular Ca2+ levels, induced calcineurin (CaN) activity, decreased cAMP response element-binding protein (CREB) transcriptional activity and resulted in calcineurin-dependent death of human neuroblastoma cells. Similarly, CaN induction and CREB inhibition were

The signature event in synucleinopathies such as Parkinson’s disease, dementia with Lewy bodies (DLB) and multiple system atrophy is the misfolding, aggregation and intracellular accumulation of a-synuclein (aSyn) (Goedert 2001). aSyn is a 140 amino acid long protein abundant in pre-synaptic terminals of neurons where it may play a role in vesicular trafficking (Burre et al. 2010). Under normal physiological conditions, aSyn occurs as a a-helix rich tetramer that is not prone to aggregation (Bartels et al. 2011). However, under disease conditions aSyn misfolds into b-sheet-rich conformations and aggregates into fibrils that are the main components of Lewy bodies (LB) and Lewy neurites (Spillantini et al. 1998; Braak et al. 1999). These

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observed when aSyn oligomers were applied to organotypic brain slices, which opposed hippocampal long-term potentiation. Furthermore, aSyn oligomers induced CaN, inhibited CREB and evoked memory impairments in mice that received acute intracerebroventricular injections. Notably, all these events were reversed by pharmacological inhibition of CaN. Moreover, we found decreased active CaN and reduced levels of phosphorylated CREB in autopsy brain tissue from patients affected by dementia with Lewy bodies, which is characterized by deposition of aSyn aggregates and progressive cognitive decline. These results indicate that exogenously applied aSyn oligomers impact neuronal function and produce memory deficits through mechanisms that involve CaN activation. Keywords: a-synuclein, calcineurin, DLB, LTP, memory, oligomers. J. Neurochem. (2012) 120, 440–452.

Received September 14, 2011; revised manuscript received November 3, 2011; accepted November 3, 2011. Address correspondence and reprint requests to Giulio Taglialatela, PhD, 301 University Blvd, Galveston, TX 77555-1043, USA. E-mail: [email protected] 1 Present address: Department of Pathology, Oregon Health & Science University, Portland, Oregon, USA. Abbreviations used: aSyn, a-synuclein; aCSF, artificial CSF; Ab, amyloid beta; CaN, calcineurin; CREB, cAMP response element-binding protein; CS, conditioned stimulus; DLB, dementia with Lewy bodies; EPSC, excitatory post-synaptic current; ICV, intracerebroventricular; IP, intraperitoneal; LB, Lewy bodies; LDH, lactate dehydrogenase; LTP, long-term potentiation; PBS, phosphate-buffered saline; PD, Parkinson’s disease; PMI, postmortem interval; SDS, sodium dodecyl sulphate; SEAP, secreted alkaline phosphatase activity; US, unconditioned stimulus.

2011 The Authors Journal of Neurochemistry 2011 International Society for Neurochemistry, J. Neurochem. (2012) 120, 440–452

Cognitive effects of a-synuclein oligomers | 441

deposits are accompanied by progressive neuronal dysfunction and eventually death of affected neuronal populations (Braak and Del Tredici 2008). Behavioral and cognitive deficits are concomitant with these pathological changes (Braak et al. 2005; Caviness et al. 2011). The discovery of three missense mutations in the SNCA gene (encoding aSyn) that are linked to rare forms of familial PD (Polymeropoulos et al. 1997; Kruger et al. 1998; Zarranz et al. 2004) have highlighted the potential importance of aSyn in the pathogenesis of these diseases, likely caused by toxic gain-of-function. Indeed, aSyn knock-out mice are normal, but mice over-expressing aSyn display a synucleopathic phenotype (Kahle 2008). However, aSyn fibrils are thought to be protective because they may sequester more toxic intermediate aggregates such as oligomers (Olanow et al. 2004). aSyn oligomer toxicity is exemplified by a juvenile form of PD in which extensive neurodegeneration occurs in the absence of LB formation (Takahashi et al. 1994; Mori et al. 1998; Olanow et al. 2004). Also, microscopic and biochemical analysis showed that LB-bearing neurons do not exhibit cytotoxic signs (Gertz et al. 1994; Tompkins et al. 1997). More directly, in vitro and in vivo experiments recently illustrated that aSyn oligomers are toxic to neural cells whereas fibrils are not (Kayed et al. 2003; Putcha et al. 2010; Winner et al. 2011). Classically, the aggregation and deposition of aSyn has been considered an intracellular phenomenon (McNaught and Olanow 2006). However, more recent evidence demonstrates the existence of extracellular aSyn oligomers and suggests that they play key roles in disease progression (Lee 2008; Brown 2010). For example, aSyn oligomers are released from cultured cells and primary neurons (Emmanouilidou et al. 2010; Danzer et al. 2011) and are detectable in the CSF of PD patients (El-Agnaf et al. 2006) as well as in the soluble protein fraction from brains of DLB patients (Paleologou et al. 2009). In addition, aSyn can be transmitted from neuron to neuron (Desplats et al. 2009) or neuron to astroglia (Lee et al. 2010), fetal tissue grafts in the brain of PD patients acquire LB pathology (Li et al. 2008; Chu and Kordower 2009), and stem cells or fetal tissue transplanted into the CNS of transgenic mice over-expressing human aSyn show intracellular deposits formed by host aSyn (Desplats et al. 2009; Hansen et al. 2011). Collectively, these findings suggest that aSyn oligomers are found extracellularly. The presence of extracellularly released aSyn oligomers suggests that they are involved in synucleinopathic human diseases (Lee 2008). It also illustrates the importance of determining the effects of extracellular aSyn on neurons, specifically at synapses where aSyn is thought to be released from the aSyn-enriched pre-synaptic terminals (Schulz-Schaeffer 2010). With this goal in mind, the present study used in vitro, ex vivo and in vivo models to investigate the effects of exogenously applied aSyn oligo-

mers on Ca2+-related signaling, hippocampal synaptic plasticity and cognitive function. We focused on two proteins important in plasticity, learning and memory: calcineurin (CaN), a CNS-abundant Ca2+/calmodulin-dependent phosphatase (Mansuy et al. 1998; Mansuy 2003), and cAMP response element-binding protein (CREB), a transcription factor indirectly regulated by CaN (Bito et al. 1996; Pittenger and Kandel 1998; Silva et al. 1998; Kinney et al. 2009). We further report that similar alterations in active CaN and CREB as elicited in our model systems are also observed in autopsy human brain specimens from patients affected by DLB.

