Comparative And Quantitative Proteomics Study Of ...

Comparative And Quantitative Proteomics Study Of Cerebrospinal Fluid From SpinalInjured Subjects

Thesis Submitted for the Degree of Doctor of Philosophy (Science) in Biotechnology by Mohor Biplab Sengupta

Department of Biotechnology University of Calcutta 2015

To Ma and Baba

Acknowledgement It has been five years since I joined SINP and I find myself surprised at how fast all these years passed. I joined the lab with practically no knowledge of the experimental methods that I was about to use. Added to that, the fact that I would be working in a clinical setting for a major part was both exciting and intimidating. Needless to say, I started my journey with a series of mistakes and several tumbling blocks that gave me my initial Ph.D. blues. The person who guided me through this is my Ph.D. mentor Dr. Debashis Mukhopadhyay and I am immensely thankful to him for believing that I could do it and letting me be. The creative liberty I was offered in this lab was substantial and I will always be grateful to him for that. Thank you Sir! I thank my clinical collaborators Dr. Kiran Mukhopadhyay, Dr. Sourav Iswarari, Dr. Krishnapada Sardar and Dr. Biplab Acharyya and my scientific collaborators Dr. Pradeep Mohanty and Dr. Mahashweta Basu. Together we have made a daunting project take its final shape. I would also like to immensely thank Dr. Atri Chatterjee for the very useful inputs at various stages of my work. He is an amazing doctor and has helped me understand and conceptualise many clinical aspects. No words would be enough to express my gratitude to Dr. Arunabha Chakrabarti, my senior in lab. He taught me all the proteomics techniques and the invaluable ‘tricks of the trade’ that helped me troubleshoot any problems faster. Apart from being a great friend, he helped me understand how a lab runs. I hope to always be in touch with him and seek his advice whenever I am in doubt. I formally thank SINP and its Director, Dr. Mohanty. I extend my thanks to the head of Biophysics and Structural Genomics Division, Dr. Subrata Banerjee and all other faculty members of BSG which include Dr. Oishee Chakrabarti, Dr. Sangram Bagh, Dr. Soumen Kanti Manna, Dr. Chandrima Das, Dr. Kaushik Sengupta and Dr. Dipak Dasgupta. They are all acquainted with my work, some having heard my presentations and some having personally guided me. Each of them at some point of time gave me important suggestions about work. I would especially like to thank Dr. Oishee Chakrabarti for taking a lot of time out for me and guiding me through the a major part of my work. Her support meant a great deal to me and I absolutely admire and look up to her. Informally, I thank my two great friends Devika and Priyanka. They have stood by me through thick and thin and this does not only include lab related issues. They are a part of my life and I am fortunate to have them with me. I thank my past and present lab members Arunabha, Samir, Shounak, Kasturi, Sayantani, Piyali and the various summer students who have worked for a short while with me but gave a large part of their dedication to the work. That includes Preeti, Namrita, Alka and Sourav. Had it

not been for them, I would be requiring more hours in a day to get things in place. I thank all the students of BSG, Avik, Suchismita, Shilpita, Sutapa, Madhurima, Debasree, Anita, Nandini, Anindita, Srijan, Saran, Rukmini, Zenia, Debdatto and Angshu. I wish to thank all members of BSG staff and I will especially mention Sanjoy Show whose single handed dedication has ensured cleanliness of the lab and kept the tissue culture room and equipments contamination free. I thank my M.Sc. mentors Dr. Dhrubajyoti Chattopadhyay, Dr. Indubhushan Chatterjee, Dr. Koustubh Panda, Dr. Gopal Chakrabarti and Dr. Anindita Sil for helping me shape my career and understand the basics clearly. I finally thank Council of Scientific and Industrial Research (CSIR), Govt. of India, for funding my fellowship. My work was funded by the Integrative Biology on Omics Platform (IBOP) project of the Dept. of Atomic Energy (DAE), Govt. of India. Lastly I thank the close and solid circle of support around me; my family. My husband Badhon has been much more than just that. He has let me share all mundane lab news with him and comforted me when I was down. My parents in law and my sister in law Roshni have given me all their love and care. I feel lucky to have them by my side. I thank my parents who, needless to say, are the reason for everything that I have achieved so far. This thesis is dedicated to you, Ma and Baba. If I have left out anyone I apologise. It is just that there is such an immense list of people who have been there with me and for me through this journey that I am afraid I might have not mentioned everyone!

Mohor Biplab Sengupta

Abstract Spinal cord injury (SCI) is one of the major causes of mortality and morbidity worldwide. The causes of SCI range from gunshot wounds to fall from height. In India, though comprehensive epidemiological studies are lacking, the study cohort that was studied in this thesis, consisted of east Indian ethnicity people with mechanical damage to the spinal cord due to fall. Post SCI, hypoperfusion in the grey matter, spillage of neurotransmitters, glutamate excitotoxicity, ionic perturbations, damage and failure of plasma membrane, inflammatory processes, oxidative damage, lipid peroxidation and apoptosis are widely prevalent. The perturbed molecular scenario at the vicinity of the injured cord lasts across the chronic phase post SCI and restoration to normal molecular functioning is very gradual. SCI is graded into five classes (A-E) according to severity by the American Spinal Injury Association (ASIA) Impairment Scale (AIS). AIS A SCI (complete injury) is the most severe form of injury, whereas, AIS E is the least severe form of injury (classified as normal). AIS B to D are classified as incomplete injuries. The study samples were two severity groups with vastly different prognosis; complete and incomplete injury. Perturbed molecular pathways at the vicinity of the injury were deduced by first identifying the differentially abundant proteins for the two injury groups and subsequently by constructing a protein-protein interaction (PPIN) consisting of the differentially abundant proteins and their primary interactors as nodes. Protein phosphorylation, iron transport, mRNA metabolism, lipid catabolism, ATP catabolism, immune response, DNA repair and tRNA and rRNA transcription were perturbed during SCI. The lipid catabolism pathway was particularly interesting in the background of injury due to its role in clearing the huge amount of myelin debris. Apolipoprotein A1 (ApoA1) was differentially abundant in several CSF samples among the two injury severity types. ApoA1 was further found to be negatively regulated by TNF-α at a later phase of secondary injury, it was found to activate ERK1/2 and finally, it was shown to alter wound healing dynamics in an in vitro scratch injury model of neuroblastoma cell line.

Contents Chapter 1: Introduction 1 1.1. Spinal Cord Injury: Epidemiology, mechanisms, demography 2 1.2. The mammalian spinal cord 4 1.2.1. Anatomical overview 4 1.2.2. Topography of spinal cord 6 1.3. Classification of SCI 9 1.3.1. Some definitions 9 1.3.2. ASIA Impairment Scale (AIS) of injury severity 10 1.4. Autonomic dysfunctions post SCI 10 1.5. Primary injury 11 1.6. Secondary injury 11 1.6.1. Immune response 12 1.6.2. Breach of plasma membrane 12 1.6.3. Glutamate excitotoxicity 13 1.6.4. Electrolyte imbalance 13 1.6.5. Mitochondria damage 14 1.6.6. Lipid peroxidation 14 1.6.7. Demyelination of surviving axons 14 1.6.8. Apoptosis 15 1.6.9. Vascular system derangements 15 1.6.10. Neurogenic shock 16 1.6.11. Chromatolysis 16

1.7. Molecular inhibition of axon regeneration 16 1.7.1. Myelin associated inhibitory factors (MAIFs) 17 1.7.2. CSPGs and the glial scar 18 1.8. ApoA1: a probable player in healing 21 1.8.1. ApoA1 in inflammation 23 1.8.2. ApoA1 activates MAPK and Cdc42 pathways 24 1.9. Objective of the thesis 24

Chapter 2: Materials and Methods 36 2.1. Statement of Ethics 37 2.2. Patient selection and scoring 37 2.3. CSF collection and processing for proteomics experiments 40 2.4. Two-dimensional gel electrophoresis 41 2.5. Identification of proteins by MALDI-MS 41 2.6. Sample labelling for 2D-DIGE 42 2.7. Scanning of gels, analysis by DeCyder and biological variance analysis 42 2.8. Biological variance analysis 42 2.9. Construction of protein-protein network (PPIN) 42 2.10. Module detection 43 2.11. Enrichment analysis 43 2.12. Cell culture and passage 44 2.13. Infliction of mechanical injury to cells 44 2.14. Imaging and analysis of scratch closure 44 2.15. ApoA1 treatment of cells and protein extraction 44 2.16. Western blot for CSF and cell lysate proteins 45

