Reducing pathological accumulations of phosphorylated neurofilament h through modulation of pin1 activity implications in amyotrophic lateral sclerosis

REDUCING PATHOLOGICAL ACCUMULATIONS OF PHOSPHORYLATED NEUROFILAMENT-H THROUGH MODULATION OF PIN1 ACTIVITY: IMPLICATIONS IN AMYOTROPHIC LATERAL SCLEROSIS CHARLENE PRISCILLA POORE (B.Sc.(Hons.), Monash University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2013 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously CHARLENE PRISCILLA POORE i ACKNOWLEDGEMENT I would like to express my deepest gratitude to my supervisor, Dr Sashi Kesavapany for his guidance, support and much patience Thank you for mentoring me and teaching me the joy of science I would also like to thank my co-supervisor, Prof Markus Wenk for his insightful advice and guidance during my PhD I am grateful to my committee members, Prof Tang Bor Luen and Prof Soong Tuck Wah for sharing their knowledge and counsel during this journey I am also grateful to GlaxoSmithKline, Neural Pathways Discovery Performance Unit, Singapore for providing an excellent environment to research, where the staff are friendly and willing to help and I am glad for the opportunity to have been part of the team A special thanks to the staff and students at the Neurobiology Programme, Centre for Life Sciences and Department of Biochemistry (NUS) for their support and care I would also like to thank my past and current lab mates from NUS and GSK: Jeyapriya Sundaram, Noor Hazim, Chua Hui Wen, Peh Khong Ming, Tan Kin Hup, Vinupriya Ganapathy, Cornie Chua, Poh Kay Wee, Cheryl Tay and Alexander Stephan for their advice, encouragement, help and support as well as good company I would also like to thank the BRC staff, Yasmin and Yusuf who have helped me during my research I would also like to thank my parents and brother, who have always been supportive and encouraging Last but not least, I would like to thank Jared Lee for his patience and understanding, as well as for his strength when mine was failing ii TABLE OF CONTENTS DECLARATION PAGE……………………………………………… ……… i ACKNOWLEDGEMENTS……………………………………………… ……ii TABLE OF CONTENTS………………………………………………… … iii ABSTRACT…………………………………………………………………… ix LIST OF TABLES …………………………………………………………… x LIST OF FIGURES… ……………………………………………………….xiii ABBREVIATIONS……………………………………….………………… xvi CHAPTER 1: INTRODUCTION……………………………….…… ……….1 1.1 PEPTIDYL-PROLYL ISOMERASE PIN1……………… .1 1.1.1 Characterization of Pin1 Activity………………………… …………… 1.1.2 Biological Functions of Pin1…………………………… ……………….3 1.1.2.1 Pin1 and the coordination of the cell cycle……………………… 1.1.2.2 Pin1 role in oncogenesis……………… 1.1.2.3 Pin1 regulation of cellular stress…… 1.1.2.4 Pin1 in germ cell development……… 1.1.2.5 Role of Pin1 in telomere regulation ageing … 1.1.2.6 Pin1 modulation of the immune response……………….