Methods Cell culture Cultured human neuroblastoma (SY5Y) cells from American Type Culture Collection (ATCC, Manassas, VA, USA) were maintained at 37C in a humidified cell incubator under a 5% CO2 atmosphere in T-125 culture flasks containing 10 mL of Dulbecco’s modified Eagle’s medium/F12 supplemented with 10% heat-inactivated fetal bovine serum (Sigma, St Louis, MO, USA). Every two days, half of the medium was replaced; when cultures reached confluence the cells were dislodged by vigorous shaking and divided into two flasks. SEAP transfection Transfections were performed using liposome-mediated plasmid introduction in SY5Y cells. The liposome used was DMRIIE-C (Invitrogen, Carlsbad, CA, USA). Cultures at 40–50% confluence received 1.2 pmole/mL of DNA coupled to DMRIIE-C at a ratio of 1 : 3 diluted in serum-free OptiMEM medium (Invitrogen). The liposome–DNA mix was replaced with fresh culture medium with serum after 3 h, and the cells were allowed at least 48 h of recovery prior to addition of aSyn with or without 10 lM forskolin (Sigma). Secreted alkaline phosphatase activity (SEAP; Clontech, Palo Alto, CA, USA) was used to measure the effects of test compounds on transcription factor activity. Plasmids containing the SEAP gene coupled to either a control promoter (pTAL-SEAP) or enhancer sequences specific to binding CRE (CRE-SEAP) were transiently transfected as described above. SEAP activity was assayed directly from the culture medium using the Great EscAPe Chemiluminescent Detection Kit according to manufacturer’s instruction (Clontech). LDH assay Release of lactate dehydrogenase (LDH) from cultured cells was used as an indirect measure of cell death. Extracellular LDH levels were determined using a sample of the culture medium and a commercially available kit (Roche, Indianapolis, IN, USA) according to the manufacturer’s instructions. Phosphatase activity assay The activity of CaN (PP2B) and combined activity of PP1/PP2A were assayed from lysed cytosolic cells and tissue extracts using a commercially available colorimetric kit (EMD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions.


442 | Z. S. Martin et al.

Ca2+ imaging SY5Y cells grown on poly-D-lysine coated coverslips were loaded with fura-2 acetoxymethyl ester (Invitrogen) according to manufacturer’s instruction. Slides were then mounted in a stage containing 1 mL of serum-free media. Ca2+ imaging was performed on an inverted Nikon Eclipse TE200 microscope with a Nikon S Fluor 20X, 0.75 numerical aperture objective at the Optical Microscopy Core, UTMB-Galveston. The samples were excited with a DG4/ DG5 xenon source of illumination at 340 and 380 nm using MetaFluor software (Universal Imaging, Dowingtown, PA, USA). Emission spectra were collected with a 71000av2 filter cube (Chroma Technology Corp., Bellows Fall, VT, USA) equipped with a fura-2 emitter D510/80 m. Experiments were performed at 21C. The basal Ca2+ ratios were recorded (i). At t = 5 min, 4 lM of monomeric aSyn was added (ii). At t = 15 min, 4 lM of fibrillar aSyn was added (iii), and at t = 25 min, 4 lM of oligomeric aSyn was added. Finally, at t = 45 min, 2 lM of ionomycin was added as a positive control. Ratiometric measurements and calculations were performed with MetaFluor software. The trace shown is the averaged 340/380 ratio response of 20 cells after background subtraction. Preparation of aSyn oligomers and fibrils aSyn oligomer and fibrils were prepared using standardized continuous stirring methods as previously described (Kayed et al. 2003). aSyn aggregation into oligomers and fibrils was initiated by seeding monomeric aSyn with a small amount (1 : 140 v : v) of amyloid beta (Ab) oligomers. The final concentration of contaminant Ab oligomers in the aSyn solution used in these experiments was 250 times lower than the minimum effective Ab concentration in similar experimental models (Reese et al. 2008). Seed Ab oligomers were prepared by dissolving 0.3 mg of lyophilized Ab42 (W.M. Keck Facility, University of Yale) in 200 lL of hexafluoro2-propanol (Sigma-Aldrich) for 10–20 min at 21C. The resulting Ab solution was added to distilled, deionized H2O in a siliconized Eppendorf tube to a final concentration of 66 lM. A magnetic stir bar was placed in the tube and holes were made in the cap to promote hexafluoro-2-propanol evaporation, and the solution was stirred for 48 h. For preparation of aSyn oligomers or fibrils, 0.1 mg of lyophilized powder of aSyn (rPeptide, Bogart, GA, USA) was dissolved in 230 lL H2O and seeded with the Ab oligomers (1 : 140 v : v) and stirred 20 min for oligomers and 5 days for fibrils. Quality and consistency of the resulting preparations were assessed by western and dot blots using the antibody 4D6, which recognizes aSyn in any aggregated or monomeric state, and A11, which specifically recognizes amyloid oligomers but not monomers or fibrils (Figure S1). Animals C57BL/6 mice were used in the intracerebroventricular (ICV) and fear conditioning paradigms. Male Sprague–Dawley rats (150– 250 g) were employed for electrophysiological experiments. Animals were maintained at the UTMB vivarium under USDA standards (12 h light/dark cycle, food and water ad libitum). Animals were killed by exposure to halothane vapors followed by decapitation at the end of each experiment. Brains were rapidly removed and either frozen whole (for immunohistochemistry) or dissected into individual areas including hippocampus, cerebellum,

frontal cortex, and occipital cortex, then stored at )80C until further biochemical analyses.

