Molecular Pathogenesis of Charcot-Marie-Tooth disease caused by mutations in SIMPLE Open Access

Abstract

Mutations in SIMPLE cause autosomal dominant, Charcot-Marie-Tooth disease (CMT) type 1C. The cellular function of SIMPLE is unknown and the pathogenic mechanism of SIMPLE mutations remains elusive. Results described in this dissertation show that SIMPLE is highly expressed in the peripheral nerves and Schwann cells. Our analysis has identified a transmembrane domain (TMD) embedded within the cysteine-rich (C-rich) region for anchoring SIMPLE to the membrane. We found that SIMPLE associates with endosomal sorting complex required for transport (ESCRT) components STAM1, Hrs, and TSG101 on early endosomes and functions with the ESCRT machinery in the control of endosome-to-lysosome trafficking. Our analyses reveal that SIMPLE is required for efficient recruitment of these ESCRT components to endosomal membranes and for regulating endosomal trafficking and signaling attenuation of ErbB receptors.

Results described in this dissertation show that CMT1C-linked pathogenic mutations are clustered within or around the TMD of SIMPLE, and these mutations cause mislocalization of SIMPLE from the early endosome membrane to the cytosol. The CMT1C-associated SIMPLE mutant proteins are unstable and prone to aggregation, and they are selectively degraded by both the proteasome and aggresome-autophagy pathways. We find that the ability of SIMPLE to regulate ErbB trafficking and signaling is impaired by these SIMPLE mutations, resulting in the prolonged activation of ERK1/2 signaling. We also examined the effects of a CMT1C-linked mutation in vivo by generating transgenic mice expressing a CMT1C-linked human SIMPLE mutant protein. Mice expressing mutant SIMPLE develop a progressive motor and sensory neuropathy that recapitulates some of the key clinical features of CMT1C disease, including behavioral impairments, nerve conduction defects, and myelin pathology. Together, our findings support that SIMPLE mutation disrupts myelin homeostasis and causes progressive peripheral neuropathy via a combination of loss-of-function, toxic gain-of-function and dominant-negative mechanisms.Furthermore, our results suggest that dysregulation of endocytic trafficking and receptor signaling could contribute to the pathogenesis of demyelinating CMT1C neuropathy.