Chapter 3: The severity and time based differential proteome of SCI CSF 47 3.1. Patient details 48 3.1.1. Group I (1-8 days post injury) 48 3.1.2. Group II (15-60 days post injury) 49 3.2. Eight CSF proteins show differential abundance in complete and incomplete SCI at 1-8 days post injury 50 3.3. Abundance levels of the eight proteins at 15-60 days post injury 56 3.4. Temporal changes in abundance of Haptoglobin and Zinc alpha 2 glycoprotein in both injury severities 57

Chapter 4: The perturbed biological pathways in SCI 59 4.1. Enrichment analysis revealed six functional modules with perturbed members 60

Chapter 5: Modulation and effect of ApoA1 in injury 64 5.1. ApoA1 is more abundant in complete injury CSF 65 5.2. HDL shows significantly greater abundance in complete injury at 1-8 days post injury 65 5.3. Serum amyloid A is increased at 15-60 days post injury 66 5.4. TNF-alpha is increased at 15-60 days post injury 67 5.5. ApoA1 activates ERK1/2 in neuroblastoma cells 68 5.6. ApoA1 causes marginally faster closure of scratch injury in SHSY5Y model 69

Chapter 6: Discussion 72

Appendix 1 I Appendix 2 LIV

Abbreviations

(Those not already mentioned in the text. Sequence follows their appearance order in the text)

SCI Spinal Cord Injury CSF Cerebrospinal Fluid ASIA American Spinal Injury Association ISNCSCI International Standards for Neurological and Functional Classification of Spinal Cord Injury IMSOP International Medical Society of Paraplegia AIS ASIA Impairment Scale CNS Central Nervous System IL Interleukin TNF Tumour Necrosis Factor NMDA N-methyl-D-aspartate ATP Adenosine Tri-Phosphate PNS Peripheral Nervous System DRG Dorsal Root Ganglion LINGO Leucine rich repeat and Ig domain containing (protein) TGF Transforming Growth Factor GFAP Glial Fibrillary Acidic Protein ECM Extra Cellular Matrix PI3K Phosphatidylinositol 3-Kinase mTOR Mammalian Target of Rapamycin PTP Protein Tyrosine Phosphatase ERK Extracellular Signal-Regulated Kinase SR-B1 Scavenger Receptor class B type 1 ABCA1 ATP Binding Cassette Transporter member 1 JNK c-Jun N-terminal Kinase Cdc Cell Division Cycle MAPK Mitogen-Activated Protein Kinase MKK MAPK Kinase Kinase

IPG Immobilised pH Gradient BSA Bovine Serum Albumin DIGE Difference Gel Electrophoresis CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate DTT Dithiothreitol SDS Sodium Dodecyl Sulphate MALDI Matrix-Assisted Laser Desorption Ionisation ACN Acetonitrile TFA Trifluoroascetic acid TOF Time Of Flight NCBI National Center for Biotechnology Information EDTA Ethylenediaminetetraacetic Acid PMSF Phenylmethylsulfonyl Fluoride TBST Tris-Buffered Saline with Tween 20 MHC Major Histocompatibility Complex APC Antigen presenting cell

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Chapter 1: Introduction

SCI; Mechanical and Molecular Trauma 2 1.1. Spinal Cord Injury: Epidemiology, mechanisms, demography SCI is defined as an acute traumatic injury to the spinal cord that leads to sensory and motor function deficits in various degrees. Depending on the injury severity, it might lead to paralysis. Persons afflicted might experience a plethora of other conditions and there is a general decline in the quality of life (1). SCI incurs tremendous health care costs for the affected individuals and several studies recently have iterated the cumulative financial burden it creates on the system. For cervical complete and incomplete injuries, the first year cost was $157,718 and $56,505 respectively, according to a Canadian study between 1997 to 2007 (2). According to another study from Canada, the lifetime economic burden incurred per person with SCI is approximately $1.5 million and $3.0 million for incomplete paraplegia and complete tetraplegia respectively (3). In USA, a recent study with Medicaid beneficiaries shows that the adjusted mean expenditures for SCI patients with neuropathic pain are US$47,518 and US$30,150 for those without neuropathic pain, within 12 months after the first SCI diagnosis (4). Another study cites the average per person annual cost in the first year post SCI as $334,170 and $40,589 for subsequent years for incomplete injury versus $1023,924 for the first year and $177,808 for subsequent years for complete tetraplegic patients in 2011 (5). The same study also mentions a model system in which 57.1% people who reported SCI were employed before their injury, and this number fell to 11.8% within one year post injury. With physical rehabilitation over time many of the patients regained their ability to work and twenty years post SCI the same group had a 35.2% employment rate (5). Typically, SCI is one of the leading causes of mortality and morbidity. Global average incidence is highest in Asia with 43.8 per million people afflicted and the lowest is in Africa with 27.85 per million people afflicted annually (1). Relative annual incidences of SCI vary widely with the geographical region and the time period of study (6). Highest incidences have been recorded from central Portugal and Taiwan with both countries having an annual average incidence of more than 50 per million (6). On the higher side, Iran, New Zealand, several states of USA like Utah, Alaska, California and Mississippi and Alberta in Canada have recorded more than 40 per million incidences of SCI. On the lower side, Australia, Finland, France, Ireland, Jordan, Norway, the Netherlands, Sweden, Spain, Switzerland and Turkey report incidences below 20 per million, whereas, Fiji and Denmark have reported incidences of less than 10 per million (6). Prevalence is defined as the proportion of population afflicted with the condition at a certain time. Some prevalence studies reports 906 per million in USA (7), 280 per million in Helsinki during 1953-1998 (8), 250 per million in the Rhone-Alpes region of France during 1970-1975

SCI; Mechanical and Molecular Trauma 3 (9), 526 per million in Iceland during 1975-2009 (10), 65 per million in western Norway during 1952-2001 (11), 440 per million in Iran during 2007-2008 (12) and 681 per million in Australia during 1986 to 1997 (13). Prevalence studies have shown that prevalence is highest in the USA with 1800 per million afflicted people and the lowest in south Asia with 236 per million people afflicted (1). However these data correspond to different durations of observation and in different times, so prevalence cannot be conclusively established. Average age at the time of injury worldwide is 41.15 years (1). It has been observed that the age at injury has increased over time which is attributed to increase in general life expectancy and mean age of the general population over time (6). For example, the average age at injury in 1970 was 29 years and in 2005 it was 37 years according to the “National Spinal Cord Injury Statistical Center Database (1). In our study with eastern Indian cohort, we sampled patients in the age group of 19 to 55 years (14). The life expectancy of people with SCI are significantly reduced when compared with people without SCI and has improved since the 1980s (15). Specifically, the life expectancy is low during the first year of severe injury and increases subsequently. Some common causes of death post SCI are renal failure, pneumonia and septicaemia (15). The gender ratio of the afflicted is very country specific. On an average, roughly 4.2 men per women are afflicted (1). Studies in Canadian states revealed the male-female ratio was 4.4:1 during 1995-2004 in British Columbia (16), 2.5:1 during 1997-2000 in Alberta (17) and 1.53.5:1 in Ontario during 1994-1999 (10). In the states of USA, Alabama, Mississippi and Oklahoma, roughly during a 2 year period of study, the gender ratio was 4:1 (18-20), whereas, roughly 5 males per female were afflicted in Alaska (21) and 3.5 males per female in Utah (22). Studies from Europe and Asia revealed a wide range of male-female ratio of the afflicted with 7:1 in Thessaloniki, Greece and 3:1 in Stockholm, Sweden, both in 2006 (23). In other areas in Europe, Russia and east Asia, studies revealed 3.3:1 in Denmark during 1975-1984 (24), 5.5:1 in Estonia during 1997-2007 (25), 4.5-5.1:1 in Finland during 1976-2005 (26), 2.6:1 in Iceland during 1975-2009 (27), 3.35:1 in Romania during 19751993 (28), 2.5:1 in 1992 in Turkey (29), 5.8:1 during 1988-1993 in Jordan (30), 4.3:1 in 1990 in Japan (31), 3.2:1 during 1998-1999 in Australia (32) and 6.7:1 in Fiji during 1985-1994 (33). The most common cause of injury worldwide is traffic accidents (34). The second major cause of injury is fall from a height (35) . Falls include falls from buildings or trees, as we have observed in our study (14) or falls in the house or workplace. Epidemiological studies in different countries site several causes of SCI. Traffic accidents are the most common cause of injury in Denmark, Spain, Iceland, Switzerland, Turkey, Jordan, Japan, Australia and New