… .10 1.1.3 Role of Pin1 in neurons………………………… 11 1.1.4 Pin1 in neurodegeneration…………………… .12 1.1.4.1 1.1.4.2 Parkinson’s Disease…………………………………………… 15 1.1.4.3 1.2 Alzheimer’s Disease…………………………………………… 12 Amyotrophic Lateral Sclerosis……… 16 AMYOTROPHIC LATERAL SCLEROSIS……………… 17 1.2.1 Familial ALS…………………………………… 17 1.2.2 Molecular Pathways In ALS Pathogenesis…… 18 1.2.2.1 Oxidative damage in ALS…………… 20 1.2.2.2 Excitotoxicity………………………… .22 1.2.2.3 Mitochondrial dysfunction…………… .23 1.2.2.4 Protein Aggregation…………………… .25 1.2.2.5 Endoplasmic Reticulum Stress……… 27 iii 1.2.2.6 1.2.2.7 1.3 Impaired Axonal Transport………… 28 Non-cell Autonomous toxicity in ALS 30 NEUROFILAMENTS……………… .33 1.3.1 Molecular Biology of Neurofilaments… 33 1.3.1.1 Structure and assembly of neurofilaments… .33 1.3.1.2 Transport of Neurofilaments………………… 34 1.3.2 Neurofilament function…………… .36 1.3.2.1 Biological function of neurofilaments 36 1.3.2.2 Function of neurofilaments is regulated by phosphorylation… 36 1.3.3 Neurofilaments and Neurological Diseases…… .39 1.3.3.1 1.3.3.2 Alzheimer’s Disease…………………… 40 1.3.3.3 Parkinson’s Disease…………………… .40 1.3.3.4 1.4 Charcot-Marie-Tooth………………… .39 Amyotrophic Lateral Sclerosis……… 41 RNA INTERFERENCE THERAPEUTICS……………… 46 1.4.1 RNAi Pathway……………………………… 46 1.5 ADENO-ASSOCIATED VIRUS……………………………………… 49 1.5.1 AAV Delivery of shRNA…… 49 1.5.2 Clinical Trials using AAV…………… 51 1.6 OBJECTIVE OF STUDY 52 CHAPTER MATERIALS AND METHODS…………………………… 53 2.1 MATERIALS…………………………………………………… 53 2.1.1 Preparation of Plasmid Construct ……………… 53 2.1.2 Mammalian Cell Cultures …………………………… 54 2.1.3 Adeno-associated virus (AAV) Production…… .56 2.1.4 Immunostaining and Biochemical analyses… 58 2.1.5 Animal Studies……………………………………………… 65 2.2 METHODS…………………………………………………… 68 2.2.1 Preparation of Plasmid Construct……………… 68 2.2.1.1 Plasmid Construct………………………………………… 68 iv 2.2.1.2 Transformation of competent cells with plasmids……… 68 2.2.1.3 Plasmid DNA purification…………………………… .68 2.2.2 Cell cultures……………………………………………… .69 2.2.2.1 Preparation of primary cortical neuronal cultures…… .69 2.2.2.2 Preparation of HEK 293T/17 cultures 71 2.2.2.3 Cell transfections……………………………………… … 72 2.2.2.4 Transduction of primary cultures and HEK 293T/17 cells…… 73 2.2.3 Adeno-associated virus (AAV) Production……………… .74 2.2.3.1 Production of recombinant AAV (rAAV) using the AAV Helper- Free System……………………… .74 2.2.3.2 rAAV production performed by Vector Core Lab, UPENN………………………………………………………… .76 2.2.4 Immunostaining and Biochemical analyses ……………… …… 81 2.2.4.1 Immunocytochemistry…………………………………… 81 2.2.4.2 Immunohistochemistry…………………………………… 81 2.2.4.3 SDS-PAGE and Western Blotting…………………… .83 2.2.4.4 Real-time reverse transcription polymerase chain reaction (RTPCR)…… … 85 2.2.4.5 Protein Quantification (BCA assay)…………………… 88 2.2.5 Animal Studies…………………….………………………… 89 2.2.5.1 Animal models………………….………………………… 89 2.2.5.2 C57BL/6J WT mice ………………… .89 2.2.5.3 B6.Cg-Tg(SOD1*G93A)1Gur/J transgenic mice….…… 89 2.2.5.4 Genotyping of transgenic mice…………………….…… 89 2.2.5.5 Intramuscular injections…… ……………………… 90 2.2.5.6 Cardiac Perfusion……………………………… 91 2.2.5.7 Motor Function Studies…………… ………………… .92 2.2.6 Statistical analysis and presentation of data…………………… … 93 v CHAPTER VALIDATION OF PIN1 KNOCKDOWN USING THE PIN1 shRNA CONSTRUCT………… .…………….