Ex vivo rat brain slices According to IACUC-approved protocols, rats were decapitated, and the brain was quickly removed and blocked in cold (4C) artificial CSF (aCSF) containing (in mM): 117 NaCl, 4.7 KCl, 1.2 Na2PO4, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 11 glucose, was oxygenated and equilibrated to pH 7.4 with 95% O2/5% CO2. Coronal brain slices (500 lm) containing the hippocampus were prepared using a Vibroslice (Camden Instruments, London, UK) and equilibrated in aCSF at 21C for 30 min prior to the addition of test compounds. At the end of the experiments, slices were rapidly frozen in liquid nitrogen and stored at )80C until further analyses were performed. Patch-clamp recordings Whole-cell recordings using the blind-patch technique or differential interference contrast-enhanced infrared (IR)-videomicroscopy were done as described previously (Fu and Neugebauer 2008; Li et al. 2011). Recording pipettes (3–5 MW tip resistance) made from borosilicate glass (1.5 mm o.d., 1.12 mm i.d.; Drummond) were filled with intracellular solution containing (in mM): 122 K-gluconate, 5 NaCl, 0.3 CaCl2, 2 MgCl2, 1 EGTA, 10 HEPES, 5 Na2-ATP, and 0.4 Na3-GTP; pH was adjusted to 7.2–7.3 with KOH and osmolarity to 280 mOsm/kg with sucrose. A single brain slice was transferred to the recording chamber and submerged in aCSF (31C), which superfused the slice at 2 mL/min. A single neuron was recorded in each slice, with a fresh slice used for each new experimental protocol. As the hippocampal regions are easily visible with the aid of light microscopy, recording electrodes were visually positioned in CA1. After tight (> 2GW) seals were formed, and the whole-cell configuration was obtained, neurons were included if their resting membrane potential was at least )50 mV, and the injection of depolarizing current evoked action potentials overshooting 0 mV. Data acquisition and analysis of voltage and current signals was performed with a dual 4-pole Bessel filter (Warner Instr., Hamden, CT, USA), low-noise Digidata 1322 interface (Axon Instr., Union City, CA, USA), Axoclamp-2B amplifier (Axon Instr.), Pentium PC, and pClamp9 software (Axon Instr.). Headstage voltage was monitored continuously on an oscilloscope to ensure precise performance of the amplifier. Neurons were voltage-clamped at )60 mV. Seal and series resistances were checked throughout the experiment (using pClamp9 membrane test function) to ensure high-quality recordings. Synaptic transmission and LTP induction Synaptic transmission was measured at the Schaffer collateral/ commissural pathway that provides glutamatergic input to CA1. Using a concentric bipolar stimulating electrode (Kopf Instr., Tujunga, CA, USA) of 22 kW resistance, excitatory post-synaptic currents (EPSCs) were evoked in CA1 neurons by focal electrical stimulation (Grass S88 stimulator, Grass Technologies, West Warwick, RI, USA) of the Schaffer collateral/commissural pathway. Test EPSCs were evoked by electrical stimuli (200 ls square-wave pulses) delivered at a frequency of 0.033 Hz. Stimulus intensity was set to evoke a half-maximal response (50% of EPSC amplitude). Long-term potentiation (LTP) was induced using high-frequency stimulation consisting of three trains at 100 Hz (1 s duration each;



intertrain-interval of 20 s). During high-frequency stimulation stimulus, intensity was raised to evoke an EPSC that was 75% of the maximum amplitude. ICV injection Using an IACUC-approved protocol, ICV injections were performed using a modified free-hand method as previously described (Dineley et al. 2010). Briefly, mice were deeply anesthetized with an intraperitoneal (IP) injection of ketamine/xylazine mixture (65 and 7.5 mg/kg, respectively), the scalp was shaved and an incision was made through the midline to expose the top of the skull. A 28 gauge needle held with a forceps was lowered 1 mm posterior and 1 mm lateral of the bregma. The needle was connected with 0.28 mm polyethylene tubing to a 25 lL syringe driven by an electronic programmable micro-infuser (Harvard Apparatus, Holliston, MA, USA), which was used to deliver 3 lL/mouse at a rate of 6 lL/min. After injection, the needle was left in place for 1 min and the surgical wound stitched before allowing the mouse to recover on a heated pad under a warm light. Reliability and consistency of injections was routinely tested during actual experiments by injecting India ink in parallel animals and macroscopically observing proper coloration of the ventricles (Dineley et al. 2010). 18 h after ICV, animals were given an IP injection of either 1% dimethylsulfoxide in 0.9% saline or CaN inhibitor FK506 diluted in 0.9% saline at 10 mg/kg (LC Laboratories, Woburn, MA, USA). Fear conditioning All mice were subject to fear conditioning training 6 h after FK506 (or vehicle) injection. In separate experiments, mice were treated with rapamycin (5 mg/kg IP) or vehicle (1% dimethylsulfoxide in 0.9% saline). There were 10 animals per treatment group. The protocol consisted of a training phase when the mice were placed in a particular environment (a chamber with particular lighting, geometry, or odor that constitutes the context conditioned stimulus, CS) and allowed to explore freely for 3 min. An auditory CS (80 dB white noise) was then presented for 30 s and one footshock (0.8 mA, 2 s duration; the unconditioned stimulus, US) was delivered during the final 2 sec of the auditory CS. A second presentation of the auditory CS and the US was delivered at the 5 min mark, and the animals remained in the chamber for another 2 min. Twenty-four hours later, the mice were returned to the same training chamber and the context test for fear learning was performed. The amount of freezing the mice exhibited during 5 min in the training chamber was measured. One hour later the cued test was performed in a completely novel context. The animals were placed in the testing chamber and freezing was measured for 3 min before the auditory CS was re-presented and freezing quantified over the next 3 min. Immunofluorescence Immunofluorescence was performed as described previously (Reese et al. 2011). Briefly, mouse or human brain sections (10 lm) were cut using a cryostat and affixed to Superfrost Plus slides (Thermo Fisher Scientific, Waltham, MA, USA) for storage at )80C until use. Slides were equilibrating to 21C, rinsed in 0.1 M phosphatebuffered saline (PBS) and fixed in ice-cold 4% paraformaldehyde for 15 min. After two brief washes in 0.1 M PBS, the sections were blocked and permeabilized for 1 h in 0.1 M PBS containing 10% goat serum, 0.03% Triton-X (ICN Radiochemicals, Irvine, CA,

