The role of cilia transport in oligodendrocyte development and regulation of PP2A activity 公开

Umberger, Nicole Lynn (2014)

Permanent URL: https://etd.library.emory.edu/concern/etds/st74cq73g?locale=zh
Published

Abstract

Platelet derived growth factor AA/αα (PDGF-AA/αα) signaling is essential for development of oligodendrocyte progenitors (OLPs) into mature oligodendrocytes (OLs), the cells which produce myelin to insulate axons of neurons in the central nervous system. OLPs are derived from the precursor motor neuron (pMN) domain in the neural tube, which later becomes the spinal cord, and specification of the pMN domain requires cilia. Cilia are microtubule based organelles, and are required for regulation of sonic hedgehog (Shh) signaling activity, which specifies the pMN domain. Arl13bhennin (Arl13bhnn) mouse embryos have short cilia, disrupted Shh activity, and, despite an expanded pMN domain, do not specify OLPs before embryos die during midgestation. Based upon this observation and previous connections of PDGF-AA/αα signaling to cilia, I asked if Arl13b and cilia are required for OLP development.

In this dissertation, I examine the role of Arl13b and of the cilia transport protein Ift88 in OLP development in vivo, and show that Ar13lb is not required for OLP specification or development, and that Ift88 is not required for postnatal OL development. To further dissect the role of Arl13b and cilia in PDGF-AA/αα signaling, I used an in vitro system and analyzed response to PDGF-AA stimulation in several cilia mutant cell lines. These experiments demonstrate that inhibited response to PDGF-AA stimulation in cilia mutant MEFs is due to up-regulation of mTORC1 signaling, and identify a novel role for PP2A in cilia signaling in vertebrates. Combined, my in vivo and in vitro results provide a more comprehensive understanding of the role of cilia in PDGF-AA/αα signaling.

Table of Contents

CHAPTER 1: Cilia, signaling, and oligodendrocyte development 1

1.1 Introduction 2

1.2 Cilia 2

Structure 3

Intraflagellar transport 4

1.3 IFT and cilia transport mutants 5

IFT172 5

IFT122 6

DYNC2H1 7

ARL13B 7

1.4 Sonic hedgehog 8

Signaling 8

Sonic hedgehog and cilia 10

Neural tube patterning 11

IFT, Shh, and neural tube patterning 12

1.5 Neural stem cell debate 14

Neural stem cell debate 14

1.6 PDGF-AA/αα signaling through PI3K/AKT and mTORC1 16

PDGFs and PDGFRs 16

Phosphatidylinositols and Phosphoinositide 3-kinase 17

AKT 18

mTORC1 19

PDGFAA/αα, and mTORC1 signaling in the context of cilia 21

PDGFAA/αα 21

mTORC1 21

1.7 Oligodendrocytes 23

Oligodendrocyte progenitor specification 23

Oligodendrocyte progenitor maturation 24

PDGF-AA/αα signaling in OLPs 25

Myelination 26

PDGF-AA/αα, cilia, and oligodendrocytes 26

1.8 Introduction to PP2A 27

Structure and regulation of PP2A 27

PI3K/AKT/mTOR signaling 28

Hh/Shh signaling 29

PP2A and cilia 30

1.9 Outstanding questions and preview 30

CHAPTER 2: MATERIALS AND METHODS 32

Brain tissue lysis 33

MEF lysis 33

Protein quantification 34

SDS PAGE and Western blotting analysis 35

Embryo dissections 36

Perfusion 37

Sectioning 37

Immunofluorescence 38

Mouse strains and genotyping 38

MEF preparation 41

PDGF-AA, LY294002, rapamycin, okadaic acid, and FTY720 treatments 42

PP2A activity assay 43

Chapter 3: The role of Arl13b and Ift88 in oligodendrocyte development in vivo 46