………………… … 94 3.1 BACKGROUND…………………………………………………… 94 3.2 METHODS………………………………………… .95 3.2.1 Production of Pin1 shRNA and Control shRNA plasmid DNA…… .95 3.2.2 Validation of Pin1 knockdown by transient transfections of the Pin1 shRNA construct……………………………………… .95 3.2.3 Immunocytochemistry to validate knockdown of Pin1 expression.… 95 3.2.4 SDS-PAGE and Western Blot to quantify knockdown of Pin1 protein levels………………………………………………………… 96 3.2.5 Real Time RT-PCR to determine reduction of Pin1 RNA levels…… 96 3.2.6 Stable Pin1 knockdown using AAV as the gene delivery tool…… 96 3.3 RESULTS…………………………………………………………….… 98 3.3.1 Validation of Pin1 knockdown by transient transfections of the Pin1 shRNA construct into HEK 293T/17 and primary cortical neurons… 98 3.3.2 Production of AAV2-Pin1 shRNA and AAV2-Control shRNA for efficient transduction HEK 293T/17 and primary cortical neurons… 102 3.4 DISCUSSION………………………………… .110 CHAPTER GENE DELIVERY OF AAV-PIN1 shRNA INTO THE MOTOR NEURONS OF THE SPINAL CORD…………………………….114 4.1 BACKGROUND…………………………………………………… 114 4.2 METHODS……………………………………… .…115 4.2.1 Production and purification of AAV2-Control shRNA and AAV2-Pin1 shRNA………………………………………………………… 115 4.2.2 Production and purification of AAV serotypes from Vector Core Lab…………………………………………………………………….115 4.2.3 Validation of transduction efficiency of various AAV serotypes in vitro in primary cortical neurons…………………………………………… 115 4.2.4 Intramuscular injections of AAV 115 vi 4.2.5 Immunohistochemistry to validate transduction efficiency and Pin1 knockdown in spinal motor neurons 116 4.3 RESULTS 117 4.3.1 Delivery of AAV2-Control shRNA and AAV2-Pin1 shRNA into the mouse spinal cord …………………………………………………… 117 4.3.2 Evaluation of the transduction efficiency of various AAV serotypes 119 4.3.3 Validation of stable gene expression of AAV into the spinal motor neurons in mice…………………………………… 124 4.3.4 Determination of AAV titer and Pin1 knockdown in transduced spinal motor neurons ……………………….……………………… .127 4.4 DISCUSSION………………………………….……………………… 131 CHAPTER KNOCKDOWN OF PIN1 AND REDUCTION OF p-NF-H ACCUMULATIONS IN THE ALS MOUSE MODEL………… 135 5.1 BACKGROUND…………………………………………………… 135 5.2 METHODS………………………………………… .136 5.2.1 Production and purification of AAV9-Pin1 shRNA and AAV9-Control shRNA from Vector Core Lab …………………………… .136 5.2.2 Administration of AAV9-Pin1 shRNA into WT and G93A SOD1 mice .136 5.2.3 Motor function studies………………………… 136 5.2.4 Histological analysis of pathological hallmarks in WT and G93A SOD1 mice………………………… .137 5.3 RESULTS 138 5.3.1 Evaluation on motor function dysfunction and pathological hallmarks in the G93A SOD1 mice ……………………………… 138 5.3.2 Effect of Pin1 silencing on p-NF-H accumulations and disease pathology in the G93A SOD1 mice ……… 145 5.4 DISCUSSION………………… ……….………………… ……… 161 vii CHAPTER CONCLUSION AND FUTURE STUDIES 166 BIBILIOGRAPHY……………………….……………………… 172 APPENDICES ………………………….