Table of Contents

Table of Contents

Chapter 1. Introduction and background

1.1. Opening remarks………………………………………………………………… 2

1.2. Clinical features of Charcot-Marie-Tooth disease………………………...…… 2

1.3. Demyelinating types of Charcot-Marie-Tooth disease………………………… 3

1.3.1. Peripheral Myelin Protein 22, PMP22 (CMT1A) ………………………. 4

1.3.2. Myelin Protein Zero, MPZ/P0 (CMT1B)……………………………….. 7

1.3.3. Small Integral Membrane Protein of Lysosome/Late Endosome,

SIMPLE (CMT1C)…………………………………………………………….. 9

1.3.4. Early Growth Response 2, EGR2/Krox20 (CMT1D and CMT4E)……... 12

1.3.5. Connexin 32, Cx32/GJB1 (CMT1X)……………………………………. 13

1.3.6. Ganglioside-induced Differentiation Associated-Protein, GDAP1

(CMT4A)……………………………………………………………………….14

1.3.7. Myotubularin-related protein 2 and 13, MTMR2 and MTMR13

(CMT4B1 and CMT4B2)……………………………………….……………... 15

1.3.8. SH3 domain and Tetratricopeptide repeats-Containing protein 2,

SH3TC2 (CMT4C) ……………………………………………………………. 17

1.3.9. N-myc Downstream Regulated 1, NDRG1 (CMT4D)………………….. 18

1.3.10. Periaxin, PRX (CMT4F)……………………………………………….. 19

1.3.10. FYVE, RhoGEF, and PH-domain containing protein 4, FRABIN

(CMT4H)………………………………………………………………………. 19

1.3.12. Polyphosphoinositide phosphatase, FIG4 (CMT4J)…………………... 20

1.4. Are different forms of demyelinating CMT caused by common

mechanisms? …………………………………………………………………………. 21

1.4.1. Accumulation of toxic misfolded proteins and aggregates……………… 21

1.4.2. Dysfunction in receptor trafficking and/or intracellular signaling……… 22

1.4.3. Disruption of myelin structures and axon-Schwann cell interactions…... 23

1.5. Non-demyelinating types of Charcot-Marie-Tooth disease…………………… 24

1.6. Treatment for Charcot-Marie-Tooth disease………………………………….. 26

1.7. Protein misfolding and protein quality control systems………………………. 27

1.7.1. Toxicity mediated by misfolded proteins and aggregates………………. 27

1.7.2. Ubiquitin signaling……………………………………………………… 28

1.7.3. Ubiquitin-proteasome system…………………………………………… 29

1.7.4. ER-associated degradation (ERAD)……………………………………. 30

1.7.5. Aggresome-autophagy pathway………………………………………… 31

1.8. Regulation of endocytic trafficking……………………………………………... 32

1.9. Cellular mechanisms regulating axon-Schwann cell communication………… 34

1.9.1. NRG1-ErbB receptor signaling pathway determ ines myelin sheath

thickness……………………………………………………………………….. 35

1.9.2. The effects of myelinating Schwann cells on axon caliber……………… 36

1.10. Hypotheses and organizational overview……………………………………… 37

Chapter 2. Mutations associated with Charcot-Marie-Tooth disease cause SIMPLE protein mislocalization and degradation by proteasome and aggresome-autophagy pathways

2.1. Abstract…………………………………………………………………………… 52

2.2. Introduction……………………………………………………………………… 52

2.3. Materials and methods…………………………………………………………... 54

2.3.1. Plasmids and antibodies…………………………………………………. 54

2.3.2. Cell cultures and transfections………………………………………….. 55

2.3.3. GST-tagged protein purification………………………………………… 56

2.3.4. Immunofluorescence confocal microscopy and quantification of

endosomal localization………………………………………………………… 56

2.3.5. Subcellular fractionations and membrane association analysis…………. 56

2.3.6. [³⁵S]Methionine pulse-chase analysis…………………………………… 57

2.3.7. Chemical cross-linking analysis………………………………………… 58

2.3.8. Detergent insolubility assays……………………………………………. 58

2.3.9. Analysis of aggresome formation………………………………………. 58

2.3.10. Treatment of cells with proteasome, lysosome, autophagy inhibitors

and activators………………………………………………………………….. 59

2.3.11. Statistical analysis……………………………………………………… 59

2.4. Results……………………………………………………………………………. 59

2.4.1. SIMPLE protein is highly expressed in peripheral nerves and in

Schwann cells………………………………………………………………….. 59

2.4.2. SIMPLE is a C-tailed-anchored integral membrane protein……………. 61

2.4.3. Endogenous SIMPLE is localized to early endosome but not late

endosome and lysosome……………………………………………………….. 64

2.4.4. CMT1C-associated mutations cause mislocalization of SIMPLE from

the early endosomal membrane to the cytosol…………………………………. 65

2.4.5. CMT1C-associated mutations cause SIMPLE protein to be unstable….. 68

2.4.6. CMT1C-associated SIMPLE mutant proteins are prone to

aggregation…………………………………………………………………….. 69

2.4.7. CMT1C-associated mutations promote the formation of SIMPLE-

positive aggresomes …………………………………………………………… 70

2.4.8. CMT1C-associated SIMPLE mutant proteins are degraded by both

the proteasome and autophagy pathways……………………………………… 72

2.5. Discussion…………………………………………………………………………73

2.6. Acknowledgements……………………………………………………………….78

Chapter 3. Charcot-Marie-Tooth disease-associated protein SIMPLE functions with the ESCRT machinery to regulate endosomal trafficking