USA), and 0.1% phosphatase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA). Incubation with primary antibodies in 0.1 M PBS containing 10% serum and 0.1% phosphatase inhibitor was carried out overnight at 4C. Antibodies used were CREB (mouse, 1 : 100; Cell Signaling, Danvers, MA, USA) and phospo-CREB (Ser133, rabbit, 1 : 100; Millipore, Billerica, MA, USA). Slides were washed and incubated for 1 h with Alexa Fluor 488 and 594 secondaries (1 : 600; Invitrogen) in the same solution as primaries. Slides were again rinsed twice in PBS and once in distilled, deionized H2O before Vectashield containing 4¢,6-diamidino-2phenylindole was applied (Vector Laboratories, Burlingame, CA, USA), and coverslips were mounted. Nail polish was applied to seal the edges and preserve fluorescence. Western blotting Cells or frozen tissue were lysed in sodium dodecyl sulphate (SDS) lysis buffer containing 5 mM EDTA, 50 mM Tris, 2% SDS, 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride and 1% protease cocktail inhibitors (Sigma). Following lysis, the cells were sonicated for 15 s, centrifuged at 20 000 g for 5 min, and the supernatants were collected. Protein samples (30 lg as determined by the BCA assay; Pierce, Rockford, IL, USA), were subjected to SDS-polyacrylamide gel electrophoresis using 12% gels, followed by electrophoretic transfer to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Membranes were then incubated with appropriate dilutions (usually 1 : 1000 v : v) of the primary antibody, washed in TBS-T, then incubated with a horseradish peroxidase conjugated secondary antibody (Bio-Rad) against rabbit IgG (for polyclonal primaries) or mouse IgG (for monoclonal primary antibodies). Signals were detected with Enhanced Chemiluminescence (ECL) substrate (Pierce). Human tissue Frozen frontal lobe tissue was obtained through a Materials Transfer Agreement with the Oregon Brain Bank at Oregon Health and Science University in Portland. Table 1 shows the diagnosis, age, sex, postmortem interval (PMI), Braak staging, plaque load and presence of aSyn pathology (macroscopic aggregates and Lewy bodies) in cortex, amygdala and midbrain of the cohort in this study. Collectively, these cases have PMI ranging from 3–48 hours (control: 10.55 ± 7.60; DLB: 24.43 ± 14.73; p = 0.0846, Student’s t-test) and a mean age of 77 years (control: 81.8 ± 7.66; DLB: 73.8 ± 6.57; p = 0.0823, Student’s t-test). Statistical analysis Where appropriate (two group comparisons), statistical differences were assessed by Student’s t-test. Analyses with more than two groups were subject to one-way analysis of variance (ANOVA) and, if overall p < 0.05, was followed by Fisher least significant difference. Columns and error bars represent mean ± SD, in all figures.

Results CaN is a key negative modulator of synaptic plasticity and memory function (Mansuy 2003; Lee and Ahnn 2004). We have previously reported that CaN hyperactivation mediates some of the detrimental effects that Ab oligomers exert on synapses and cognition in Alzheimer’s disease models



Table 1 Demographic characteristics of the cohort of subjects donating the brain sample specimens used in this study. PMI, postmortem interval; Braak stage 1–6; Plaque load 0–3; DLB, dementia with Lewy bodies; PD, Parkinson disease; aSyn pathology, presence of aSyn aggregates/LBs as detected by immunohistochemistry in cortex, amygdala and mid brain. aSyn pathology Case no.

State

Age

Sex

PMI (h)