SCI; Mechanical and Molecular Trauma 4 Zealand (24,27,29-33,36-38). Falls are the most common cause of SCI in Estonia (25), Finland (26), Greenland (39), the Netherlands (40), Romania (28) and Fiji (33). Other major causes are sports, violence and suicide attempt (6,15). 1.2. The mammalian spinal cord (41) 1.2.1. Anatomical overview The ectoderm gives rise to the central nervous system. The brain and spinal cord are covered in protective layers called the “meninges”. The cells in the spinal ganglia, which are derived from the neural crest take a bipolar shape and develop two processes, one central and one peripheral. The central processes enter the marginal zone of the alar plate as dorsal root fibres and bifurcate there into ascending and descending branches. The peripheral processes of the spinal ganglion cells unite with the ventral root fibres in the region of the intervertebral foramina to form the mixed (i.e. afferent and efferent) spinal nerves. Peripherally, a spinal nerve is distributed to a segment of the body, including a myotome and a dermatome (see Section 1.3.1). Centrally, the tandem arrangement of the sites of emergence of the dorsal and ventral root fibres from the surface of the spinal cord, allow a subdivision of this organ into segments. There are usually 31 pairs of spinal nerves, grouped as eight pairs of cervical (C), 12 pairs of thoracic (T), 5 pairs of lumber (L), 5 pairs of sacral (S) and 1 pair of coccygeal (Co) spinal nerves (Figure-1.1). The central nervous system, which is of ectodermal origin is surrounded by mesodermal structures. A system of three connective tissue layers, the meninges, and a fluid compartment containing CSF are located between the bony skull and vertebral column and the nervous tissue of the brain and the spinal cord respectively. Blood vessels, themselves of mesodermal origin, are surrounded by derivatives of the meninges over their full extent, until the interface between the capillary wall and the glial basal membrane makes exchange of substances possible. CSF is produced by the choroid plexus of the ventricles. The brain is completely enclosed by the three connective tissue layers, the meninges. These are, starting from the brain’s surface, the pia mater, the arachnoid and the dura mater. The dura is composed of fibroblasts and extracellular collagen (Figure-1.2). The innermost border layer of the dura consists of flattened cells with sinuous processes connected by occasional desmosomes and extracellular spaces filled with an amorphous substance. A real subdural space does not exist; the border layers of the dura and the arachnoid are interconnected by occasional cell processes and desmosomes. The arachnoid can be subdivided into two layers. The outer layer of the arachnoid, located next to the dura, is known as the barrier layer of the arachnoid. It is impassable for lyophilic molecules, due to

SCI; Mechanical and Molecular Trauma 5 the presence of a tight junction between its constituting cells. The inner layers of the arachnoid and the pia mater actually form a single loose structure containing the smaller or larger interconnected spaces for the CSF.

Figure-1.1: Spinal cord of Homo sapiens from C3 to S5 (41).

SCI; Mechanical and Molecular Trauma 6

Figure-1.2: Cranial meninges (41). The above figure illustrates the ultrastructure of cranial meninges. The dura consists of an outer layer of fibroblast and collagen fibres and a layer of dural border cells. A subdural space does not exist. The arachnoid consists of an outer barrier layer, where the cells are connected by tight junctions (small arrows) and an inner layer that fuses with the pia mater. The subarachnoid space is formed from the coalition of intercellular spaces of the arachnoid/pia mater. D, desmosome; N, nucleus.

1.2.2. Topography of spinal cord The spinal cord with its meninges is located within the vertebral canal (Figure-1.3). The cord occupies the entire length of the vertebral canal during early stages of development. Due to the continued growth of the vertebral column it gradually lags behind, and in the adult only

SCI; Mechanical and Molecular Trauma 7 reaches to the upper level of the second lumbar vertebra. This process is known as the ascensus medullae. The caudal tip of the spinal cord tapers into the conus medullaris, and

Figure-1.3: (A) Cross sections of C5, T7, T11 and L5 and (B) Vertebral structure (41).

continues as a thread (filum terminale) to the level of the sacrum. The spinal cord can be subdivided into 31 segments (8 cervical, 12 thoracic, 5 lumbar, 5 sacral and 1 coccygeal). Each segment gives rise to dorsal and ventral rootlets, which unite into a pair of dorsal and ventral roots. The dorsal roots contain the spinal ganglia (Figure-1.1). The ganglia are

SCI; Mechanical and Molecular Trauma 8 located within the intervertebral foramina, where the dorsal and ventral roots combine into a pair of spinal nerves. Due to the ascensus of the cord, the spinal segments are located more rostral than their corresponding vertebrae. As a consequence, the dorsal and ventral roots descend over some distance through the vertebral canal to their exit through the intervertebral foramen, caudal (i.e., rostral for the cervical roots 1–7) to the corresponding vertebra. The bundle of lumbar, sacral and coccygeal roots surrounding the filum terminale, caudal to the cord, is known as the cauda equina (horse tail). At cervical and lumbar levels the cord is enlarged (Figure-1.4). These enlargements (intumescentiae) innervate the extremities.

Figure-1.4: The anterior and posterior portions of the spinal cord (41).

SCI; Mechanical and Molecular Trauma 9 1.3. Classification of SCI The American Spinal Injury Association (ASIA) published the first edition of the International Standards for Neurological and Functional Classification of Spinal Cord Injury (ISNCSCI) in 1982 (42). The ASIA standard, after being endorsed by the International Medical Society of Paraplegia (IMSOP) in 1992, has been known as International Standards for Neurological and Functional Classification of Spinal Cord Injury. 1.3.1 Some definitions (42) Tetraplegia: Impairment or loss of sensory and motor functions due to injury in the cervical segment of the spinal cord. This includes the arms, trunk, pelvic region and limbs. Paraplegia: Impairment or loss of sensory and motor functions due to injury in the thoracic, lumbar or sacral regions of the spinal cord but not the cervical region. The functioning of the arms is preserved but depending on the injury point, loss of sensory and motor functions of the trunk, pelvic regions and limbs may result. Dermatome: The area of the skin innervated by the sensory axons within each nerve root. Myotome: The muscle fibres innervated by motor axons within each nerve root. Neurological level of injury: The most caudal segment of the spinal cord with normal sensory and motor functions on both sides of the body. Sensory level of injury: The most caudal segment of the spinal cord with normal sensory functions on both sides of the body. This is assessed by the sensory perception testing of a key sensory point in the 28 dermatomes on the right and 28 dermatomes on the left of the body. Motor level of injury: The most caudal segment of the spinal cord with normal motor functions on both sides of the body. This is assessed by motor function testing of a key muscle in the 10 myotomes on the left and right side of the body. Incomplete injury: Partial or complete preservation of sensory and motor functions below the neurological level and includes the lowest sacral segment. Complete injury: Absence of sensory and motor function in the lowest sacral segment.

SCI; Mechanical and Molecular Trauma 10 1.3.2 ASIA Impairment Scale (AIS) of injury severity (43) AIS Grade

Description

A

Complete; no sensory and motor functions preserved in sacral region S4-S5

B

Incomplete; sensory but not motor function preserved below neurological level

C

Incomplete; motor functions preserved below neurological level; key muscles grade < 3

D

Incomplete; motor functions preserved below neurological level; key muscles grade > 3

