Direct reprogramming of astrocytes to enhance recovery after stroke Open Access

Jiang, Qize (Fall 2017)

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Ischemic stroke is a leading cause of death and long term disability in the United States. Ischemic stroke results in death of neurons in the affected area with limited capacity for regeneration in the adult brain. Recent advances in stem cell techniques provide the possibility of ameliorating ischemic damage by replacing lost neurons with transplanted neuronal precursor cells that can terminally differentiate into mature neurons and integrate with the host circuitry. The focus of many transplantation studies currently centers on neuronal differentiation and transplantation of ES or iPS cells into the ischemic brain. More recently, studies demonstrate the panneuronal transcription factor NeuroD1 (ND1) can reprogram astrocytes directly into neurons, a process called direct reprogramming. Lentiviral vector delivery of ND1 to astrocytes results in permanently reprogrammed neurons without the need for maintained ectopic expression of the introduced transcription factor.

Astrocytes primarily provide support to surrounding neurons but also proliferate reactively in response to pathologies including ischemic stroke. Reactive astrocytes proliferate and hypertrophy in response to ischemic stroke and form a border around the site of injury forming a glial scar. Without intervention, there are an abundance of these reactive astrocytes in the peri-infarct region around the injury. Intra-lineage direct reprogramming provides an endogenous source of new neurons from existing proliferative astrocytes and has immense potential to reduce the burden of stroke. 

Compared to traditional stem cell transplantation approaches, converted neurons derived from endogenous astrocytes will have the advantage of already being “settled” in a microenvironment that is more conducive to synaptogenesis and survival. Effective reprogramming of astrocytes to neurons further acts to “melt” the glial scar which normally exerts an inhibitory barrier for synaptogenesis and axonogenesis. Finally, new neurons are autologous and post-mitotic eliminating risk associated with rejection or tumor formation. Direct reprogramming of astrocytes has not yet been explored as a therapeutic tool in a model of ischemic stroke. The following work aims to use this novel approach to directly reprogram proliferative astrocytes into neurons in vivo following ischemic stroke and to augment activity dependent repair using whisker stimulation resulting in enhanced functional recovery in mice. We hypothesize direct conversion of astrocytes to neurons in the peri-infarct area will improve functional recovery in a mouse model of ischemic stroke that can be enhanced with whisker stimulation. The following study utilizes a novel approach to cell replacement in a model of ischemic brain injury and evaluates the efficacy of intra-lineage direct reprogramming of astrocytes into neurons in vivo. Direct reprogramming with activity dependent repair may improve functional recovery and reduce the morbidity of this common but devastating disease.

Table of Contents

Chapter One: Ischemic Stroke. 1

1.1        Stroke. 1

Definition and types of stroke. 1

Stroke Treatment. 2

1.3 Pathophysiology of ischemic stroke. 4

Ischemic Core. 4

Peri-infarction.. 5

1.4 Mechanisms and features of cell death and survival 5

Apoptosis. 8

Necrosis. 8

Autophagy. 9

Hybrid cell death.. 10

Global ischemia. 14

Focal ischemia. 15

Chapter Two: Direct Reprogramming. 18

2.1 Direct Reprogramming. 18

2.2 Comparison with current cell therapy approaches: Hematopoietic stem cells 23

The mammalian hematopoietic system... 23

History of hematopoietic research.. 23

Hematopoietic stem cells and stroke. 24

Hematopoietic stem cell transplantation.. 26

The advantage and disadvantage of using BMSCs. 30

2.3 Viral delivery of transcription factors. 33

AAV.. 33

Lentivirus. 34

Chapter Three: Engaging reprogrammed cells via activity dependent repair. 35

3.1 Introduction.. 36

Functional recovery after stroke. 37

Neuroplasticity, circuits repair and functional recovery. 38

Vascular plasticity in stroke recovery: angiogenesis and arteriogenesis. 40

Enriched environment for activity-dependent repair and stroke recovery. 41

Rodent models of stroke rehabilitation.. 42

3.2 Current activity-dependent repair therapies. 42

Constraint-Induced motor therapy for stroke patients. 42

CIMT induces neurophysiological changes. 44

CIMT for chronic stroke patients. 45

Exercise and transcranial magnetic stimulation for stroke recovery. 47

Clinical assessments of stroke recovery. 47

Virtual reality. 48

Chapter Four: Rationale, Aims, and Experimental Methods. 50

4.1 Rationale and significance. 50

Direct reprogramming. 51

Barrel cortex ministroke model in mice. 52

4.2 Specific aims. 53

4.3 Materials and methods. 55

Plasmid Construction and Viral Production.. 55

Cell Culture and Viral Infection.. 57

Generate NeuroD1 plasmid by cloning NeuroD1 under GFAP promoter with mCherry tag into the FUGW plasmid. 58

NeuroD1 lentiviral production, purification, and titer calculation.. 59

Electrophysiological and histological examination of direct reprogramming in vitro using NeuroD1 lentivirus. 59

Enhancing functional recovery by activity dependent repair using whisker stimulation. 61

Animal Protocol 61

Focal ischemic stroke in mice. 61

Permanent embolic ischemic stroke in mice. 62

Local cerebral blood flow (LCBF) measurement. 63

Laser Doppler scan imaging. 63

[14C]Iodoantipyrine Autoradiography: 64

Infarct volume measurement. 65

TUNEL staining and cell death assessments. 66

Western blot analysis. 67

Immunohistochemical staining and cell counting. 68

Primary cell cultures and immunocytochemistry. 69

Transmission Electron Microscopy. 70

Statistics. 70

Stereotaxic Administration.. 71

Chapter Five: Long-term Survival and Regeneration of Neuronal and Vasculature Cells inside the Core Region after Ischemic Stroke in Adult Mice. 72

5.1 Abstract. 72

5.2 Results. 73

Focal ischemic stroke in the mouse and ischemia-induced cell death.. 73

Acute and chronic neuronal cell death in the ischemic core. 76

Mechanism of neuronal cell death in the ischemic core. 83

Endothelial cell and vasculature fate in the ischemic core. 86

Invasion of microglial/microphage into the ischemic core after stroke. 89

Astrocytes fate in the ischemic core and penumbra. 91

Ultrastructural examination of surviving cells in the ischemic core. 92

Regenerative factor expression in the ischemic core. 96

Regenerative activities in the ischemic core. 99

The possibility of protecting neuronal and vasculature cells in the ischemic core 103

5.3 Discussion.. 104

Chapter Six: Direct reprogramming of astrocytes to enhance recovery after stroke 112

6.1 Introduction.. 113

6.2 Results. 115

in vitro direct reprogramming. 115

NeuroD1 directly reprograms astrocytes into mature neurons in vitro 7 weeks post infection. 121

Animal model of direct conversion Cre-lox system... 123

Direct reprogramming of astrocytes alters gliosis following ischemic stroke. 124

Activation of microglia after stroke and direct programming. 127

Functional recovery and activity dependent repair. 128

Direct conversion of astroctyes to immature neurons in vitro. 129

Synapsin GFP reporter in vitro. 130

Direct conversion reduces the glial scar formation 6 weeks after injury. 130

Tracking Direct Conversion of Astrocytes to Neurons in vivo. 131

Direct conversion reduces activation and fate of microglia after stroke. 132

Direct reprogramming improves functional outcomes in mice after stroke. 132

6.3 Discussion.. 133

Reprogramming reactive astrocytes after ischemic stroke. 134

Direct reprogramming of astrocytes alters stroke pathophysiology. 135

Conclusions. 136

References. 138

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