Braak

Plaque

Cortex

Amyg

MdBrain

Clinical Dx

1720 1731 1761 1052 1728 198 727 887 1764 1783 1850 1859

Control Control Control Control Control DLB DLB DLB DLB/PD DLB/HS DLB/PD DLB/PD

73 74 86 87 > 89 72 76 67 73 78 85 66

M F M M F M F F M M M M

5.25 7.5 < 24 8 8 < 24 19 12 33 48 31.5 3.5

2 2 1 2 4 4 6 5 2 2 1 2

1 1 0 1 0 3 3 3 1 1 1 0

No No No No No Yes Yes Yes Yes Yes Yes Yes



Dementia Dementia/PD Dementia/PD Dementia/PD Dementia/PD Dementia/PD Dementia/PD

(Dineley et al. 2007, 2010; Reese et al. 2008). These neurotoxic aggregate species share structural and functional similarities with aSyn oligomers (Glabe and Kayed 2006). As such, we chose to examine whether specific aggregates of aSyn also induce CaN activity and subsequent detrimental effects on synaptic and cognitive function. Consistent with previous reports on other oligomeric amyloid proteins (Demuro et al. 2005), treatment of SY5Y cells with oligomeric aSyn augmented intracellular Ca2+ levels (Fig. 1). Monomeric and fibrillar aSyn were ineffetive, even though they were added to the very same cells that were responsive to oligomeric aSyn. Increased intracellular Ca2+ may result in activation of the Ca2+/calmodulin-dependent phosphatase CaN, which directly dephosphorylates the proapoptotic protein BAD (Wang et al. 1999). Therefore, we determined phosphatase activity and cell survival in SY5Y cells treated with monomeric, oligomeric, and fibrillar aSyn (Fig. 2a and b). Consistent with previous results (Kayed et al. 2003), we observed that only oligomeric aSyn increased the activity of CaN and induced cell death as measured by the amount of LDH in the culture medium. Conversely, the combined activity of the phosphatases PP1 and PP2A, which are related to CaN but are not activated by Ca2+/calmodulin, did not increase, suggesting that the observed cell death is coincident with increased CaN activity. Indeed, we found that cytotoxicity was attenuated by treatment with the CaN inhibitor FK506, but not by its analog Rapamycin, which has no effect on CaN activity although both act as immunosuppresants (Abraham and Wiederrecht 1996). Incubation with monomeric and fibrillar aSyn preparations did not cause an appreciable increase of cytotoxicity. Through either direct or indirect dephosphorylation of the transcription factor CREB, CaN exerts negative effects on synaptic plasticity and LTP (Josselyn et al. 2004; Hotte et al.

Fig. 1 aSyn oligomers, but not monomers or fibrils, increases intracellular Ca2+ levels in SY5Y cells. Graph showing a 35 min time course of Ca2+-dependent fluorescence recorded and averaged from 20 fluo-3 loaded SY5Y cells in response to consecutive application of aSyn monomers, fibrils, and oligomers (2 lM for 10 min each). Cells were challenged with ionomycin (2 lM) at the conclusion of the experiment. Each point represents the average of the 340/380 nm readings from 20 individual cells. Representative images (top) depict the response of four individual cells at the time points indicated by the corresponding number on graph. Warmer colors correspond to a higher level of fluorescence.

2007). To ascertain if these events might be triggered by aSyn oligomers, we first sought to determine if the oligomeric and fibrillar forms of aSyn had any effect on CREB transcriptional activity (Fig. 3). SY5Y cells were transiently transfected with a cDNA construct encoding a secreted alkaline phosphatase (SEAP) reporter gene driven by a CREB-sensitive promoter (pCRE-SEAP). All cells were additionally transfected with a vector encoding renilla luciferase to ensure equal transfection efficiency among samples (data not shown). Twenty-four hours after transfec-



Fig. 3 CREB-promoted transcriptional activity is inhibited by oligomeric aSyn in both basal and AC/PKA-stimulated conditions in SY5Y cells. Release of the reported gene product SEAP in SY5Y cells transiently transfected with the CREB-sensitive CRE-SEAP construct and treated for 3 h with oligomeric or fibrillary aSyn (2 lM) in the presence or absence of the AC/PKA activator forskolin (10 lM). n = 6 replicates per group; *p < 0.01 versus control cells (ANOVA).

Fig. 2 Selective induction of CaN activity and CaN-dependent cell death by oligomeric aSyn in SY5Y cells. (a) CaN (top) and combined PP1 + PP2A (bottom) activity in SY5Y cells treated for 24 h with 2 lM aSyn monomers, oligomers or fibrils. Graph is representative of two independent experiments returning similar results. n = 3 independent measurements per condition; *p < 0.05 versus control group (ANOVA). (b) LDH release in the culture medium of SY5Y cells treated for 24 h with 2 lM aSyn monomers, oligomers or fibrils. Separate dishes of oligomer-treated cells were additionally treated with FK506 (10 lM) or rapamycin (10 lM). Graph is representative of two independent experiments returning similar results. n = 4–6 independent measurements per condition; *p < 0.01 versus control group (ANOVA).

tion, cells were treated with 2 lM of either oligomeric or fibrillar aSyn, in the presence or absence of the adenylate cyclase activator forskolin (10 lM). The amount of SEAP in the culture medium, assayed 3 h after treatment, (well before the observance of any cell death) is indicative of CREBdriven transcription. Under basal conditions, release of SEAP was significantly diminished by treatment with oligomeric aSyn whereas cells treated with fibrillar aSyn were unaffected. Because the level of CREB-driven transcription in SY5Y cells is quite low under basal conditions, we performed a second experiment using cells treated with forskolin, which greatly induced PKA-promoted CREB transcription activity, evidenced by the increased SEAP

release (Fig. 3, right). Under these conditions, treatment of the stimulated cells with oligomeric aSyn significantly opposed the release of SEAP; those treated with fibrillar aSyn were not affected. Next, we expanded on the initial findings from SY5Y cells in ex vivo rat brain slices. Figure 4a shows a time course experiment in acutely cultured rat brain slices to assess pCREB by western blot. pCREB was maximally decreased in total protein extracts from brain slices treated with oligomeric aSYn for 15 min but recovered by the 30- to 60-min time points. Conversely, monomeric and fibrillar aSyn did not affect the phosphorylation of CREB. In fact, treatment with monomer appeared increase pCREB (although not statistical significant). The effect of oligomeric aSyn on pCREB was closely mirrored by a dramatic rise in CaN activity that also peaked at 15 min and remained significantly increased up to 60 min (Fig. 4b). Previous studies have shown that aberrant CaN activity is sufficient to oppose expression of LTP (Winder et al. 1998; Belmeguenai and Hansel 2005). To determine if oligomeric aSyn could affect LTP through inducing CaN activity, we performed electrophysiological recordings in hippocampal (CA1) brain slices from wild-type rats exposed to aSyn. In control slices, as well as those pre-treated for 1 h with 0.5 lM monomeric and fibrillar aSyn, Schaffer collateral high frequency stimulation resulted in enhanced CA1 EPSCs (Fig. 5a). However, when slices were incubated with 0.5 lM oligomeric aSyn, LTP expression was clearly opposed. This deficit in LTP expression could be rescued by adding the CaN inhibitor FK506 to the superfusion buffer (10 lM) 15 min before recording, whereas rapamycin had no effect. Immediately following electrophysiological recordings under these various conditions, slices were assayed for CaN activity (Fig. 5b). Consistent with results from cultured cells and hippocampal slices, only aSyn oligomers significantly increased CaN activity. This was prevented in slices



