Aberrant microRNA Expression in Spinal Muscular Atrophy Motor Neurons The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Wertz, Mary Helene. 2015. Aberrant microRNA Expression in Spinal Muscular Atrophy Motor Neurons. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:17464519 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Aberrant!microRNA!Expression!in!Spinal!Muscular!Atrophy!Motor!Neurons! ! A!dissertation!presented! by! Mary!Helene!Wertz! to! The!Division!of!Medical!Sciences! In!partial!fulfillment!of!the!requirements! for!the!degree!of! Doctor!of!Philosophy! in!the!subject!of! Neurobiology! ! Harvard!University! Cambridge,!Massachusetts! ! April!2015! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ©!2015!'!Mary!Helene!Wertz! All!rights!reserved. ! Dissertation!Advisor:!Mustafa!Sahin!! ! ! ! Mary!Helene!Wertz! ! Aberrant!microRNA!Expression!in!Spinal!Muscular!Atrophy!Motor!Neurons! ! Abstract! ! Spinal! Muscular! Atrophy! (SMA)! is! a! devastating! autosomalMrecessive! pediatric!neurodegenerative!disease!characterized!by!loss!of!spinal!motor!neurons.! It!is!caused!by!mutation!in!the!survival!of!motor!neuron!1,!SMN1,!gene!and!leads!to! loss! of! function! of! the! fullMlength! SMN! protein.! SMN! has! a! number! of! functions! related! to! RNA! processing! in! neurons,! including! RNA! trafficking! in! neurites,! and! RNA!splicing!and!snRNP!biogenesis!in!the!nucleus.!While!previous!work!has!focused! on!the!alternative!splicing!and!expression!of!traditional!mRNAs,!our!lab!has!focused! on! the! contribution! of! another! RNA! species,! microRNAs! (miRNAs),! to! the! SMA! phenotype.!! miRNAs! are! ~22! nucleotide! small! RNAs! that! are! involved! in! postM transcriptional! regulation! of! gene! expression.! They! function! by! translational! repression! or! mRNA! decay! of! target! RNAs.! Interestingly,! dysregulation! of! RNA! processing! and! miRNA! expression! has! been! identified! in! motor! neuron! diseases! including!SMA!and!Amyotrophic!Lateral!Sclerosis.!Our!lab!previously!discovered!a! miRNA,! miRM183,! that! is! dysregulated! in! SMA! and! impacts! its! targets! in! cortical! neurons!and!SMA!mouse!spinal!cords.!! However,!spinal!motor!neurons!are!the!cell!type!most!affected!by!SMN!loss.! We!hypothesized!that!motor!neuron!specific!miRNA!changes!are!involved!selective! ! iii! vulnerability!in!SMA.!Therefore,!we!sought!to!determine!the!effect!of!loss!of!SMN!on! spinal!motor!neurons.!To!accomplish!this,!I!used!microarray!and!RNAseq!to!profile! both! miRNA! and! mRNA! expression! in! primary! spinal! motor! neurons! after! acute! SMN! knockdown.! By! integrating! the! miRNA:mRNA! profiles! we! identified! dysregulated!miRNAs!with!enrichment!in!differentially!expressed!putative!targets.! miRM431!was!the!most!substantially!increased!miRNA!and!a!number!of!its!putative! targets!were!downregulated!after!SMN!loss.!Further,!I!confirm!that!miRM431!directly! regulates!its!target!chondrolectin!and!impacts!neurite!length.! This!work!is!critical!to!understanding!the!cellMtype!specific!effect!of!aberrant! miRNA! expression! in! SMN! knockdown! motor! neurons.! It! demonstrates! the! contribution! of! dysregulated! RNA! processing! in! motor! neurons! to! neurodegeneration.! Furthermore,! this! work! highlights! the! impact! of! nonMcoding! RNAs! in! human! disease! and! points! to! specific! miRNA! whose! dysregulation! potentially!impacts!motor!neuron!health.! ! ! ! iv! Table!of!Contents! Abstract!........................................................................................................................................................................!iii! Table!of!Figures!..........................................................................................................................................................!vi! Table!of!Tables!...........................................................................................................................................................!vi! Acknowledgements!.................................................................................................................................................!vii! Chapter!1!Introduction!to!Spinal!Muscular!Atrophy!and!microRNAs!.................................!1! Spinal!Muscular!Atrophy!a!clinical!perspective!............................................................................................!2! Multifaceted!functions!of!SMN!in!RNA!processing!.......................................................................................!5! In!vitro!and!in!vivo!models!of!SMA!......................................................................................................................!8! Development!of!therapies!for!SMA!..................................................................................................................!11! Commonalities!of!SMA!with!other!motor!neuron!diseases!...................................................................!15! microRNA!....................................................................................................................................................................!16! miRNA!in!spinal!motor!neurons!........................................................................................................................!25! Neurodegenerative!diseases!and!miRNA!expression!...............................................................................!27! The!intersection!of!SMA!and!miRNAs!.............................................................................................................!27! Chapter!2!Methods!..............................................................................................................................!30! In!vitro!acute!shSMN!knockdown!model!.......................................................................................................!31! Exiqon!LNA!miRNA!microarray!data!analysis!...........................................................................................!39! mRNA!expression!profiling!by!RNAseq!..........................................................................................................!40! Identification!of!putative!miRNA!target!mRNA!interactions!...............................................................!43! Characterization!of!miRNA!target!interaction!..........................................................................................!46! SMA!patient!samples!..............................................................................................................................................!49! Chapter!3!Integration!of!miRNA:mRNA!profiling!reveals!the!impact!of!miRNA! dysregulation!on!SMA!motor!neurons!.........................................................................................!51! Introduction!...............................................................................................................................................................!52! Results!..........................................................................................................................................................................!53! Discussion!...................................................................................................................................................................!79! Chapter!4!miRN431!targeting!of!Chondrolectin!is!dysregulated!in!SMA!...........................!85! Introduction!...............................................................................................................................................................!86! Results!..........................................................................................................................................................................!89! Discussion!...................................................................................................................................................................!96! Chapter!5!Discussion!.......................................................................................................................!100! Chapter!6!References!......................................................................................................................!109! Appendix!1!..........................................................................................................................................!129! Appendix!2!..........................................................................................................................................!135! Appendix!3!..........................................................................................................................................!150! Appendix!4!..........................................................................................................................................!157! Appendix!5!..........................................................................................................................................!168! Appendix!6...........................................................................................................................................180! ! ! ! ! v! Table!of!Figures!