E

Normal

1.4. Autonomic dysfunctions post SCI SCI afflicted persons regard autonomic dysfunctions as a bigger downside to the quality of life than loss of sensory and motor perceptions (44). Tetraplegic SCI subjects give highest priority to improvement of hand functions (45), whereas paraplegic persons prioritise normal sexual functions. When both groups are combined, the priority is normal bowel and bladder functions. At the moment of injury, if the lesion is above C3, the afflicted person may suffer an autonomic nervous system dysfunction and may need assisted respiration. Deaths due to cardiac arrest in the first few minutes post SCI was common but there has been some data to suggest its gradual decline over time (44). A mean arterial blood pressure of 80mm Hg or above is recommended in the acute phase of SCI (46) and urine output is low due to improper function of anti diuretic hormone. Some common issues arising from autonomic dysfunctions in the acute phase of SCI are bradycardia and respiratory system failure, which further leads to development of pneumonia and because of a loss of temperature regulation, the body is unable to lose excess heat, as a result of which cardiac arrest may occur (44). After the injury, low blood pressure arises due to disruption in the rennin-angiotensin system (47) and this along with the inability to raise blood pressure by vasoconstriction, makes mobilization of the patient difficult. There is also a loss of bladder and bowel movement control. The SCI patient develops pressure sores on the skin if same position is maintained for more than a couple of hours (44). Loss of erectile function in males and lubrication in females is a major problem after SCI and these problems may persist. These are some of the basic manifestations of autonomic deregulation after spinal cord injury with tend to last during the window of a couple of weeks but sometimes manifest even after years. Spinal cord injury can be grossly divided into two stages: primary injury and secondary injury. Primary injury stars within minutes of spinal cord lesion. The condition includes immediate damage of blood vessels causing hypotension and disruption of axons causing spillage of

SCI; Mechanical and Molecular Trauma 11 neurotransmitters. Because regulation of blood flow is lost, the spinal cord swells up (43). This series of events makes way for the secondary injury which starts hours after the primary injury and have a widespread molecular deregulation. 1.5. Primary injury The most initial trauma to the spinal cord is called primary injury and it is the basis on which the AIS grade of severity is determined. Primary injury therefore is the prognostic indicator of SCI (48). There can be four types of primary injury: 1. “Impact and persistent compression”, as with burst fractures and acute disc ruptures; 2. “Impact with transient compression”, as is seen in people with degenerative cervical spine disease; 3. “Distraction”, as can be seen with flexion and extension of the spinal cord during exercise. This type of injury may present no radiological evidence and is common in persons with degenerative spine giving rise to SCI. Finally, 4. “Laceration and transection”, which is partial or complete cutting of the spinal cord. Primary injury initially affects only the softer and more vascular grey matter and spares the peripheral white matter (49). There is irreversible damage to the grey matter within the first five hours after injury, whereas the irreversible damage to the white matter is complete 72 hours post injury. The mechanical damage to the grey matter disrupts blood vessels which in turn causes hypoxia and ischemia in the grey matter (48). The primary injury consists of the acute injury, which are the physiological and molecular changes that immediately follow the initial trauma (50). These include necrotic cell death, oedema and shifts in the electrolyte balance (51). These events continue into and herald the secondary injury phase, which is also called the sub acute injury phase (50). New physiological and molecular changes occur in the secondary phase, like delayed calcium influx, free radical formation, inflammatory responses and apoptosis (51). 1.6. Secondary injury While primary injury consists of the time window of seconds to minutes after the trauma, secondary injury begins minutes after the trauma and lasts for weeks to months (52). The secondary injury phase witnesses tremendous molecular activity with several processes being initiated and each giving rise to further molecular changes, which makes the secondary injury phase self propagating (50). We will look at some of the major secondary phase cellular and molecular events in brief.

SCI; Mechanical and Molecular Trauma 12 1.6.1. Immune response The immune response is mediated within minutes after injury and persists for days to months post injury (53). Immune response is essential for recovery after SCI as it clears the debris around the injury site and promotes axon growth. However, cell death and demyelination of axons result from excess inflammatory responses at the SCI site (54). Therefore uncontrolled immune response can be damaging (55). The resident innate immune system cells of the CNS are microglia. These cells have varied effects on the system based on the context and time (56). The immune response involves a biphasic leucocyte infiltration at the site of injury. Initially, there is an infiltration of neutrophils which release lytic enzymes and exacerbate injury to neurons, glia and blood vessels. In the second phase, there is recruitment of the macrophages which clean up the damaged tissues by phagocytosis (57). This biphasic response causes demyelination of the spared axons, cavitation in the grey and white matter and Wallerian degeneration (57). Contusion injury also sensitizes the immune system against the CNS myelin (58). Traumatic SCI also induces nuclear factor- kappa B which transcribes a number of genes regulating immune response, cell death and proliferation (59). Inflammatory response arising after SCI brings four types of cells at the injury site: neutrophils, monocytes, microglia and T-lymphocytes (60,61). The neutrophils are brought to the injury site from the circulatory system by molecular guidance of the vascular endothelial cells. The neutrophils release cytokines like interleukin (IL)-1β, interleukin-6 and tumour necrosis factor-α (TNF-α) and these cytokines activate other inflammatory and glial cells, giving rise to an inflammatory cascade of events, ultimately harming the system. Neutrophils are required to make the injury site free of microbial intrusion and also for the removal of tissue debris (51). Monocytes infiltrate into the spinal cord from the circulatory system and differentiate into macrophages. These activated macrophages and resident microglia secrete inflammatory cytokines, free radicals and growth factors (62). The growth factors are pro survival molecules promoting tissue repair and regeneration, whereas, the cytokines and free radicals contribute to lesion expansion (63). Some researchers believe that autoreactive T-lymphocytes induce demyelination of axons (54), while others argue that these cells protect the myelin insulated neurons (64,65). Summarizing these facts, it can be said that the immune response is both beneficial and destructive after SCI and would only be of net benefit if exogenously controlled (55). 1.6.2. Breach of plasma membrane Non specific breaches in the cell membrane are one of the foremost outcomes after a traumatic SCI (66) and are common in many neuronal injury models (67-70). This breach in

SCI; Mechanical and Molecular Trauma 13 plasma membrane results in unregulated ionic flux (71) and is the cause of several detrimental outcomes in the cell including proteolysis, tissue degradation and apoptosis (72). 1.6.3. Glutamate excitotoxicity Glutamate is the major neurotransmitter of the central nervous system (73) and there is an excessive release of glutamate after injury (74) and accumulation in the extracellular space around the injury site (75). Glutamate binds to its receptor, the NMDA receptor which also acts as potassium and calcium gate and there is a huge influx of K+ and Ca2+ ions into the cell, which is further aggravated due to the failure of Na+-K+ ATPase (76) activity and the activation of Na+-Ca2+ exchanger (77). Influx of Ca2+ ions into the cell causes widespread apoptosis and necrosis, a process commonly called excitotoxic cell death (78). Since neurons express glutamate receptors abundantly, increased extracellular glutamate causes extensive demyelination of axons and a resultant conduction block (50) which contributes to subsequent sensory and motor function deficits. In addition, glutamate excitotoxicity causes generation of ROS (reactive oxygen species) (79) and RNS (reactive nitrogen species) (80,81) and subsequent failure of the mitochondrial electron transport chain (82), inactivation of glyceraldehydes 3 phosphate dehydrogenase, lipid peroxidation and oxidative modifications of proteins (48). In addition, it causes perturbations in the microcirculatory system and secondary ischemia (74,83,84). 1.6.4. Electrolyte imbalance After acute injury, there is an initial inward leakage of sodium ions in the cells. This causes sodium-calcium exchangers to import damaging levels of Ca2+ into the cells (85,86). Once inside the cell, Ca2+ activates calpains and caspases which breakdown proteins in the immediate vicinity on injury, leading to the breakdown of the axoplasm (87). Furthermore, Ca2+ dissolves in the surrounding area and there is a widespread activation of calpains which lead to break down the cell membranes allowing more Ca2+ to enter the damaged cell (88), thereby propagating the process. Calpains degrade axon-myelin structural units (89). Calcium also activates phospholipase A2, lipoxygenases and cyclooxigenases, which in turn leads to the formation of certain thromboxanes, prostaglandins and leucotrienes from arachidonic acid (90,91). These substances cause platelet aggregation and vasoconstriction leading to reduced blood flow (92). Rise in arachidonic acid levels is associated with inhibition of Na+-K+ ATPase leading to tissue edema (93). Excessive accumulation of K+ ions in the extracellular space causes depolarisation of neurons and spinal shock (94). Intracellular magnesium ions are depleted during the secondary injury phase (48). This affects glycolysis, oxidative phosphorylation, protein synthesis and several enzymatic reactions where Mg2+ serves as a cofactor. Furthermore, decreased magnesium levels