3.1. Abstract…………………………………………………………………………… 102

3.2. Introduction……………………………………………………………………… 102

3.3. Materials and methods………………………………………………………….. 105

3.3.1. Plasmids and antibodies…………………………………………………. 105

3.3.2. Yeast two-hybrid screens……………………………………………….. 106

3.3.3. Recombinant protein purification, in vitro binding assays, and GST

pull-down assays………………………………………………………………. 106

3.3.4. In vitro ubiquitination assays……………………………………………. 107

3.3.5. Cell transfections and immunoprecipitation…………………………….. 107

3.3.6. Subcellular fractionation………………………………………………… 108

3.3.7. Immunofluorescence confocal microscopy and quantification of

colocalization, endosome size and dispersion…………………………………. 108

3.3.8. EGF endocytic trafficking assays……………………………………….. 109

3.3.9. EGFR and ErbB3 degradation assays…………………………………… 110

3.3.10. SIMPLE tyrosine phosphorylation assays…………………………….. 111

3.3.11. ERK1/2 phosphorylation assays…………….………………………… 111

3.3.12. Statistical analysis…………………….……………………………….. 111

3.4. Results……………………………………………………………………………. 112

3.4.1. SIMPLE interacts and colocalizes with ESCRT-0 and ESCRT-I

subunits on early endosomes………………………………………………….. 112

3.4.2. SIMPLE does not function as an E3 ubiquitin-protein ligase…………… 113

3.4.3. SIMPLE functions in the regulation of endosome-to-lysosome

trafficking and signaling attenuation ………………………………………….. 114

3.4.4. SIMPLE promotes recruitment of STAM1, Hrs, and TSG101 to

membranes……………………………………………………………………...116

3.4.5. SIMPLE interaction with TSG101 is required for endosome-to-lysosome

trafficking………………………………………………………………………. 117

3.4.6. CMT1C-linked SIMPLE mutants impair endosomal trafficking via a

loss-of-function and dominant-negative mechanism…………………………... 119

3.4.7. CMT1C-linked SIMPLE mutants cause dysregulation of NRG1-ErbB

signaling in Schwann cells…………………………………………………….. 121

3.5. Discussion…………………………………………………………………………122

3.6. Acknowledgements……………………………………………………………… 127

Chapter 4. Motor and sensory neuropathy due to myelin infolding and paranodal damage in a transgenic mouse model of Charcot-Marie-Tooth disease type 1C