…………………… …A1 viii ABSTRACT One of the pathological hallmarks of motor neuron death in Amyotrophic Lateral Sclerosis (ALS) is the abnormal accumulations of phosphorylated neurofilaments in the neuronal cell body Previous reports suggested that Pin1, a prolyl-isomerase that catalyses the cis-trans isomerisation of the phosphorylated serine/threonineproline motifs, may be responsible for the aberrant accumulations of phosphorylated neurofilament heavy chain (p-NF-H) in neurons, which are observed during neurotoxicity The aim of this thesis is to investigate the reduction of aberrant p-NF-H accumulations through knockdown of Pin1 activity using recombinant adeno-associated virus (AAV)-mediated transduction of Pin1 shRNA in vivo in the G93A SOD1 ALS mouse model This was achieved using the Pin1 shRNA construct, which showed effective and stable knockdown of Pin1 in HEK 293T/17 cells and in primary neuronal cultures To produce an in vivo gene delivery system into the spinal motor neurons by intramuscular route of administration, the transduction efficiencies of AAV serotypes 1, 2, 5, 6, and were compared AAV9 yielded the highest level of motor neuron transduction in the spinal cord and was used for Pin1 shRNA gene delivery Accumulations of pNF-H in the spinal motor neurons of G93A SOD1 transgenic mice were observed prior to motor neuron cell death, astrocyte activation and behavioural deficits This suggested that early prevention of p-NF-H accumulations may be beneficial The spinal motor neurons transduced with AAV9-Pin1 shRNA showed significant reduction in aberrant p-NF-H accumulations indicating the Pin1 knockdown strategy may be of therapeutic potential in reducing the neurotoxic accumulations in ALS ix regulation of Pin1 in 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(2007) Post-phosphorylation prolyl isomerisation of gephyrin represents a mechanism to modulate glycine receptors function Embo J 26:1761-1771 Zolotukhin S, Byrne BJ, Mason E, Zolotukhin I, Potter M, Chesnut K, Summerford C, Samulski RJ, Muzyczka N (1999) Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield Gene Ther 6:973-985 198 APPENDICES Sma I (32) Sma I (43) ITR human U6 promoter BsrBI (5033) BsrBI (4792) NdeI (379) TATA Origin BsrBI (450) shRNA Pin1 Mlu I (503) NdeI (748) CMV promoter 5103 bp EGFP Amp-R BsrBI (2991) Origin I (4219) Bpm SV40 late polyadenylation signal ITR Sma I (2241) Sma I (2252) BsrBI (2546) SmaI (32) SmaI (43) ITR MscI (107) SmaI (203) human U6 promoter EcoRI (658) hairpin loop CMV promoter NcoI (1137) 5558 bp NcoI (1403) Amp-R EGFP Bpm I (1849) Bpm I (2089) SV40 late polyadenylation signal MscI (2632) ITR SmaI (2696) SmaI (2707) A1 Appendix Figure Construct backbone of Pin1 shRNA (top) and Control shRNA (bottom) indicating restriction digest sites, ampicillin resistance (Amp-R), ITRs, promoter regions and polyadenylation site Appendix Figure Immunostaining of lumbar of G93A SOD1 Pin1 shRNA treated mouse at 135 days old with anti-GFP (green) and co-stained with antiGFAP (A) and anti-p-NF-H antibodies (B) Nuclear counterstaining was performed using DAPI (blue) Arrows indicate transduced motor neurons and arrowheads indicate non-transduced motor neurons Scale bars represent 200m Appendix Figure Small portion of astrocytes were transduced by AAV9 intramuscular injections into mice Immunostaining of lumbar