SCI; Mechanical and Molecular Trauma 14 contribute to additional calcium accumulation into the cell. Magnesium also blocks NMDA receptors decreasing excitotoxicity and therefore it is neuroprotective to the cell (95). Decreased levels of extracellular Mg2+ therefore antagonise this effect. 1.6.5. Mitochondria damage Mitochondria are the seat of cellular redox reactions and Ca2+ homeostasis. CNS trauma perturbs the mitochondrial functions like oxidative phosphorylation and cellular respiration (96,97). Respiration dependent Ca2+ uptake and sequestration is also inhibited in CNS injury leading to disturbances in mitochondrial Ca2+ transport and deregulation of Ca2+ homeostasis (96,98). Ca2+ mediated permeability changes on the inner mitochondrial membrane leads to osmotic imbalance and lysis of the mitochondria. Studies suggest that the mitochondria sequester incoming Ca2+ and excitatory neurotransmitters and that increased accumulation of mitochondrial Ca2+ and not cytosolic Ca2+ is the cause of excitotoxic cell death post SCI (99-101). 1.6.6. Lipid peroxidation As a sequel to calcium influx, mitochondrial dysfunction and arachidonic acid activation, there is activation of inducible nitric oxide synthase (iNOS) (102,103), starting the pathological cascade of reactive oxygen species (ROS) and reactive nitrogen species (RNS) formation (104). ROS and RNS cause oxidative damage to proteins and nucleic acids apart from causing lipid peroxidation (78) and these free radicals cause damage to organelles and cytoskeletal structures. Lipid peroxidation is the process in which free radicals absorb an electron from the lipid molecule making it less stable. This unstable lipid molecule launches a chain redox reaction leading to cell membrane lysis, necrosis and mitochondrial dysfunction (105). 1.6.7. Demyelination of surviving axons The spared axons traversing the site of injury are the main connections from the brain to the region caudal to the injury (103). Therefore, demyelination of these, which is a major event in the sub acute phase of SCI (106), causes significant sensory and motor function deficits known as ‘conduction block’. Demyelination occurs because of the loss of oligodendrocytes at the injury site immediately after injury (107). Oligodendrocytes farther away from the vicinity of the injury (rostral and caudal white matter) also undergo apoptosis following months after injury (108). Myelin loss causes the axons to be directly exposed to free radicals and cytokines. The neurons eventually undergo apoptosis and necrosis (103,109).

SCI; Mechanical and Molecular Trauma 15 1.6.8. Apoptosis A large body of work shows that after the necrotic cell death of primary or acute injury phase, apoptosis sets in. Apoptosis occurs in neurons, microglia, oligodendrocytes and perhaps astrocytes (48). It is usually triggered by inflammatory responses, cytokine release, free radicals and excitotoxicity occurring during the secondary injury phase (110). Apoptosis of the oligodendrocytes result in steady demyelination of axons that begin as the secondary phase progresses (111) and apoptotic oligodendrocytes can be found away from the vicinity of injury (112). Released tumour necrosis factor initiates receptor dependent apoptosis of neurons, microglia and oligodendrocytes (113). High intracellular Ca2+ ions in secondary injury cause mitochondrial damage, cytochrome c release and activation of caspases and calpains, giving rise to the alternative receptor independent apoptosis (87,113). Interestingly, apoptosis of cortical motor neurons occur even when the axons are far removed from the site of injury and the hub of Ca2+ release (114) but this is believed to be induced by inflammatory cytokines, free radicals and excitotoxic molecules (115). 1.6.9. Vascular system derangements The microcirculatory system comprising capillaries and venules suffer haemorrhages immediately post traumatic SCI. The larger blood vessels are relatively spared and the damage is progressive (116,117). The haemorrhage after traumatic SCI is most predominant in the grey matter leading to necrotic death (118). Alternatively, ischemia or reduction in blood flow is also predominant in the microcirculatory system rather than the major blood vessels and this phenomenon is also one of the earlier events post traumatic SCI (119-121). The ischemia worsens progressively (120). Vasospasm (121,122) and intravascular thrombosis (123) lead to post traumatic ischemia. Ischemia causes oedema around the injury site (124). Endothelial cell function loss is an early event in the vascular derangements post SCI and increases vascular permeability leading to oedema. This may occur due to the formation of craters in the endothelial lining, accumulation of cell debris and disruptions of endothelial cell junctions (118). The autoregulatory homeostasis i.e. the ability to maintain constant blood flow over a wide range of pressures, takes a backseat. This condition may worsen ischemia during systemic hypoperfusion (neurogenic shock) or alternatively, worsen haemorrhage during high systemic blood pressures (121). The state of hypoperfusion may be succeeded by a temporary period of reperfusion also known as “luxury perfusion”. This occurs due to accumulation of lactate around the blood vessels leading to a consequent decrease of pH (125). The loss of microcirculation, oedema, haemorrhage, ischemia and endothelial damage is dependent on the severity of injury and also progresses over time (50).

SCI; Mechanical and Molecular Trauma 16 1.6.10. Neurogenic shock Neurogenic shock is defined as tissue perfusion because of paralysis of vasomotor input (48). It is characterised by bradycardia, hypotension, hypothermia, decreased peripheral resistance and decreased cardiac output (50). Due to loss of sympathetic tone after SCI, hypotension results, which is exacerbated by neurogenic shock. When injury is higher up in the cord, the sympathetic supply to the heart may be disrupted due to unopposed vagal activity. This results in bradycardia (126). 1.6.11. Chromatolysis When injury to the cord is sustained and irreparable, the nucleus of the cell body moves towards the periphery, Nissl bodies disappear and the cell body becomes tumourous. This phenomenon is called chromatolysis and it is an axonal reaction to changes in cell metabolism and promotes axonal regeneration (127,128). Chromatolysis is the direct effect of ischemia of the microcirculatory system and this phenomenon causes degeneration of myelin sheath both in the central and peripheral nervous system (103). Onset and progress of chromatolysis thus causes severe disruption in neuronal communications giving rise to a conduction block. 1.7. Molecular inhibition of axon regeneration The ability of damaged CNS axons to regenerate after injury is severely compromised, whereas, those of the PNS are able to regenerate well. The injured axon in the CNS may survive through long periods of time but due to a number of extrinsic and intrinsic inhibitory factors, these are unable to regenerate (129). Back in 1928 Cajal described that the end of an injured axon fails to grow and forms a “dystrophic endball”. In the following years, several studies have shown that these dystrophic growth cones are structures of high molecular activity but unable to grow as they are surrounded by an environment essentially hostile to growth (130). The DRG neurons have axons in both CNS and PNS but they are able to regenerate only the axons in the PNS. Furthermore, studies have revealed that implants of peripheral nervous tissue around an injured CNS axon is conducive to growth (131). However, these axons failed to grow into the spinal cord.(132). This finding in fact was a milestone as it started a search for extrinsic axon growth inhibitors in the CNS. The lack of “observed” regeneration in CNS is supported by the theory of restricted plasticity to stabilise established connections in the CNS. In the developing CNS, the environment of axons is highly growth conducive and all the neural connections are formed at this time. The repulsive guidance cues that are present at the time of development, persist into adulthood (129). However, post natal restructuring of CNS axons would lead to rewiring of functional

SCI; Mechanical and Molecular Trauma 17 established neuronal connections and to prevent it, the adult CNS has evolved mechanisms. Three most well established intrinsic molecular roadblocks to regeneration are myelin associated inhibitory factors (MAIFs) (133), chondroitin sulphate proteoglycans (CSPGs) and glial scar (129,134). It is still debatable whether the lack of regenerative capacity of the CNS is actually a fact or a temporary observation, considering that axons take excruciating amount of time to grow (1mm/day) and would have to grow 100-800 mm to reach their former connections (134). The review by Young (2014) excellently places the concept that mature CNS is intrinsically capable of regeneration after injury and many molecular events post injury that are perceived as growth inhibitory might actually be guidance cues for the regenerating axons (134). 1.7.1. Myelin associated inhibitory factors (MAIFs) In 1989 Schwab and Schnell showed that myelin is inhibitory to axon growth (135). A couple of years later Nature published the groundbreaking study of IN-1, an antibody against a myelin component, being able to promote regeneration of corticospinal tracts in rats (136). Investigation of the antigen against IN-1 led to the discovery of Nogo A (137,138), a reticulon family protein (139). Nogo has three isoforms, Nogo A, Nogo B and Nogo C, among which Nogo A is the best studied because of its abundant expression in oligodendrocytes (140,141). Nogo proteins are products of the reticulon 4 protein (RTN4) (138,139,142). Nogo A is not expressed in astrocytes or Schwann cells (141) and these groups have shown that the expression of Nogo A is not altered after injury. Nogo A has two structurally distinct growth inhibitory domains. A 66 amino acid growth inhibitory loop called Nogo 66 is found in all three Nogo isoforms (137), whereas the growth inhibitory amino terminal of Nogo A is not shared by the other Nogo isoforms and are encoded by different RTN genes (139,142). Nogo proteins are present at the cell surface and endoplasmic reticulum and they show varied membrane topologies with regard to cytoplasmic and extracellular location of the N terminus (143). The receptor for Nogo 66 christened as NgR1 was the first Nogo receptor to be identified (144). NgR1 does not have a transmembrane domain (144) and therefore it interacts with membrane bound proteins to transduce signals. These membrane proteins are the low affinity neurotrophin receptor, p75 and its family member, tumour necrosis factor –α (TNFα) receptor superfamily member 19 (TROY) (145). The leucine rich repeat (LRR) protein LINGO1 also binds NgR1 extracellularly (146). Nogo 66 also binds to paired immunoglobulin-like receptor B (PirB) (147) Subsequently, it was studied that anti Nogo antibodies promote axon growth in some animal models (148,149). NgR1 also binds to myelin associated glycoprotein (MAG) (150) and oligodendrocytes myelin glycoprotein (OMGP) (145). MAG and OMGP have synergistic

