Cell-type-specific profiling of defects in translation and neurogenesis in a human induced pluripotent stem cell model of Fragile X syndrome Open Access

Raj, Nisha (Spring 2019)

Permanent URL: https://etd.library.emory.edu/concern/etds/qr46r187b?locale=en


Aberrant translation and disrupted signaling are molecular defects common to several neurodevelopmental disorders, including Fragile X syndrome (FXS), the most common monogenic cause of autism and inherited intellectual disability. Studies in animal models of FXS have shown that in the absence of the RNA-binding protein FMRP, there is an increase in global translation and dysregulation of key signaling pathways. However, the molecular pathogenesis of FXS in humans has remained understudied. Here we use induced pluripotent stem cell (iPSCs)-derived neural progenitor cells (NPCs) and differentiating neurons from multiple control and FXS patients to characterize molecular defects in a human disease-relevant model. We found that a subset of FXS patient derived NPCs show increased protein synthesis, increased proliferation and altered differentiation profiles. FXS patient derived cerebral organoids also showed an increase in KI67+/SOX2+ proliferating cells. Furthermore, genome-wide splicing analysis of patient cerebral organoids revealed alternative splicing of a key cell-cycle regulated kinase in FXS. We developed a multi-parametric flow cytometry based assay, NeuroMIP, to quantify protein synthesis and proliferation within specific neural subpopulations in our patient cells. Our results suggest that increased proliferation is a core molecular phenotype in FXS, and defects in protein synthesis may contribute to altered proliferation. The loss of FMRP also affects the differentiation profile and timing of neural progenitor cell subtypes during development, and we see increased protein synthesis in specific neural subtypes in FXS patient cells. We anticipate that this study using a human disease-relevant cellular model of FXS will provide new insight into molecular and cellular phenotypes in FXS, as well as the future development of therapeutic strategies. Our results provide strong support for an approach that involves patient stratification based on cellular and molecular phenotypes, enabling pre-clinical testing of treatments in a more tailored and precise manner.

Table of Contents

CHAPTER 1: General Introduction. 1

1.1. Fragile X syndrome. 2

1.1.1. The Fragile X phenotype. 2

1.1.2. FMR1. 4

1.1.3. Fragile X mental retardation protein (FMRP) 6

1.2. Animal models of FXS. 7

1.2.1. Insight from the FXS drosophila model 8

1.2.2. Insight from the Fmr1-knockout mouse. 8

1.3. Preclinical rationale for developing targeted therapies interventions in FXS. 11

1.3.1. mGluR theory of fragile X syndrome. 11

1.3.2. Altered signaling via membrane receptors in FXS. 13

1.3.3. Altered intracellular signaling in FXS. 14

1.3.4. Development of clinical trials in FXS. 15

1.4. Induced pluripotent stem cells (iPSCs) as a tool to model neurological disorders. 16

1.5. Dissertation rationale and objectives. 17

CHAPTER 2: Materials & Methods. 22

CHAPTER 3: Development of a human cellular model of Fragile X syndrome. 33

3.1. Introduction. 34

3.1.1. Modeling FMR1 silencing and CGG repeat expansion in FXS hPSCs. 34

3.1.2. Modeling cellular and molecular deficits in FXS hPSCs. 35

3.1.3. Chapter 3 rationale and objectives. 37

3.2. Results. 38

3.2.1. Generation of induced pluripotent stem cells (iPSCs) from human dermal fibroblasts. 39

3.2.2. Differentiation of iPSCs into neural precursor cells (NPCs), neurons and cerebral organoids 39

3.3. Discussion. 41

3.4. Figures and Tables. 44

CHAPTER 4: Dysregulated global translation in Fragile X patient derived cells. 51

4.1. Introduction. 52

4.1.1   Overview of translation. 52

4.1.2   Defects in signaling and translation in neurological disorders. 54

4.1.3   FMRP as a translational regulator 54

4.1.4   Techniques to measure protein synthesis. 55

4.1.5. Chapter 4 rationale and objectives. 56

4.2. Results. 57

4.2.1. BONCAT and SUnSET show increased global translation in FXS patient cells. 57

4.2.2. Evidence of increased PI3K signaling in FXS. 59

4.2.3. Increased translation in FXS patient NPCs may be PI3K signaling-dependent 59

4.3. Discussion. 60

4.4. Figures. 62

CHAPTER 5: Altered cell fate and proliferation profiles in FXS patient cells. 69

5.1. Introduction. 70

5.1.1. An overview of cortical neurogenesis. 70

5.1.2. Altered cell fate and proliferation in neurodevelopmental disorders. 71

5.1.3. FMRP as a regulator of neurogenesis. 71

5.1.4. Chapter 5 rationale and objectives. 73

5.2. Results. 75

5.2.1.  Development of a multiparametric assay to measure cell-type specific phenotypes. 75

5.2.2. Increased proliferation in FXS patient NPCs and cerebral organoids. 76

5.2.3. Altered cell fate in FXS. 77

5.2.4. Cell-type specific translational dysregulation in FXS patient NPCs. 78

5.2.5. Processes regulating neuronal fate commitment and proliferation are disrupted in FXS patient-derived cerebral organoids 78

5.2.6. Novel splicing defect in FXS patient cerebral organoids. 79

5.3. Discussion. 79

5.4. Figures & Tables. 81

CHAPTER 6: General Discussion. 93

6.1. Summary. 94

6.2. Induced pluripotent stem cell models of disease. 94

6.3. The ever-expanding functions of FMRP. 96

6.4. Lost in Translation: cell fate and protein synthesis. 98

6.5. Finding a “cure” for fragile X syndrome. 99

6.6. Future directions and concluding remarks. 101

6.7. Figures. 103

References 105

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