Fig. 4 Oligomeric aSyn reduces pCREB levels and coincidentally increases CaN activity in organotypic rat brain slices. (a) Representative western blot (top) detecting pCREB in total protein extracts from organotypic brain slices treated with 0.75 lM of monomeric, oligomeric and fibrillar aSyn for the time length shown. The blot was stripped and re-probed for total CREB to control for sample gel loading. Densitometry band quantification (bottom) confirmed that pCREB levels were significantly reduced by aSyn oligomers but not monomers or fibrils. The graph shows the average from 3 independent experiments. *p < 0.05 versus time 0 control (ANOVA). (b) CaN activity assay in homogenates from rat brain slices treated with 0.75 lM oligomeric aSyn for the times indicated. n = 3 replicates per time point. *p < 0.05 versus time 0 control (ANOVA).

that were co-treated with FK506, but not in those co-treated with rapamycin. Given that hippocampal slice LTP is a cellular model for learning and memory (Barnes 1995; Goosens and Maren 2002; Howland and Wang 2008), we tested the hypothesis that aSyn oligomers could induce CaN-dependent memory deficits in vivo. Our behavioral paradigm employed a 2-pair rodent fear conditioning paradigm in which a white noise cue (CS) was paired with a foot shock (US) (Dineley et al. 2007). Twenty-four hours after training the mice were tested for both contextual and cued fear learning. In the contextual test, there are no stimuli, and the animal instead relies on visuospatial and olfactory cues acquired during the training session and consolidated during the previous 24 h. Freezing behavior during a 5-min test phase is a reliable and robust

measure of hippocampus-dependent associative learning and memory and therefore especially appropriate for studying putative adverse effects of oligomeric aSyn on memory function (Freichel et al. 2007). Indeed, we found that mice treated with oligomeric aSyn have a marked reduction in freezing behavior in each epoch of the contextual test (Fig. 6b). However, these mice performed as well as control animals if they received FK506 6 h prior to training. FK506 treatment was designed to optimize the CNS drug concentration, which remains relatively stable 5–24 h following injection (Butcher et al. 1997; Fukudo et al. 2005) and does not affect freezing behavior per se (Dineley et al. 2007). Mice injected ICV with aSyn oligomers exhibited no significant differences in the amygdala-dependent cued fear conditioning (Figure S2), indicating that the hippocampus may be more sensitive to aSyn-induced alterations of CaN activity. After testing, these mice were killed and brain regions rapidly dissected. The hippocampus, amygdala, medial cortex, anterior cortex, basal forebrain/septum, and cerebellum were assessed for CaN activity and combined PP-1/PP2A activity on freshly homogenized tissue (Fig. 6d). We found that CaN activity was significantly elevated in hippocampus, amygdala, medial cortex, and basal forebrain/septum of mice subjected to icv injection of aSyn compared to saline-treated mice. While ineffective when administered alone (Dineley et al. 2007), FK506 restored CaN activity to control levels in these brain areas of aSyntreated mice. Conversely, combined PP-1/PP-2A enzymatic activity was not significantly affected by oligomeric aSyn or FK506 treatment in any of the CNS areas assayed, although there was a general trend toward increased values following aSyn oligomer treatment. CREB phosphorylation at Ser133 was measured in the same brain areas described above and we found that mice treated ICV with aSyn oligomers exhibited significantly reduced pCREB in the same brain regions that showed increased CaN activity (Fig. 6e). FK506 abolished aSyninduced reduction in CREB phosphorylation, suggesting that it was indeed CaN-dependent. It is worth noting that total CREB levels remained unchanged, illustrating that reduction of pCREB by aSyn oligomers could not be ascribed to an overall decrease of CREB, but rather reflected a decrease in protein phosphorylation. Immunofluorescent examination of the hippocampi of mice injected ICV with either saline or aSyn oligomers (Figure S3) confirmed that neuronal pCREB was significantly decreased by aSyn oligomers. As well, expression of total CREB remained unchanged after ICV injection of aSyn oligomers, although a trend toward reduced expression was noticeable. However, ICV injection of a parallel set of mice with aSyn fibrils did not elicit any change in pCREB or CREB expression as determined by immunohistochemistry (Figure S3), further illustrating that, consistent with the



Fig. 5 Oligomeric aSyn decreases LTP expression in hippocampal neurons in a CaN-dependent fashion. (a) HFS-induced expression of LTP in the hippocampus (CA1) of rat brain slices treated for 1 h with vehicle (control) or with 0.5 lM monomeric, oligomeric or fibrillar aSyn. A parallel set of oligomer-treated slices were additionally treated (45 min after addition of aSyn) with FK506 (10 lM) or rapamycin (10 lM), which remained in the perfusion buffer throughout the recording. Each symbol is the average of 10 monosynaptic EPSCs. Peak amplitudes were measured and expressed as percent of baseline values before HFS. Individual

traces in inserts show averaged EPSCs recorded before and 30 min after HFS. Monosynaptic EPSCs were evoked by electrical stimulation (200 ls square-wave pulses at 0.033 Hz) of the Schaffer collateral/commissural pathway. LTP was evoked by three highfrequency trains (100 Hz, 1 s duration each; intertrain-interval 20 s). For illustration purposes, all scales were set to a maximum of 400%. (b) CaN (top) and PP1 + PP2A combined activity (bottom) assayed in the same brain slices shown in A at the end of the LTP recording. Columns represent mean ± SD; n = 3 per group; *p < 0.05 versus control (ANOVA).