SCI; Mechanical and Molecular Trauma 18 effects on axon growth inhibition when they work along with Nogo A (151). The following findings established Nogo as an axon growth inhibitory protein. Disruption of Nogo-NgR1 interaction enabled axon growth in vitro (144,152). Soluble Nogo receptor improves axon regeneration (153,154). NgR1 phosphorylation by extracellular casein kinase 2 inhibits its interaction with Nogo 66, MAG and OMGP and promoted neurite outgrowth in myelin coated culture dishes in vitro (155). NgR1 blockage promotes regeneration after CNS injury in adult rats (156). Another active fragment unique to Nogo A is the region coded by amino acids 544-725 of exon 3 and known as Nogo–Δ20. This fragment of Nogo A is highly growth inhibitory in vitro and also restricts the migration of non neuronal cells like fibroblasts (138,139,142). Nogo A is also known to cause autoimmune demyelination (157) and multiple sclerosis (158). Active fragments of Nogo A, MAG and OMGP activate the small GTPase Ras homolog gene family member A (RhoA) and it’s effector protein Rho-associated coiled coil containing protein kinase (ROCK) (159). Rho proteins control the cell motility (160) by controlling cytoskeletal rearrangements.(161). Rac, another small GTPase with cytoskeletal regulatory functions antagonist to Rho, is inactivated in the presence of Nogo–Δ20 (162). RhoA activates phospholipase D (163), C (164), A2 (165) and serine-threonine kinases.(166). Downstream of ROCK, the phosphatase slingshot, LIM domain kinase 1 (LIMK1) and cofilin act to depolymerise actin and cause the axon growth cone collapse (167,168) (Figure-1.5). 1.7.2. CSPGs and the glial scar The inception of gliar scar stems from the disruption of the blood brain barrier (BBB) and infiltration of scar generating molecules like IL-1 (169), TGFβ isoforms (170,171) and fibrinogen (172). Moreover there is leucocyte extravasation and accumulation of the secreted inflammatory proteins at the injury site so much so that imaging studies have shown that the density of inflammatory cells is 40 fold higher in the lesioned white matter and 9 fold higher in the lesioned grey matter (173). Activated macrophages and microglia that infiltrate the site of injury secrete matrix metalloproteases which increases the permeability of the blood vessels at the injury site (174). The astrocytes at the injury site undergo proliferation and hypertrophy. The proliferated astrocytes form a thin layer at the periphery of the lesion (175) and the astrocyte density is 2-4 fold of that in non injured tissue (173). Astrocyte hypertrophy is the main reason for reactive astrogliosis rather than astrocyte proliferation. The hypertrophic astrocytes are enlarged and express intermediate filament proteins like nestin, vimentin and GFAP abundantly (176). The hypertrophic axons undergo restructuring to form a mesh like entangled mass posing as a growth barrier to regenerating axons (177) (Figure-1.6).


Figure-1.5: Nogo mediated signaling cascade (143).

Also, these astrocytes synthesise chondroitin sulphate proteoglycans (CSPGs) and secrete these into the extracellular matrix. CSPG secretion begins at 24 hours post injury and lasts for months after injury (178,179). The ECM of the CNS is rich in proteoglycans, which contain core proteins attached to glycosaminoglycan (GAG) chains. GAGs are repeating disaccharide units (180) and the functional components of the CSPGs. CSPGs function as guidance cues in the developing CNS, assist the neurons in forming a boundary (181) and form perineuronal net (PNN) which are essential for the synapse formation and maintenance of stable neuronal connections by limiting synaptic plasticity (182,183). Upregulation of CSPG following injury is well known to limit axonal sprouting (184), regeneration (185) and conduction (186). CSPGs restrict the maturation of oligodendrocytes precursor cells (OPCs) in vitro and also drive the neural precursor cells (NPCs) towards selective maturation to astrocytic lineage. This results in lack of replacement of the damaged oligodendrocytes from the injury site and therefore translates into ineffective myelin repair (187). CSPG synthesis peaks at 2 weeks post injury and persists chronically (188). This includes several types of CSPGs like neurocan, versican, brevican and Ng2 (189).


Figure-1.6: Cartoon of the glial scar around the injury lesion (190). The growing axon forms a dystrophic end bulb into the scar.

Infiltration of blood at the site of injury increases the concentration of fibrinogen, which is a carrier of TGFβ. TGFβ induces Smad2 phosphorylation leading to the production of CSPGs (172). Recently it was found that CSPGs are also upregulated by TGFβ mediated PI3K/Akt pathway via mTOR activation (191). Several approaches have been made to limit the CSPG mediated growth inhibitory signaling cascades (192). Decorin, a small leucine rich proteoglycan has shown to inhibit CSPG deposition. Xyloside inhibits attachment of GAGs to the core protein during CSPG formation in the cell. The bacterial enzyme chondroitinase ABC (ChABC) cleaves GAG chains off the core protein rendering CSPGs useless. Recently DNA enzymes have been developed which block xylotransferase-1, an enzyme essential for the biosynthesis of GAGs. Recently ADAMTS4 (A Disintegrin like And Metalloproteinase with Thrombospondin type 1 Motif 4) was also developed and this destroys the core proteins of CSPGs. While ChABC and ADAMTS4 act from outside the cell, the rest act inside (see Figure-1.7).


Figure-1.7: Sequence of events following injury, which ultimately give rise to the glial scar. TGFβ initiates Smad and Akt pathways which independently lead to CSPG synthesis. The yellow boxes denote the common and recent ways to inhibit CSPGs (192).

Several mechanisms have been suggested about how CSPGs act to promote growth inhibition. It can cause steric hinderance of growth promoting molecules such as integrins and laminin by the formation of perineuronal nets. Perineuronal nets are formed in the developing CNS and are crucial in controlling neural plasticity at this time (193). Additionally, the cell surface receptors PTPσ and LAR phosphatase, members of the leucocyte common antigen-related (LAR) phosphatase subfamily, act as functional receptors of CSPGs (194,195). The Nogo receptors NgR1 and NgR3 also bind to CSPGs mediating downstream growth inhibitory signals (196). CSPGs mediate the growth inhibitory effects by activating Rho and deactivating Akt and Erk (Figure-1.8) (197-200). Recent evidences suggest the converging idea that the reactive astrocytic scar at the lesion penumbra serves to alienate intact axons from the lesioned molecular niche. This is an evolutionarily conserved mechanism to contain the inflammatory damage at the lesion site. (201,202). 1.8. ApoA1: a probable player in healing The CNS secretes apoliproteins and these can be found in the CSF in protein level abundantly (203). Apolipoproteins have been commonly defined as proteins that combine with a lipid to form lipoproteins such as (high density lipoprotein) HDL or (low density lipoprotein) (204) LDL or protein components of HDL/LDL. The basic function of lipoproteins is to transport lipids through the aqueous plasma compartment (203,205). The major role of lipoprotein molecules is reverse cholesterol transport (RCT) (203).


Figure-1.8: Cell surface receptors for CSPGs and the downstream growth inhibitory signals (200).