3.1 Summary 47

3.2 Introduction 47

3.3 Results 49

3.3.1 Generation of Arl13bΔOlig1-Cre mice, birth incidence, and post-natal weight 49

3.3.2 Deletion of Arl13b in Arl13bΔOlig1-Cre embryos 50

3.3.3 Arl13bΔOlig1-Cre embryos and pups fail to display phenotypes indicative of disrupted OL development 51

3.3.4 Repopulation of the pMN domain with Arl13b expressing Olig2+ progenitors (in Arl13bΔOlig1-Cre embryos at e12.5) 52

3.3.5 Generation and characterization of Ift88ΔOlig1-Cre mice 52

3.3.6 Rational for Nestin-Cre54

3.3.7 Generation of Arl13bΔNestin1-Cre mice and turnover of Arl13b in the neural tube 54

3.3.8 Arl13bΔNestin1-Cre mice do not display defects in prenatal OL development or early postnatal myelination 55

3.3.9 Arl13bΔNestin1-Cre mice do not display defects in postnatal myelination and develop cystic kidneys 56

3.3.10 Generation and characterization of Ift88ΔNestin1-Cre mice 56

3.4 Discussion 57

3.4.1 Benefits and disadvantages of Olig1-Cre and Nestin-Cre58

Table 3.1 58

3.4.2 Arl13b and Ift88 are not essential for OL development in vivo59

3.4.3 Support for a sequential progenitor model from Arl13bΔOlig1-Cre embryos 60

Chapter 4: Cilia transport regulates PDGF-AA/αα signaling via elevated mTOR SIGNALING AND DIMINISHED PP2A activity 62

4.1 Introduction 63

4.2 Results 65

4.2.1 PP2Ac localizes to the basal body of MEFs 65

4.2.2 P-AktT308 is Increased in Cilia Transport Mutant MEFs 66

4.2.3 PP2A Function is Disrupted in Cilia Transport Mutant MEFs 67

4.2.4 Total PP2A Activity is Similar in WT and Cilia Transport Mutant MEFs 69

4.2.5 mTORC1 Pathway Activity is Increased in Cilia Transport Mutant MEFs 69

4.2.6 Abnormal Response to PDGF Signaling in Cilia Transport Mutant MEFs 70

4.2.7 Rapamycin Treatment Restores PDGFRαα Levels and Response to PDGF-AA Stimulation 71

4.3 Discussion 73

Chapter 5: Perspectives77

5.1 Arl13b and Ift88 in oligodendrocyte development 78

5.2 PP2A and cilia trafficking 79

5.3 PDGF-AA/αα, Akt, and mTORC1 82

5.4 Final summary 84

FIGURES AND TABLES

Figure 1.2.1 Ciliary structure 85

Figure 1.2.2 IFT 86

Figure 1.4 Neural tube patterning 87

Figure 1.5 Mixed progenitor and sequential models of pMN domain specification 89

Figure 1.6 PDGF ligands and receptors 91

Figure 1.7.1 Stages of oligodendrocyte development 93

Figure 1.7.2 Myelination 95

Figure 1.8 PP2A 96

Figure 3.1 Arl13bΔOlig1-Cre observed vs expected, survival, postnatal weight 97

Figure 3.2 e10.5 neural tube of Arl13bΔOlig1-Cre embryos 100

Figure 3.3 MBP expression from p11 optic nerve of Arl13bΔOlig1-Cre pups 101

Figure 3.4 e16.5 neural tube of Arl13bΔOlig1-Cre embryos 102

Figure 3.5 e15.5 neural tube of Arl13bΔOlig1-Cre embryos 103

Figure 3.6 Ift88ΔOlig1-Cre observed vs expected 104

Figure 3.7 e12.5 neural tube of Ift88ΔOlig1-Cre embryos 106

Figure 3.8 Observed vs expected for Arl13bΔNestin-Cre and Ift88ΔNestin-Cre 107

Figure 3.9 e12.5 neural tube of Arl13bΔNestin-Cre embryos 108

Figure 3.10 e14.5 neural tube of Arl13bΔNestin-Cre embryos 109

Figure 3.11 e14.5 whole mount of Arl13bΔNestin-Cre embryos 110

Figure 3.12 PDGFRαα and Olig2 expression from p7 optic nerves of Arl13bΔNestin-Cre pups 111

Figure 3.13 e18.5 neural tube and Olig2, PDGFRα, and CNP expression from optic nerves of Ift88ΔNestin-Cre pups 112

Figure 4.1 PP2Ac localization and P-AktT308 levels in WT and cilia transport mutant MEFs 113

Figure 4.2 PP2A activity assay and un-methylated and methylated PP2Ac 115

Figure 4.3 mTORC1 signaling in WT and cilia transport mutant MEFs 116

Figure 4.4 Response to PDGF-AA stimulation with and without rapamycin 117

Figure S1 P-AktT308 localization in WT and cilia transport mutant MEFs 119

Figure S2 P-AktT308 levels of Dync2h1lln MEFs grown in 0.5% serum 120

Figure S3 Ift172wim MEFs do not form cilia during rapamycin treatment 122

Figure 5.3 PDGF-AA/αα, Akt, mTORC1, and PP2A interactions 123

REFERENCES 125

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