in vitro and ex vivo experiments described above, these effects were selectively induced only by oligomeric aSyn. Collectively, the results described above illustrate that CaN activation and the subsequent decrease in pCREB consistently characterize the impact of extracellular-applied aSyn oligomers on neurons, regardless of the model system. To extend these experimental observations to the actual human disease, we measured active CaN and pCREB levels in the brains of patients with clinically diagnosed DLB and age-matched controls. Immunofluorescence analysis of frontal cortex demonstrates the presence of highly immunoreactive macroscopic aSyn aggregates (Lewy bodies and Lewy neurites) in DLB brains (Figure S4). To determine CaN activation we measured levels of the truncated 57 kDa form of subunit A of CaN (CaN-A). Upon activation, subunit A is cleaved to reveal the active site (Liu et al. 2005). Therefore, levels of truncated CaN-A are an indirect measure of CaN activity. This approach is necessary when using human autopsy specimens because CaN is

rapidly deactivated by postmortem oxidation, making direct measurements of CaN enzymatic activity highly unreliable (Wang et al. 1996). Western blot analysis revealed that truncated CaN was increased in the DLB samples relative to age-matched control (Fig. 7a), suggesting increased CaN activity in the DLB brains. Along with increased truncation of CaN, levels of phosphorylated CREB were dramatically reduced in the nuclear fraction from the DLB samples as compared to controls, whereas levels of total CREB were relatively unchanged between the two groups (Fig. 7b). Confocal imaging confirmed the marked reduction of pCREB in the samples from DLB patients (Figure S5). Notably, double staining with NeuN, a neuronal marker, showed that pCREB was reduced in both neuronal (NeuN+) and non-neuronal (NeuN)) cells. It is unlikely that the observed changes were due to differential protein degradation in samples collected at different PMI. Indeed, CaN and pCREB levels did not correlate with PMI (CaN: r2 = 0.158, p = 0.852; pCREB: r2 = )0.390, p = 0.387).



Fig. 6 An acute ICV injection of aSyn oligomers coincidentally induces CaN activity, decreases pCREB, and impairs FC memory in mice. (a) A schematic illustrating the experimental design used in these experiments. aSyn oligomers (100 pmole/3 lL/mice) were injected ICV 24 h prior to FC training (48 h prior to FC test and sacrifice). Prior to FC training half of the mice were treated with FK506 (10 mg/kg IP) and half received saline. (b) Contextual FC test scores in naı¨ve mice and mice treated with saline ICV followed by saline IP (Saline/Saline) or aSyn ICV followed by saline ip (Oligo/Saline) or FK506 ip (oligo/FK506). Percent freezing (reflecting memory recollection) for each group is shown for each 60 s block of a 240 s test period (upper panel) or as cumulative data for the entire test period (lower panel). n = 15 mice per group. **p < 0.01 versus Saline/Saline

Discussion Taken together, our results indicate that the extracellular application of aSyn oligomers to in vitro, ex vivo and in vivo experimental models has profound effects on neuronal signaling that result in loss of neuronal viability, altered functional markers, decreased synaptic plasticity and memory deficits. In every model system, these effects were specifically attributable to oligomeric aSyn, rather than monomeric or fibrillar concoctions. A model emerges whereby extracellularly applied oligomeric aSyn increases intracellular Ca2+, which activates CaN, resulting in the net dephosphorylation of key signal transduction molecules that are required for synaptic plasticity, learning and memory. We further observe that higher levels of truncated, active CaN and lower levels of pCREB co-exist in the DLB human brain. It is therefore prudent to propose that aSyn oligomers

or oligo/FK506 (ANOVA). (c) CaN (top) and PP1 + PP2A combined activity (bottom) in the hippocampus (HIPP), amygdala (AMYG), medial cortex (MCTX), anterior cortex (ACTX), basal forebrain area (BFA) and cerebellum (CB) of the same mice killed at the end of the FC memory tests. (d) Phosphorylated (active) CREB levels assayed by western blot in the same brain areas from the same mice as shown in panel (d). Membranes were stripped and re-probed for total CREB and final values were expressed as ratio of pCREB/CREB calculated for each sample. Representative western blots from this experiment detecting pCREB and CREB in the hippocampus and amygdala are shown at the bottom. n = 5 mice per group (randomly chosen from the 15 per group used in behavioral studies). *p < 0.05 versus Saline/ Saline or oligo/FK506 (ANOVA).

are capable of disrupting signaling pathways that underlie synaptic function and may play a role in cognitive decline that accompanies certain synucleopathies such as DLB and possibly PD (Schulz-Schaeffer 2010). As previously noted, the crucial roles of CaN and pCREB in LTP expression have been well established, and there is ample consensus that LTP constitutes the cellular basis of hippocampus-dependent memory formation. As such, and in light of the LTP and behavioral experiments described here, we propose that at least one functional outcome of extracellular-applied aSyn oligomers on neuronal networks is CaNdependent deficits in synaptic plasticity and cognitive function. It is important to note that in both LTP and behavior experiments, the CaN inhibitor FK506 was applied minutes to hours after aSyn oligomers, respectively, indicating that the impairments in LTP or memory elicited by aSyn oligomers under these experimental conditions are reversible and further



Fig. 7 Increased truncated (active) CaN and decreased pCREB in frontal cortex of DLB brains. (a) (Top) Representative western blot detecting CaN-A in protein extracts from frontal cortex autopsy specimens from DLB patients (D) and age-matched controls (C). Membrane was stripped and re-probed for b-actin to ensure equal loading. (Bottom) Densitometric analysis of immunoreactive bands comparing the levels of the truncated CaN-A (corrected to b-actin) in frontal cortices from DLB patients (n = 7) and age-matched controls (n = 5). The average density value in control cases was arbitrarily set at 100. *p < 0.05 versus control (ANOVA). (b) (Top) Representative western blot detecting pCREB in protein extracts from the frontal cortex of DLB patients (D) and age-matched controls (C). Membranes were stripped and re-probed for total CREB, NeuN (neuronal marker), and b-actin (loading control). (Bottom) Densitometric analysis of immunoreactive bands comparing CREB and pCREB levels (both corrected to b-actin) in frontal cortices from DLB (n = 7) and age-matched controls (n = 5). The average density value in control cases was arbitrarily set at 100. *p < 0.001 versus control (ANOVA).