4.1. Abstract……………………………………………………………………………157

4.2. Introduction……………………………………………………………………… 158

4.3. Materials and methods………………………………………………………….. 159

4.3.1. Generation and genotyping of SIMPLE WT and SIMPLE W116G

transgenic mice………………………………………………………………… 159

4.3.2. Antibodies………………………………………………………………. 160

4.3.3. Teased nerve fibers, Nile red staining, and immunofluorescence confocal

microscopy…………………………………………………………………….. 161

4.3.4. Schwann cell culture and Western blot analysis………………………… 161

4.3.5. Behavioral tests…………………………………………………………. 162

4.3.6. Electrophysiology………………………………………………………. 162

4.3.7. Histological analysis and electron microscopy…………………………. 163

4.3.8. Morphometric analyses…………………………………………………. 163

4.3.9. Statistical analysis…………………….………………………………… 164

4.4. Results……………………………………………………………………………. 165

4.4.1. Generation of transgenic mice expressing human SIMPLE WT or

W116G mutant………………………………………………………………… 165

4.4.2. SIMPLE is localized to early endosomes in myelinating Schwann cells

but is absent in myelin sheath or axons……………………………………….. 166

4.4.3. SIMPLE W116G mutant mice, but not SIMPLE WT transgenic

mice, exhibit motor and sensory impairments ………………………………… 167

4.4.4. SIMPLE W116G mutant mice, but not SIMPLE WT transgenic mice,

have motor and sensory nerve conduction defects……………………………. 169

4.4.5. SIMPLE W116G mutation causes peripheral nerve dysmyelination with

myelin infolding and reduced axon caliber……………………………………. 170

4.4.6. Myelin infoldings originate from the paranodal regions and near

Schmidt-Lanterman incisures…………………………………………………. 172

4.4.7. SIMPLE W116G mutation disrupts the integrity of Schwann cell-axon

units and nodes of Ranvier…………………………………………………….. 173

4.5. Discussion…………………………………………………………………………175

4.6. Acknowledgements……………………………………………………………… 180

Chapter 5. Summary of findings, discussions, and future directions

5.1. Summary of findings…………………………………………………………….. 201

5.2. The cell type distribution and subcellular localization of SIMPLE suggests a

role in regulating Schwann cell myelination………………….….…………………. 202

5.3. SIMPLE recruits ESCRT-0 and ESCRT-I to the endosomal membrane and is

required for efficient endosome-to-lysosome trafficking………………………….. 204

5.4. Protein misfolding as a mechanism in CMT pathogenesis: therapeutic

implications ……………………………………………………………………………206

5.4.1. Protein misfolding is a common feature in demyelinating CMT.………. 206

5.4.2. The proteasome and aggresome-autophagy pathways protect Schwann

cells against toxic build-up of misfolded proteins …………………………….. 207

5.4.3. Impairment in the proteasome and aggresome-autophagy pathways

contributes to peripheral neuropathies………………………………………… 208

5.4.4. Proteasome and aggresome-autophagy pathways are potential therapeutic

targets in CMT…………………………………………………………………209

5.5. Dysregulation of axonal NRG1-activated Schwann cell ErbB receptor signaling

is involved in CMT pathogenesis: therapeutic implications……………………….. 210

5.5.1. Dysregulation of NRG1-ErbB receptor trafficking and signaling

contribute to the pathogenesis of demyelinating CMT………………………… 210

5.5.2. Modulation of NRG1-ErbB receptor trafficking and signaling as possible

strategies for treating CMT……………………………………………………. 212

5.6. Schwann cell dysfunction affects nodal gap width, disrupt axonal transport,

and cause axonal degeneration in demyelinating CMT disease…………………... 214

5.7. Focally folded myelin and increased nodal gap length are pathological

findings of age-dependent demyelinating CMT mouse models and patients……... 215

5.8. Future directions………………………………………………………………… 217

5.8.1. Regulation of myelin protein synthesis and trafficking by SIMPLE.…… 217

5.8.2. Identification of the E3 ligase(s) and adapter protein(s) responsible for

targeting misfolded SIMPLE to the proteasome and aggresome-autophagy

pathways for degradation …………………………………………………….. 218

5.8.3. Ubiquitination by NEDD4 may regulate the endocytic function of

SIMPLE ………………………………………………………………………219

5.8.4. SIMPLE W116G transgenic mice as a CMT1C mouse model for

studying pharmacological interventions……………………………………….. 220

5.8.5. Generation of Schwann cell-specific disease-linked mutant SIMPLE

transgenic mice ………………………………………………………………... 220

5.8.6. Examination of peripheral nerve biopsies from CMT1C patients……… 221

5.9. Final words……………………………………………………………………….. 221

5.10. References……………………………………………………………………… 226

List of Figures

Chapter 1. Introduction and background

Figure 1.Domain structure of SIMPLE………………………………………….. 44

Figure 2.Ubiquitin-mediated signal transduction: signal generation, recognition,

and transmission………………………...……………………………… 45

Figure 3. Endocytic trafficking of cargo proteins………………………………... 47

Figure 4.Diverse roles of ubiquitin in regulating multivesicular body (MVB)

sorting of cargo proteins……………………………………………….. 49

Chapter 2. Mutations associated with Charcot-Marie-Tooth disease cause SIMPLE protein mislocalization and degradation by proteasome and aggresome-autophagy pathways

Fig. 1.SIMPLE is widely expressed in multiple tissues and is highly expressed

in the peripheral nerves and Schwann cells………………………...….. 79

Fig. 2. SIMPLE is a transmembrane protein that requires its cysteine-rich

(C-rich) domain for membrane association………………………...….. 80

Fig. 3.Endogenous SIMPLE is localized to the early endosome but not to

other organelles………………………………………………………… 82

Fig. 4. CMT1C-associated SIMPLE mutants are mislocalized from the early

endosomal membrane to the cytosol…………………………………… 84

Fig. 5. CMT1C-associated mutations reduce the stability of SIMPLE protein in

cells…………………………………………………………………… 85

Fig. 6. CMT1C-associated mutations promote aggregation of SIMPLE……… 87

Fig. 7. CMT1C-associated SIMPLE mutants are accumulated in

aggresomes…………………………………………………………….. 89

Fig. 8. Clearance of CMT1C-associated mutant SIMPLE proteins by both the

proteasome and autophagy pathways………………………………….. 91

Fig. S1.Generation and characterization of a highly specific antibody for

detecting SIMPLE protein……………………………………………... 92

Fig. S2.Comparison of the extraction profiles of membrane-associated

SIMPLE and Thy-1.……………………………………………………. 93

Fig. S3.Effects of Rab5-Q97L mutant expression and chloroquine treatment

on subcellular localization of endogenous SIMPLE…………………… 94

Fig. S4. SIMPLE mutant proteins are not localized to the ER or subjected to

p97/VCP-dependent ERAD…………………………………………… 95

Fig. S5. Mutant SIMPLE-positive aggresomes are sites of autophagy…………. 97

Fig. S6.Formation of mutant SIMPLE-positive aggresomes is microtubule-

dependent………………………………………………………………. 99

Fig. S7. Rapamycin promotes degradation of SIMPLE mutant proteins but not

SIMPLE WT protein…………………………………………………… 100

Chapter 3. Charcot-Marie-Tooth disease-associated protein SIMPLE functions with the ESCRT machinery to regulate endosomal trafficking