of AAV9-Pin1 shRNA treated G93A SOD1 mouse with anti-GFP (green) and co-stained with anti-GFAP (red) Nuclear counterstaining was performed using DAPI (blue) Arrowheads indicate transduced astrocytes Scale bars represent 200m A2 Appendix Table Mean values and SEM of immunoblot quantification and levels of Pin1 mRNA of Control shRNA and Pin1 shRNA transfected HEK 293/T17 samples as presented in the graphs in Figure 3.3.1 Mean values and SEM of percentage Pin1 shRNA/tubulin protein levels normalized Control shRNA/tubulin (%) Days Mean values SEM 82.5 10.1 79.4 9.0 37.2 3.6 56.9 10.1 Mean values and SEM of percentage Pin1 RNA expression of Pin1 shRNA/Control shRNA (%) Days Mean values SEM 51.7 3.3 54.0 9.4 29.5 1.2 36.4 4.2 Appendix Table Mean values and SEM of immunoblot quantification and levels of Pin1 mRNA of AAV2-Control shRNA and AAV2-Pin1 shRNA transduced HEK 293/T17 samples as presented in the graphs in Figure 3.3.3 Mean values and SEM of percentage Pin1 shRNA/tubulin protein levels normalized Control shRNA/tubulin (%) Days Mean values SEM 47.4 14.7 54.2 9.2 48.6 14.2 11 71.1 16.9 14 Mean values and SEM of percentage Pin1 RNA expression of Pin1 shRNA/Control shRNA (%) Days Mean values SEM 39.1 6.1 48.8 11.3 59.8 8.7 11 64.9 4.8 14 A3 Appendix Table Mean values and SEM of percentage number of AAV2Control shRNA and AAV2-Pin1 shRNA transduced cortical neurons over total number of neurons as presented in the graph in Figure 3.3.4B Mean values and SEM of percentage number of transduced Control shRNA and Pin1 shRNA cortical neurons over total number of neurons (%) Days AAV2-Control shRNA AAV2-Pin1 shRNA Mean values SEM Mean values SEM 11 14 36.7 39.4 52.2 48.5 2.6 3.1 2.5 1.5 25.8 28.8 43.0 45.9 0.8 3.3 4.2 2.4 Appendix Table Mean values and SEM of immunoblot quantification and levels of Pin1 mRNA of AAV2-Control shRNA and AAV2-Pin1 shRNA transduced cortical neuron samples as presented in the graphs in Figure 3.3.4D and E Mean values and SEM of percentage Pin1 shRNA/tubulin protein levels normalized Control shRNA/tubulin (%) Days Mean values SEM 51.3 8.8 69.0 1.8 50.9 3.8 11 46.4 5.0 14 Mean values and SEM of percentage Pin1 RNA expression of Pin1 shRNA/Control shRNA (%) Days Mean values SEM 57.3 6.8 30.4 7.7 52.9 3.6 11 32.8 5.4 14 A4 Appendix Table Mean values and standard deviation of percentage transduction efficiency of different AAV serotypes in vitro in cortical neurons as presented in Figure 4.3.2B Mean values of percentage transduction efficiency (%) Days AAV1 AAV2 AAV5 AAV6 AAV8 AAV9 42.8 12.4 6.4 22.1 36.0 34.9 64.0 21.3 7.8 54.4 49.2 37.1 86.9 33.0 11.7 55.8 71.6 64.9 11 77.9 51.4 17.6 69.8 80.8 77.2 14 Standard deviation of percentage transduction efficiency (%) Days AAV1 AAV2 AAV5 AAV6 AAV8 AAV9 1.7 3.5 2.5 7.5 5.5 3.9 7.9 8.8 4.9 9.4 3.8 5.5 4.9 0.9 8.2 8.3 5.5 1.8 11 1.0 7.9 4.8 5.0 2.3 11.0 14 Appendix Table Mean values and standard deviation of percentage transduction efficiency of AAV1- and AAV9-Pin1 shRNA at different virus titers as presented in Figure 4.3.5B AAV particles/injection 5x1011 1x1012 2x1012 AAV1-Pin1 shRNA Mean values Standard (%) deviation 6.9 5.8 9.2 8.9 14.5 10.5 A5 AAV9-Pin1 shRNA Mean values Standard (%) deviation 0.7 1.4 8.6 5.1 23.4 12.6 Appendix Table Mean values and standard deviation for rotarod, grip strength and gait analysis graphs presented in Figure 5.3.1 Mean values and standard deviation of stride time (ms) from gait analysis WT G93A SOD1 Days old Mean values Standard Mean values Standard deviation deviation 221 27.5 240 27.3 60 205 30.6 201 23.9 90 230 43.5 220 46.5 120 205 20.0 341 36.5 150 Mean values and standard deviation of stride length (mm) from gait analysis WT G93A SOD1 Days old Mean values Standard Mean values Standard deviation deviation 66 2.3 69 4.4 60 70 7.1 68 4.3 90 71 11.3 65 6.2 120 65 11.1 36 6.7 150 Mean values and standard deviation of grip strength (g) of forelimb WT G93A SOD1 Days old Mean values Standard Mean values Standard deviation deviation 45 9.2 49 11.7 60 58 17.6 62 12.9 90 57 7.9 45 14.9 120 63 11.3 35 6.5 150 Mean values and standard deviation of grip strength (g) of forelimb/hindlimb WT G93A SOD1 Days old Mean values Standard Mean values Standard deviation deviation 158 24.2 144 25.8 60 137 24.8 140 27.6 90 183 11.4 93 11.1 120 199 36.3 68 10.1 150 Mean values and standard deviation of latency to fall (sec) from rotarod WT G93A SOD1 Days old Mean values Standard Mean values Standard deviation deviation A6 60 90 120 150 300 279 283 294 0.0 32.2 13.5 10.2 280 257 158 74 16.1 45.6 48.7 12.4 Appendix Table Mean values and standard deviation for quantification of average number of large motor neurons per section presented in Figure 5.3.2E WT Days old Mean values 60 90 120 150 20 23 25 27 Standard deviation 3.7 2.9 1.0 1.0 G93A SOD1 Mean values Standard deviation 20 5.0 13 1.4 12 3.5 1.5 Appendix Table Mean values and standard deviation for quantification of the percentage of ventral horn motor neurons with p-NF-H accumulation in the cell bodies presented in Figure 5.3.3B WT Days old 60 90 120 150 Mean values (%) 0.0 1.3 2.1 2.5 Standard deviation 0.0 2.8 3.6 5.0 G93A SOD1 Mean values Standard (%) deviation 6.5 5.5 32.0 16.4 33.0 8.9 37.0 9.3 Appendix Table 10 Mean values and standard deviation for rotarod, grip strength and gait analysis studies performed in the WT saline, G93A SOD1 saline, WT Pin1 shRNA, G93A SOD1 Control shRNA and G93A SOD1 Pin1 shRNA treatment groups presented in Figure 5.3.4 Days old 60 75 90 105 120 Mean values of grip strength (g) of forelimb WT Saline G93ASOD1 WT G93ASOD1 Saline Pin1 shRNA Control shRNA 73 64 62 72 77 56 79 58 74 61 69 51 75 54 59 51 76 45 66 51 A7 G93ASOD1 Pin1 shRNA 60 54 59 54 51 135 150 Days old 60 75 90 105 120 135 150 74 35 50 44 63 32 61 39 Standard deviation of grip strength (g) of forelimb WT Saline G93ASOD1 WT G93ASOD1 Saline Pin1 shRNA Control shRNA 23.3 12.4 17.2 13.6 13.3 12.1 15.1 8.6 12.4 12.7 16.3 13.2 35.0 13.8 18.9 14.8 4.6 1.2 5.9 2.7 23.0 6.1 9.1 7.0 11.3 4.2 2.9 2.9 30 26 G93ASOD1 Pin1 shRNA 8.1 12.8 14.6 20.7 4.9 10.7 6.4 Mean values of grip strength (g) of forelimb/hindlimb WT Saline G93ASOD1 WT G93ASOD1 G93ASOD1 Days old Saline Pin1 shRNA Control Pin1 shRNA shRNA 