ABERRANT ALTERNATIVE SPLICING IN SKELETAL MUSCLE OF R6/2 HUNTINGTON’S DISEASE MICE A Thesis Presented to the Faculty of California State Polytechnic University, Pomona In Partial Fulfillment Of the Requirements for the Degree Master of Science In Biological Sciences By Elizabeth Munguia 2016 SIGNATURE PAGE THESIS: ABERRANT ALTERNATIVE SPLICING IN SKELETAL MUSCLE OF R6/2 HUNTINGTON’S DISEASE MICE AUTHOR: Elizabeth Munguia DATE SUBMITTED: Fall 2016 Biological Sciences Department Dr. Robert J. Talmadge Thesis Committee Chair Biological Sciences Dr. Andrew D. Steele Biological Sciences Dr. Sepher Eskandari Biological Sciences ii ACKNOWLEDGEMENTS First and foremost, I would like to thank my thesis advisor, Dr. Robert J. Talmadge for the amazing opportunity to join his lab and be part of a great research project. I could not have completed my Master’s thesis without his help. I am gratefully indebted to him for his time, patience, expertise, and guidance. I would also like to thank my thesis committee, Dr. Sepher Eskandari and Dr. Andrew D. Steele for their time and intellectual contributions. Finally, I want to thank my family and friends for their constant support and encouragement. Gracias mamá y papá por su amor y apoyo. Thank you Adam, Helen, Steve, and Jenny for your love and countless adventures. iii ABSTRACT Huntington’s disease (HD) is a fatal trinucleotide-repeat disorder that is characterized by neurodegeneration, which leads to motor and cognitive impairments. The motor impairments include chorea, rigidity, dystonia, and muscle weakness. Cognitive impairments include subcortical dementia, depression, mania, and suicide. These impairments increase in severity until the individual loses the ability to talk, walk, or reason. Previous research by our lab (Waters et al., 2013) demonstrated that in a transgenic mouse model for HD, i.e., the R6/2 mouse line, impairments in muscle function, including alterations in muscle excitability, chloride channel function, and chloride channel mRNA levels, were in part due to aberrant splicing of the chloride channel pre-mRNA. The R6/2 mouse line expresses the expanded CAG trinucleotide repeat from exon 1 of the human huntingtin (HTT) gene observed in human HD patients and represents a model of early onset HD. The data from Waters et al. (2013) are reminiscent of another trinucleotide-repeat disorder, myotonic dystrophy (DM), in which skeletal muscles have dramatic debilitations in the regulation of pre-mRNA splicing. Therefore, this study investigated the changes in pre-mRNA splicing in multiple muscles of R6/2 mice for several genes that are known to be aberrantly spliced in DM, and may be aberrantly spliced in HD. These genes include the chloride channel (CLCN1), insulin receptor (INSR), titin (TTN), sarco(endo)plasmic reticulum Calcium ATPase (SERCA1), z-line associated protein (ZASP), troponin (TNNT3), and α-actinin 1 (ACTN1). Reverse transcriptase-polymerase chain reactions using primers designed to detect multiple splice variants were used to detect changes in alternative splicing in R6/2 and age-matched wild-type (WT) mice. Multiple muscles were assessed for alterations in mRNA splicing iv including the tibialis anterior (TA), interosseous (IO), diaphragm (Dia), and soleus (Sol) muscles from late-stage R6/2 mice and WT controls. We found a significant difference in aberrantly spliced TTN mRNA in the TA of R6/2 mice relative to WT (p < 0.001) (n=12/group). We also found a significant difference in aberrantly spliced CLCN1 (p < 0.05) and INSR (p < 0.05) mRNAs in the IO of R6/2 mice relative to WT (n=6/group). However, we did not find significant differences in aberrantly spliced CLCN1 (p = 0.286), INSR (p = 0.716), TTN (p = 0.195), and ZASP (p = 0.496) mRNAs in the Dia of R6/2 mice relative to WT (n=5/group). There were also no significant differences in aberrantly spliced CLCN1 (p = 0.500) and INSR (p = 0.264) mRNAs in the Sol of R6/2 mice (n = 6) relative to WT (n = 7). There were also no significant differences in aberrantly spliced TTN (p = 0.0774) mRNA in the IO muscle of R6/2 mice relative to WT (n=6/group). We also found no significant differences in aberrantly spliced SERCA1, TNNT3, and ACTN1 mRNAs in TA, IO, Dia, and Sol skeletal muscles of R6/2 mice relative to WT. These results suggest the effect of HD on aberrant splicing is muscle specific and less pronounced than in DM. Thus, slower muscles, such as the Dia and Sol, may be less affected by HD in terms of aberrant alternative splicing, than faster muscles, such as the TA and IO. v TABLE OF CONTENTS SIGNATURE PAGE ......................................................................................................... iii ACKNOWLEDGEMENTS. .............................................................................................. iii ABSTRACT ....................................................................................................................... iv LIST OF TABLES ............................................................................................................. ix LIST OF FIGURES ............................................................................................................ x INTRODUCTION .............................................................................................................. 1 Huntington's Disease ....................................................................................................... 1 Huntington's Disease: Previous Research ....................................................................... 5 Alternative Splicing ........................................................................................................ 7 Animal Models of HD .................................................................................................... 8 Myotonic Dystrophy ..................................................................................................... 10 Myotonic Dystrophy: Aberrant Alternative Splicing ................................................... 12 Chloride Channel (CLCN1) ...................................................................................... 12 Insulin Receptor (INSR) ........................................................................................... 12 Sarco(endo)plasmic reticulum Ca2+-ATPase pump (SERCA1) ............................... 13 Skeletal Muscle ............................................................................................................. 13 Skeletal Muscle Composition ................................................................................... 13 Skeletal Muscle Contraction ..................................................................................... 15 Muscle Proteins ............................................................................................................. 17 vi Titin (TTN) ............................................................................................................... 17 Z-line associated protein (ZASP) .............................................................................. 17 Troponin T3 (TNNT3) .............................................................................................. 18 Alpha Actinin 1 (ACTN1) ........................................................................................ 18 Thesis Objectives .......................................................................................................... 19 Study Questions ........................................................................................................ 20 Hypotheses ................................................................................................................ 20 MATERIALS AND METHODS ...................................................................................... 21 Animal Model ............................................................................................................... 21 Experimental Procedures .............................................................................................. 22 RNA Isolation ........................................................................................................... 22 RNA Quantification and Dilution ............................................................................. 23 Splicing Analysis ...................................................................................................... 24 Genes of Interest ........................................................................................................... 25 Statistical Methods ........................................................................................................ 26 RESULTS ......................................................................................................................... 27 Chloride Channel (CLCN1) ...................................................................................... 27 Insulin Receptor (INSR) ........................................................................................... 30 Sarco(endo)plasmic reticulum Ca2+-ATPase Pump (SERCA1) ............................... 33 Titin (TTN) ............................................................................................................... 35 vii Troponin T3 (TNNT3) .............................................................................................. 38 Alpha Actinin 1 (ACTN1) ........................................................................................ 40 Z-line associated protein (ZASP) .............................................................................