Development of an In Vitro Neuronal Model of Myotonic Dystrophy Type 1 Open Access

Chao Lin (Summer 2019)

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Myotonic dystrophy (dystrophia myotonica, DM) is an autosomal-dominant multisystemic genetic disease. It has two subtypes, myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2). DM affects not only muscles but also other tissues and systems including the central nervous system (CNS). DM1 is caused by the expansion of CTG repeats in the 3’UTR of dystrophia myotonica protein kinase (DMPK) gene, which are transcribed and form intranuclear RNA foci and sequestrate proteins. The sequestration of Muscleblind-like (MBNL) into RNA foci disrupts its function in regulating target RNA processing, including alternative splicing and localization. Previous studies have made significant progress in understanding the mechanism of RNA mis-splicing in DM1. However, a major gap exists in our knowledge of how RNA localization is disrupted in DM1. To investigate how DM1 affects MBNL-regulated RNA localization in neurons, I developed a novel in vitro model of DM1 through exclusively expressing expanded CTG repeats in primary mouse cortical neurons using AAV9 virus with a neuronal-specific synapsin promoter. My model successfully reproduced DM1 features including RNA foci and MBNL sequestration in the nuclei of AAV-transduced neurons expressing CTG repeats. While no obvious mis-splicing events were observed, the localization of Snap25 mRNA in neuronal processes of transduced neurons was disrupted. I detected reduced dendritic arbor complexity in our DM1 model, which could be rescued by antisense oligonucleotides (ASO) treatment to degrade RNA foci and liberate MBNLs. Taken together, my results suggest that MBNL sequestration by RNA foci can impair distinct aspects of neuronal development. The mis-localization of MBNL target mRNAs like Snap25 may contribute to the early neurodevelopmental defects independent of RNA mis-splicing in DM1.

Table of Contents

Chapter 1. Introduction. 1

1.1 Myotonic Dystrophy. 2

1.2 Clinical presentation. 2

1.2.1 Subtypes of DM1 and clinical features. 2

1.2.2 Clinical features of classic DM1. 4

1.2.3 The CNS in DM1. 5

1.2.4 Clinical features of DM2. 6

1.3 Molecular mechanism.. 7

1.3.1 DMPK and CTG repeats. 7

1.3.2 Instability of CTG repeats 8

1.3.3 Molecular mechanism of DM2. 9

1.3.4 Accumulation of Intranuclear Ribonucleic acid (RNA) foci 9

1.3.5 Muscleblind-like (MBNL) proteins. 11

1.3.6 Intracellular function of MBNLs in RNA processing and localization. 13

1.3.7 Target RNA of MBNL: Snap25. 18

1.4 Research models for DM1. 23

1.4.1 In vitro model 23

1.4.2 In vivo model 27

1.4.3 Limitations of DM1 models. 29

1.5 Treatment for DM1. 30

1.5.1 Symptomatic treatments for DM1. 30

1.5.2 Antisense oligonucleotides (ASOs). 30

1.5.3 Other therapeutic strategies. 33

1.6 Dissertation rationale and objectives 34

1.6.1 Rationale. 34

1.6.2 Objectives. 36

1.6.3 Hypotheses. 37

1.7 Table and Figures 38

Chapter 2. Materials and Methods. 43

2.1 Acquire and culture primary cortical neurons 44

2.2 Magnetofection. 44

2.3 AAV9 construct and virus transduction. 45

2.4 Immunofluorescence (IF) 45

2.5 Fluorescent in situ hybridization (FISH) 46

2.6 FISH combined with IF. 47

2.6 ASO treatment 47

2.7 Microscopy and Imaging. 48

2.8 Statistics. 48

Chapter 3. Development of an In Vitro Model of DM1. 49

3.1 Establishment of novel in vitro neuronal DM1 model 50

3.1.1 AAV9 with human synapsin promoter can exclusively transduce primary cortical neurons 51

3.1.2 RNA foci formed in AAV9 transduced primary cortical neurons 52

3.1.3 Sequestration of MBNLs by RNA foci in DM1 model 53

3.2 RNA alternative splicing and localization in DM1 model 53

3.2.1 Alternative splicing is normal in DM1 model 54

3.2.2 Mis-localization of Snap25 in DM1 model 55

3.3 Morphological phenotype of DM1 model: reduced dendritic arbor complexity. 56

3.3.1 Dendritic defects in DM1 model 57

3.4 ASO treatment 58

3.4.1 ASO treatment eliminated RNA foci in DM1 model 58

3.4.2 ASO treatment increased dendritic outgrowth in DM1 model 59

3.5 Figures 61

Chapter 4. Conclusion and Discussion. 73

4.1 Summary. 74

4.2 Advantage of our in vitro model of DM1. 75

4.3 MBNL-regulated RNA localization in DM1. 76

4.4 ASO treatment 79

4.5 Future works 80

4.6 Figure. 82

Reference 83

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