RCT is the process of removal of cellular cholesterol from peripheral tissues and its transport to liver for conversion into bile acids. Lipoproteins are also the main mediators of lipid catabolism and are intimately linked to cardiovascular diseases (CVD), inflammation and neurological disorders (206-208). Apart from ApoD and Apo(a), apolipoproteins contain a highly conserved alpha helical domain that is functional in lipid binding (209,210). Most of the aplolipoproteins also contain a signal sequence for secretion and a receptor binding domain (RBD). Common receptors for the apolipoproteins in the CNS are LDL receptor (LDLR) family of receptors (211,212). Apolipoproteins are involved in lipid delivery to brain cell types and also initiate many signal transduction pathways (213). While the brain is 2% of the body mass, its total unesterified cholesterol content is 25% of total cholesterol content in the body, making it the most cholesterol rich organ (214). Because of the non permeable nature of the BBB to large molecules, the peripheral cholesterol in plasma compartment is separate from the cholesterol in the CNS (215) and this has been shown by studies injecting radiolabeled cholesterol in the blood stream (216). Cholesterol is the main component of the myelin sheath and the plasma membranes of glial cells (217). Lipoproteins of HDL like density are the major carriers of lipids in the CNS, in contrast to those of varying range of densities in the peripheral circulation (204,218). The cholesterol metabolism in brain is distinct from the rest of the body because of the presence of BBB (215). The half life of cholesterol metabolism in brain is 1-5 years compared with a

SCI; Mechanical and Molecular Trauma 23 few hours in the periphery (203,219). While cholesterol requirement in brain is highest during embryogenesis, in the adult organism, cholesterol is synthesized and secreted by the glial population (220). ApoA1 is an abundant protein of the human CSF as has been studied by our lab (14,221) and several other groups (204,222,223) but ApoA1 mRNA has remained virtually undetectable in brain (203). ApoA1 is not synthesized in the CNS and it enters the CNS by SR-B1 receptor mediated uptake of HDL in the brain capillary endothelial cells (224). ApoA1 in CSF is probably a fraction of the plasma ApoA1 crossing the BBB through the choroid plexus (204,222). Brain endothelial cells also express ApoA1, which contributes to the ApoA1 protein content in the CNS (225). Preliminary results from in our lab has shown that ApoA1 and HDL are differentially abundant in different severity grades of SCI and also show variation with time. Results from our group has also pointed out lipid catabolism to be a perturbed pathway in the secondary phase of SCI (14). 1.8.1. ApoA1 in inflammation A large body of research shows that HDL/ApoA1 has anti-inflammatory roles. ApoA1 is linked to innate and adaptive immunity. It is involved in lipopolysaccharide (LPS) neutralisation and clearance, making HDL an important player in innate immunity (226). ApoA1 also mediates activation and recruitment of monocytes and neutrophils to the site of inflammation (227,228). Recent evidences point that activation of Toll like receptors (TLR) in macrophages is prevented by HDL (229). ApoA1 induces IL-10 and prostaglandin E2, both of which prevent differentiation of dendritic cells, which are antigen presenting cells (APCs) (230). ApoA1 mediated cholesterol efflux via ABCA1 disrupts lipid rafts, thereby abolishing the initiations of inflammatory signals which center in lipid rafts (228,231,232). The same phenomenon also compromises antigen presentation to T cells (233) and neutrophil activation, migration and spreading (234). During acute inflammatory responses, inflammatory cytokines (TNF)-α and (IL)-1β negatively regulate the expression of ApoA1 in hepatocytes in a JNK, p38 and NFκB dependent manner (235,236). (TNF)-α and IL-6 also increase the expression of acute phase proteins such as serum amyloid A (SAA) (237) and group IIA secretory phospholipase A2 (sPLA2-2A) (238). SAA, which has a higher binding affinity to HDL than ApoA1, displaces the latter from HDL (239), whereas, sPLA2 modifies HDL composition by hydrolysing phospholipids from HDL (240) and mediates proteolysis of lipid free ApoA1 by direct interaction (241). Therefore the relationship of ApoA1 with inflammatory responses is a feed-forward cycle of events. On one hand, inflammatory responses decrease ApoA1 expression and increase its catabolism and ApoA1 on the other hand plays a role in mitigating immune responses by lipid raft disruption.

SCI; Mechanical and Molecular Trauma 24 1.8.2. ApoA1 activates MAPK and Cdc42 pathways ERK1/2 increases ABCA1 expression and activity in mouse cells (242). In human fibroblasts, ApoA1 has been shown to activate the small GTPase Cdc42 followed by MKK4/JNK pathway activation (243). Inhibition of these pathways negatively regulated ApoA1 mediated cholesterol efflux. Interaction of ApoA1 with ABCA1 during cholesterol efflux, causes Cdc42 activation by its binding to C terminal of ABCA1.(244,245). Subsequent to Cdc42 activation, its downstream effectors PAK-1 and p54JNK are activated, leading to actin polymerisation (243,245). 1.9. Objective of the thesis The thesis focuses on perturbed molecular pathways in the secondary phase of SCI and subsequently, implication of ApoA1/HDL as probable mediators in healing. The first part of the work is based on a clinical study employing proteomics methods to identify differentially abundant proteins in CSF from different severity grades of SCI. A bioinformatics analysis was performed to identify the biological pathways in which the differentially abundant proteins are members. The analysis of perturbed pathways is important in this scenario because the secondary phase of SCI witnesses a plethora of molecular changes which are a determinant of functional recovery in the long run. The second part of the work is a foray into the domain of the role and regulation of Apolipoprotein A1 in neuronal injury. The scenario of ApoA1 catabolism was studied in the context of secondary phase spinal cord injury. As lipid catabolism pathway was found to be perturbed in the secondary phase of SCI, it was followed up in an injury model of neuroblastoma by employing ApoA1 as a growth promoting test molecule and growth parameters were checked in injury and non injury model. References 1.

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P a g e | 36

Chapter 2: Materials and Methods

Materials and Methods 37 2.1. Statement of Ethics The study was conducted as a joint collaboration of Saha Institute of Nuclear Physics (SINP) and Nil Ratan Sircar Medical College and Hospital (NRSMCH) after being cleared by the Institutional Ethical Clearance committees of both institutions (1). Informed consent was obtained by the subjects as per Helsinki Declaration 2013. 2.2. Patient selection and scoring The study was conducted for two time periods post injury, 1-8 days and 15-60 days. The patient cohort for the two temporal groups was distinct. Patients afflicted with traumatic spinal cord injury and admitted to the spinal injury ward of Dept. of Orthopaedic Surgery, NRSMCH, were considered for the study and enrolled after screening by an orthopaedist and a physiatrist from the Dept. of Orthopaedic Surgery and Dept. of Physical Medicine and Rehabilitation respectively (Table-2.1). Type of study

Cohort study

Period of study

December 2011-July 2014

No of participants

45

Total sample drawn

45 ml

Included samples

20

Table-2.1: Details of the study.

Patients were evaluated according to the International Standards for Neurological Classification of SCI (ISNCSCI) (2) and those who confirmed to the set of inclusion and exclusion criteria (Table-2.2) were selected for the study. Any patient with factors that have the possibility to alter the regenerative and degenerative process in the injured area as mentioned in Table-2.2 was excluded from the study. No

Inclusion criteria

1

Patient with SCI due to fall or crush with AIS-A, C and D grade injuries

2

Patient should be at 24 hrs to eight days post injury for the first study group

3

Patient should be at 15-60 days post injury for the second study group

Materials and Methods 38 Exclusion criteria 1

Patient in spinal shock stage

2

Other neurodegenerative diseases

3

SCI with lacerated cord or due to electrical injury

4

Associated poly trauma

5

Prior surgical stabilization of spine

6

Infectious diseases

7

Metabolic disorders

8

Patients on molecules that may inhibit Rho-ROCK pathways

Table-2.2: Patient inclusion and exclusion criteria.

First, the completeness of the injury was determined assessment for loss of anal sensation and contraction. Then tests for sensory perception (pin prick and feather touch) and motor activity for upper and lower limbs were done to ascertain the ASIA grade of injury. Motor and sensory levels were determined and scored clinically. Clinical level was matched with radiological level determined with non contrast MRI and X-rays. MRI showing oedema in the cord was considered for complete injury. In incomplete injuries of lower grade where MRI did not show oedema, X-ray was considered for determining radiological level. In case of different sensory and motor levels on clinical examination, the highest clinical level was fixed that matched with MRI or X-ray. This spinal segment was considered for collecting CSF (Figure-2.1 and Figure-2.2). Before collection of CSF a complete hemogram, ESR, CRP, serum fasting sugar, post prandial sugar, electrolyte, calcium, urea, creatinine, total protein, albumin and globulin were done along with a lipid profile, liver function test and thyroid profile. A routine urine examination with ultrasound check of lower abdomen was done to assess the bladder and to look out for hidden injury because infection, metabolic disorder, electrolyte imbalance and bladder abnormality could have potential effect on the environment of injured spine under study.