suggesting that these effects of aSyn oligomers are not the result of aSyn-induced cell death. Indeed, we detected no change in the level of phosphorylation of the pro-apoptotic

BAD assayed in brain tissue samples from the same animals receiving ICV injection of aSyn oligomers and behavioral analysis (Supplementary Figure S6), and CaN-mediated dephosphorylation/activation of the pro-apoptotic protein BAD has been described as a central event in neuronal apoptosis (Wang et al. 1999; Springer et al. 2000; Shou et al. 2004; Yang et al. 2004). Our autopsy specimen results are highly suggestive that similar mechanisms linking aSyn aggregates and synaptic plasticity signaling elements may occur in brains from humans affected by clinically diagnosed DLB. Specifically, we found significantly higher levels of truncated, active CaN and lower levels of pCREB. Given the growing evidence for a central role of aSyn oligomers in the clinical manifestation and progression of synucleopathic disorders (Brown 2010; Brundin et al. 2010; Gadad et al. 2011; Winner et al. 2011), and in light of our current results suggesting CaN-dependent synaptic and memory effects of aSyn oligomers in experimental models, it is tempting to hypothesize that aSyn oligomers, through affecting levels of synaptic plasticity and memory proteins such as CaN and pCREB (Silva et al. 1998; Mansuy 2003; Josselyn and Nguyen 2005), may play a role in cognitive decline accompanying DLB progression (Schulz-Schaeffer 2010). As cognitive impairments often occur in several synucleopathies including PD (Braak et al. 2005; Caviness et al. 2011; Ferrer 2011), it is plausible that the present observations may extend beyond the specific mechanisms of DLB pathology. Indeed, we observed a dramatic decrease in CREB phosphorylation in samples from patients affected by DLB. This decrease was tightly associated with increased levels of truncated, active CaN, suggesting that a similar mechanism driven by aSyn oligomers (possibly acting extracellularly) linking increased CaN activity and decreased pCREB may also occur in diseased human brain. To that end, it is interesting that the decrease of pCREB in DLB brains was observed in both neuronal (NeuN+) and non-neuronal (NeuN)) cells, suggesting a global, rather than neuron-specific, phenomenon. Indeed, extracellular misfolded aSyn oligomers have recently gained much attention because of their potential role in disease progression (Lee 2008; Schulz-Schaeffer 2010). Recent investigations have suggested that aSyn oligomers are released via an atypical secretory pathway. aSyn oligomers are released in the culture medium of cells over-expressing human aSyn (Emmanouilidou et al. 2010), and aSyn oligomers have been found in plasma of PD patients (El-Agnaf et al. 2006). Like all neurodegenerative diseases, PD has a typical neuroanatomical progression that affects the brainstem forward to the frontal cortex (Braak et al. 2003), suggesting prion-like intercellular transmission of misfolded aSyn species (Angot et al. 2010; Brundin et al. 2010). Indeed, neuron-to-neuron and neuron-to-astroglia transmission of misfolded aSyn species has been demonstrated in vitro and in vivo (Desplats et al. 2009; Lee et al. 2010). Furthermore,



aSyn pathology deriving from the host aSyn pool has been found in fetal tissue grafted into the CNS of PD patients (Li et al. 2008; Chu and Kordower 2009), as well as in dopaminergic neurons grafted in the CNS of aSyn transgenic mice (Hansen et al. 2011). This collectively implies that aggregated aSyn species may gain access to the extracellular space where they are taken up by neighboring neurons. This mechanism may be of particular significance at the synapse, where aSyn oligomers can be released pre-synaptically and may impact post-synaptic elements. Indeed, abundant aSyn aggregates are found at pre-synaptic terminals in PD and DLB (Schulz-Schaeffer 2010) and their pathological presence is associated with loss of pre- and post-synaptic markers (Kramer and Schulz-Schaeffer 2007) and dendritic spine retraction (Zaja-Milatovic et al. 2005, 2006; Revuelta et al. 2008). This neuro-anatomical evidence suggests that significant synapse loss occurs in the brains of PD and DLB patients and may underlie the cognitive symptoms of these diseases; it also introduces a loss-of-function component to the pathology of diseases like PD. where overall clinical manifestations have classically been ascribed to overt neuronal death in specific brain regions. Therefore, our current results reveal a previously undocumented mechanism whereby small oligomeric aSyn species possibly present within the extracellular space may disturb synaptic plasticity, thus detrimentally affecting memory function and cognitive ability. Our results further suggest that such pathological mechanisms may be effectively targeted by pharmacological inhibition of CaN and possibly by future immunotherapies aimed at scavenging extracellular oligomeric aSyn.

Acknowledgements This work was supported by NINDS grant R01NS059901 (GT), Alzheimer’s Association grant IIRG-90755 (GT), and a Mitchell Center Neurodegenerative Center Collaborative Grant (GT). The authors wish to thank Dr Randall Woltjer at the Department of Pathology, Oregon Health & Science University, Portland, Oregon, for generously providing us the human autopsy brain specimens and for critical feedback.

Supporting information Additional supporting information may be found in the online version of this article: Figure S1. Quality control of aggregated aSyn species used in the present studies. Figure S2. Amygdala-dependent cued FC is not affected by an acute ICV injection of aSyn oligomers in mice. Figure S3. aSyn oligomers, but not fibrils, decrease hippocampal pCREB. Figure S4. aSyn macroscopic aggregates in frontal cortex from DLB patients.

Figure S5. Immunohistochemistry confirms a reduction of pCREB in both neurons and other cell types in DLB frontal cortex. Figure S6. An acute ICV injection of aSyn oligomers does not alter phosphorylation of BAD in mice. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

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