Figure 1.SIMPLE associates and colocalizes with STAM1 and Hrs……………. 129

Figure 2.SIMPLE depletion alters endosomal morphology and EGFR endosomal

sorting and signaling…………………………………………………… 131

Figure 3.SIMPLE is required for efficient association of STAM1, Hrs and TSG101

with membranes……………………………………………………….. 134

Figure 4. SIMPLE interaction with TSG101 is required for ligand-induced EGFR

degradation and EGF endosome-to-lysosome trafficking……………... 136

Figure 5. CMT1C-linked mutations cause a loss of SIMPLE function in

facilitating EGFR degradation and EGF endosome-to-lysosome

trafficking………………………………………………………………. 138

Figure 6. CMT1C-linked SIMPLE mutants have dominant-negative effects on

EGFR degradation and EGF endosome-to-lysosome trafficking……… 140

Figure 7. CMT1C-linked SIMPLE mutants interact with SIMPLE WT, STAM1 and

TSG101………………………………………………………………… 142

Figure 8. CMT1C-linked SIMPLE mutants inhibit the association of STAM1, Hrs,

and TSG101 with membranes ………………………………………… 144

Figure 9. CMT1C-linked SIMPLE mutants have dominant-negative effects on

NRG1-ErbB signaling in Schwann cells ……………………………… 145

Figure S1.SIMPLE does not interact directly with Hrs and does not regulate the

stability of STAM1 and Hrs…………..…………………………....….. 147

Figure S2. SIMPLE associates with STAM1 and TSG101 on the early

endosomes……………………………………………………………... 148

Figure S3.SIMPLE has no E3 ligase activity and does not interact with E2 enzymes

UbcH5, UbcH7, and UbcH8.…………………………………………... 150

Figure S4.Hrs and SIMPLE are not functionally redundant in mediating EGFR

degradation…………………………………………………………….. 152

Figure S5. Analysis of endogenous and exogenous SIMPLE protein expression in

stably transfected HeLa and MSC80 cells……………………………... 154

Chapter 4. Motor and sensory neuropathy due to myelin infolding and paranodal damage in a transgenic mouse model of Charcot-Marie-Tooth disease type 1C

Figure 1.Generation of SIMPLE WT and SIMPLE W116G transgenic mice…... 181

Figure 2.Endogenous SIMPLE is localized to early endosomes in non-compact

myelin cytoplasmic regions of myelinating Schwann cells but is absent in

myelin sheath or axons…………..………………………………....….. 183

Figure 3. SIMPLE^W116G/W116G mice show no abnormality in myelinated structures in

the brain and spinal cord………………………………………………. 184

Figure 4.Impaired motor and sensory performance in SIMPLE W116G mutant

mice.…………………………………………………………………….186

Figure 5.SIMPLE WT mice show normal motor and sensory performance…….. 188

Figure 6. Motor nerve conduction defects in SIMPLE W116G mutant mice…… 189

Figure 7. Sensory nerve conduction defects in SIMPLE W116G mutant mice….. 190

Figure 8. Normal motor and sensory nerve conduction in SIMPLE WT transgenic

mice…...……………………………………………………………….191

Figure 9. Cross-section analysis of 3-month-old SIMPLE^W116G/W116G mice does not

show obvious abnormality in the peripheral nerves…………………… 192

Figure 10. Histological analysis reveals myelin infoldings in 1-year-old SIMPLE

W116G mutant mice…………………………………………………… 193

Figure 11. Electron microscopic analysis of myelin abnormalities and axonal

degeneration in SIMPLE W116G mutant mice……………………….. 194

Figure 12. Quantitative analyses of myelin infoldings, axon morphometry, myelin

thickness, G-ratio, axon degeneration, and demyelination……………. 196

Figure 13. Paranodal myelin infoldings and widened nodes of Ranvier in SIMPLE

W116G mutant mice………………………………………………….. 198

Chapter 5. Summary of findings, discussions, and future directions

Figure 1.Protein quality control systems are potential targets for mechanism-based

treatments of demyelinating CMT……………………………………... 224

List of Tables

Chapter 1. Introduction and background

Figure 1.Identified genes and proteins associated with demyelinating CMT…… 42

Figure 2.Identified genes and proteins associated with axonal CMT…………… 43

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience
Keyword	SIMPLE endosomal trafficking CMT1C protein misfolding peripheral neuropathy Charcot-Marie-Tooth disease
Committee Chair / Thesis Advisor	Li, Lian, Emory University Chin, Lih-Shen, Emory University
Committee Members	Wichmann, Thomas, Emory University Feng, Yue, Emory University Faundez, Victor, Emory University Smith, Yoland, Emory University Glass, Jonathan D, Emory University

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