Materials and Methods 39

Figure-2.1: The figure shows sagittal MRI sections of dorsolumbar spine. There is anterior wedging of D12 vertebra with compression over the spinal cord. Signal intensities in the other vertebrae, intervertebral discs and the paraspinal muscles are normal.

Figure-2.2: The figure shows sagittal MRI sections of dorsal spine. There is traumatic disruption of the body of D7 vertebra with anterior dislocation of the superior part. The inferior part is displaced posteriorly, extremely compressing the spinal cord. Signal intensities in the other vertebrae, intervertebral discs and the paraspinal muscles are normal.

Materials and Methods 40 2.3. CSF collection and processing for proteomics experiments All the vital parameters were checked. The patient’s heart rate and ECG recording were noted. Patient’s blood pressure, oxygen saturation and signs of postural hypotension were noted in a sterile operation theatre with all resuscitation equipment. This is considered vital because hemodynamic alteration can affect the internal environment of the injured site under study. CSF was drawn by thecal puncture with 23 G spinal needle (Spinocaine 23/G) to minimize injury. Adequate flow of CSF on spinal tapping suggested normal flow of CSF through the central nervous system bathing the spinal cord, thereby giving us CSF sample that adequately represented the deranged process in the spine area that was attempted to study. The patient was made to lie on one side with spine flexed in crouched hand to knee position. This flexed position ensures easy access into thecal space. Median or paramedian approach was taken as per convenience of the procedurist. Sample was only taken when the patient was comfortable with all parameters mentioned in acceptable physiological level. CSF was collected in sterile vials and protease inhibitor cocktail preparation (Roche Diagnostics, USA) was added. As there might be a breach in blood brain barrier at the injury site, blood infiltration in the CSF was a common occurrence. Albumin depletion was tried but most of the other proteins were lost along with it, and moreover since the aim was to look at the actual protein scenario for the two injury conditions, depletion of any abundant protein was not carried out. The first few drops of CSF were discarded and the CSF was centrifuged to remove any RBCs and cellular debris. Protein content of the CSF was determined by Bradford (Biorad, CA, USA) reagent, using BSA (Sigma Aldrich, St. Louis, MO, USA) as standard. CSF samples were not pooled for any study. All proteomics work was conducted using individual samples as per selection criteria. CSF aliquots containing 50µg and 25µg protein were acetone precipitated at 13000 rpm for 15 minutes and the pellet was dissolved in 20µl and 10µl DIGE buffer (7M urea, 2M thiourea, 4% CHAPS, 30 mM Tris pH 8.8, PI cocktail, 1:100 dilution, Roche diagnostics, USA) respectively and 1500µg protein containing CSF aliquot was dissolved in 330µl rehydration buffer (7M urea, 2M thiourea, 2% chaps, 60mM DTT, 0.2% pH 3-10 ampholyte, Biorad, CA, USA). Resuspension of pellets was carried out in ice.

Materials and Methods 41 2.4. Two-dimensional gel electrophoresis Isoelectric focussing was done with 17 cm IPG strips (Biorad, CA, USA) of pH gradient 5-8, which were first rehydrated with the experimental samples dissolved in rehydration buffer. Following isoelectric focussing, the IPG strips were equilibrated for 10 minutes in reducing equilibration buffer (50mM Tris/Cl pH 8.8, 6M urea, 30% glycerol, 2% SDS and 50mM DTT) followed by 15 minutes in alkylating equilibration buffer (composition same as above except that it contains 2.5% w/v of iodoacetamide instead of DTT). Large format 12% SDSpolyacrylamide gels were used for the second dimension separation. Gels were stained with blue silver staining solution (10% v/v orthophosphoric acid; Merck, India, 10% w/v ammonium sulphate; SRL, India, 20% v/v methanol; SRL, India, 0.12% Coomassie brilliant blue G-250; SRL, India) (3). 2.5. Identification of proteins by MALDI-MS For identification of CSF proteins, AIS A grade CSF samples were used. AIS A sample encompasses CSF proteins as well as serum proteins, due to higher serum permeation in complete injury cases. This factor does not confound the analysis because all proteins identified from incomplete injury (AIS C and D) CSF samples are also present in AIS A samples. Protein spots from blue-silver stained CSF protein 2D gels were picked using the Proteome works spot cutter (Biorad, CA, USA). Spots were de-stained, processed for MALDI sample preparation using the processing kit (Thermo Scientific, IL, USA), overnight trypsin digested (Thermo Scientific, IL, USA) and lyophilised in Heto Vacuum Centrifuge (Thermo). α-Cyano4-hydroxycinnamic acid (CHCA) matrix (Thermo Scientific, IL, USA) was mixed in 1:1 ratio with 50% ACN (Thermo Scientific, IL, USA), 0.1% TFA reconstituted lyophilised spots and spotted on 192 well tungsten MALDI plates (AB Sciex, MA, USA). 4700 MALDI TOF/TOFTM Analyser, (AB Sciex, MA, USA) was used for matrix assisted laser desorption ionisation (MALDI) mass spectrometry. Peptide mass fingerprint was obtained in positive MS reflector mode with fixed laser intensity of 5500, 2000-3000 laser hits in the range of 800-4000 Da. Signal to noise ratio was set at 10 and mass exclusion tolerance at 150 ppm (4). For internal calibration, minimum signal to noise ratio was set at 20 and a mass tolerance of ±300 ppm was set which included monoisotopic peaks only. Peptides of interest were isolated at a relative resolution of 50 (full width at half maximum) and data from 3000 to 5000 laser shots were collected (4). GPS ExplorerTM version 3.6 (Applied Biosystems, MA, USA) was used for analysis of spectral data. MASCOT database scoring algorithm and NCBI and Swiss-Prot protein databases

Materials and Methods 42 were used for peptide identification. Search settings: single missed tryptic cut, fixed carbamidomethylation, variable methionine oxidation, N-terminal acetylation and 150 ppm mass accuracy. Autolytic tryptic peaks were excluded in the MASCOT search parameter and p < 0.05 was considered significant during identification. 2.6. Sample labelling for 2D-DIGE Each AIS A CSF sample was randomly paired with an AIS C or D CSF sample and DIGE was conducted. N-hydroxysuccinimide cyanine dyes (CyDyes, Amersham Biosciences, Uppsala, Sweden) were reconstituted in dimethyl formamide (DMF) in ice and 50µg of protein was labelled with 200pM DMF reconstituted Cy5 dye. The counterpart sample was similarly labelled with Cy3 dye. The samples were reverse labelled in half of the total number of DIGE experiments. Internal standard was made for each experiment by pooling in 25µg of protein from two samples followed by Cy2 labelling. The labelling was conducted in ice for 30 minutes with gentle tapping every 10 minutes. 10 nm Lysine was used to stop the labelling reaction at the end of 30 minutes. Rehydration buffer was added to the labelled samples to make the total volume 330µl, which was used to rehydrate IPG strips and subsequently separated on second dimension, as discussed above. 2.7. Scanning of gels, analysis by DeCyder and biological variance analysis Gels were scanned using the Typhoon imager (version 3.0, Amersham Biosciences, Uppsala, Sweden) at excitation/emission values of 488/520 nm for Cy2 dye, 532/580 nm for Cy3 dye and 633/670 nm for Cy5 dye. DeCyder 2D Differential In-gel Analysis (version 6.5) software (Amersham Biosciences, Uppsala, Sweden) was used for the differential in gel analysis (DIA), where a threshold of 1.5 (volume ratio of Cy3 and Cy5 spots) was set and non-protein gel features were excluded manually. 2.8. Biological variance analysis The gels were analysed using the biological variance analysis (BVA) software (DeCyder 6.5). For BVA analysis, intensive repeated landmarking was performed for the gels selected in Cy2 filter. Automated gel to gel spot matching was applied, Student’s t-test was performed by the software and spots that showed a particular trend of increase or decrease in three or more gels out of seven, with a p-value