232 183 225 177 182 60 234 198 223 192 186 75 248 203 243 181 201 90 257 176 234 176 178 105 293 148 269 160 160 120 288 119 287 139 109 135 295 90 282 111 91 150 Standard deviation of grip strength (g) of forelimb/hindlimb WT Saline G93ASOD1 WT G93ASOD1 G93ASOD1 Days old Saline Pin1 shRNA Control Pin1 shRNA shRNA 47.4 7.0 48.6 8.2 17.6 60 35.2 2.8 24.5 8.5 13.8 75 34.9 6.9 17.4 10.2 3.2 90 33.2 22.1 15.1 22.0 9.3 105 34.0 18.9 25.6 14.3 13.1 120 27.1 3.0 44.1 11.9 41.2 135 56.0 5.8 30.6 11.3 2.9 150 Days old 60 75 90 105 Mean values of latency to fall (sec) from rotarod WT Saline G93ASOD1 WT G93ASOD1 Saline Pin1 shRNA Control shRNA 291 287 290 288 295 275 292 272 291 265 279 247 286 257 286 243 A8 G93ASOD1 Pin1 shRNA 285 279 271 263 120 135 150 Days old 60 75 90 105 120 135 150 280 197 275 200 196 285 92 270 140 106 264 29 263 78 Standard deviation of latency to fall (sec) from rotarod WT Saline G93ASOD1 WT G93ASOD1 G93ASOD1 Saline Pin1 shRNA Control Pin1 shRNA shRNA 14.1 15.4 11.8 11.8 10.2 7.7 25.0 17.1 6.7 11.6 10.6 30.5 25.5 19.5 22.8 19.0 32.7 25.9 42.7 18.0 17.8 41.3 27.8 65.2 40.7 11.9 25.5 36.9 10.6 35.4 13.8 20.5 29.2 28.0 8.7 Appendix Table 11 Mean values and standard deviation for quantification of average number of large motor neurons per section in the WT saline, G93A SOD1 saline, WT Pin1 shRNA, G93A SOD1 Control shRNA and G93A SOD1 Pin1 shRNA treatment groups presented in Figure 5.3.6B Mean values of number of large motor neurons per section WT Saline G93ASOD1 WT G93ASOD1 G93ASOD1 Days old Saline Pin1 shRNA Control Pin1 shRNA shRNA 20 11 22 13 11 90 17 22 120 20 22 150 Standard deviation of number of large motor neurons per section WT Saline G93ASOD1 WT G93ASOD1 G93ASOD1 Days old Saline Pin1 shRNA Control Pin1 shRNA shRNA 2.3 2.1 4.0 3.6 1.2 90 2.5 4.1 7.6 2.2 2.1 120 7.3 2.6 3.4 4.8 2.9 150 A9 Appendix Table 12 Mean values and standard deviation for percentage of transduced cells with no p-NF-H accumulations in the cell bodies in the WT Pin1 shRNA, G93A SOD1 Control shRNA and G93A SOD1 Pin1 shRNA treatment groups presented in Figure 5.3.7D Mean percentage of transduced cells without perikaryal p-NF-H accumulations (%) Days old WT G93ASOD1 G93ASOD1 Pin1 Pin1 shRNA Control shRNA shRNA 90 77 86 90 96 48 68 120 97 49 54 150 Standard deviation of the percentage of transduced cells without perikaryal p-NF-H accumulations (%) Days old WT G93ASOD1 G93ASOD1 Pin1 Pin1 shRNA Control shRNA shRNA 12.3 17.4 20.5 90 6.7 11.8 9.7 120 7.3 6.8 16.9 150 A10 ... S phase (Ekholm-Reed et al., 2004) Pin1 regulates the turnover of Cyclin E, where Pin1- /MEF showed higher levels of Cyclin E resulting in the impairment of the progression through the G1-S phase... to inhibit or increase the activity of Cdc25, which is the activating phosphatase of the Cdc2-Cyclin B complex that is required for the entry into the mitotic phase depending on the phosphorylation... al., 2007) The authors then hypothesized that the presence of Pin1 facilitates the accumulation of p-NF -H in the spinal cord of ALS patients, which is one of the key pathological events in ALS (Kesavapany