Purified Neuronal Populations of Regenerating Ganglion Cells reveal DNA methylation-mediated Role of Na+/K+ ATPase in Axon Regeneration
Restricted (Penn State Only)
- Author:
- Rizk, Elias
- Graduate Program:
- Neuroscience (PHD)
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 07, 2023
- Committee Members:
- James Connor, Chair & Dissertation Advisor
James Robert Connor, Special Member
Alistair Barber, Program Head/Chair
Jeffrey Sundstrom, Major Field Member
Elizabeth Proctor, Major Field Member
Jian Yang, Outside Field Member
Ibrahim Ozbolat, Outside Unit Member - Keywords:
- IR Injured regenerated INR Non-injured regenerated UA Adult cells UE Embryonic cells RT PCR Reverse transcription polymerase chain reaction TBS Tris-buffered saline SEM Standard error of the mean DMSO Dimethyl sulfoxide ELF5 ets domain transcription factor 5 DAPI 4'
6-diamidino-2-phenylindole
IGF1 Insulin-like growth factor-1 DDR1 Discoidin domain receptor NSPC Neural stem/progenitor cells RhoA Ras homolog family member A ROCK Rho-associated protein kinas Na+ Sodium K+ Potassium ATPase adenosine triphosphatase Ca2+ Calsium FXYD FXYD protein family ATP adenosine triphosphate SRC SRC protein ER Endoplasmic reticulum mRNA Messenger ribonucleic acid ROS Reactive oxygen species PKC Protein kinase C Mg2+ Magnesium Cl- Chloride AQP4 Aquaporin 4 PLC Phospholipase C GLT Glutamine PBS Phosphate buffered saline DNA Deoxyribonucleic acid RNA Ribonucleic acid CpGs Cytosine guanine sites
CNS Central nervous system RGC Retinal ganglion cell FACS Fluorescence activated cell sorting DML Differentially methylated loci DMR Differentially methylated regions PNS Peripheral nervous system WD Wallerian degeneration SARM Sterile alpha and TIR motif CCL2 The chemokine (C-C motif) ligand 2 CCL3 The chemokine (C-C motif) ligand 3 CCL4 The chemokine (C-C motif) ligand 4 FOS Fos Proto-Oncogene
AP-1 Transcription Factor Subunit JUN Jun Proto-Oncogene
AP-1 Transcription Factor Subunit Camp Cyclic adenosine monophosphate GAP43 Growth associated protein 43 ATF3 Activating transcription factor 3 STAT3 Signal transducer and activator of transcription 3 CR3 Macrophage-1 antigen is a complement receptor LIF Leukemia inhibitory factor CXCR2 . C-X-C chemokine receptor type 2 CCN cysteine-rich
connective tissue growth
protein
AND factor (CTGF) and nephroblastoma overexpressed KDa kilo Dalton - Abstract:
- Central nervous system (CNS) regeneration is complex and challenging. Extensive research has focused on understanding CNS regeneration, including mechanisms underlying axonal and synaptic regeneration, the role of glial architecture and promoting or inhibiting regeneration, and the potential therapeutic approaches for promoting regeneration. While embryonic mammalian central nervous system (CNS) axons readily grow and differentiate, only a minority of fully differentiated mature CNS neurons are able to regenerate injured axons, leading to stunted functional recovery after injury and disease. Despite dose challenges, advances in our understanding of CNS regeneration have come a long way since Ramon Y Cajal's initial observation. There has been an explosion of research involving stem cell use, gene therapy, and electrical stimulation to help with CNS regeneration. The first aspect of my thesis presents an overview of regeneration in the central nervous system. This covers a historical perspective of regeneration, the response of the cell body to an injury, then observations of regeneration in the peripheral and central nervous system. The first part also summarizes some of the mechanisms involved in regeneration and the limitation of such processes in the CNS. The second aspect of my thesis assesses the role of sodium-potassium ATPase and its function in nervous system regeneration. The purpose of this review is to better understand the pump and the available knowledge on the role of the pump in regeneration as a whole and specifically known information on its role in central and peripheral nervous system regeneration in the animal kingdom. This portion of the thesis lays the foundation for the subsequent portions of the thesis and the notable findings of the pump’s effect on mammalian central nervous system regeneration in retinal ganglion cells. We review the molecular structure and function of the pump and the known role of the pump in regeneration. Following these two portions of the thesis, we discuss the development of a Fluorescent-Activated Cell Sorting (FACS)-based methodology in a rat optic nerve transection model to segregate the injured retinal ganglion cells (RGCs) into regenerating and non-regenerating cell populations. This allows us to delineate DNA methylation changes specifically associated with axonal regeneration. We then ran whole-genome DNA methylation profiling of these purified neurons to reveal known and novel genes and pathways linked to mammalian RGC regeneration. This methodology brought to light the contribution of subunits encoding the Na+/K+-ATPase to embryonic growth and adult axon regeneration. In turn, biochemical and genetic inhibition of the Na+/K+-ATPase pump significantly reduced RGC axon regeneration. Together, these data demonstrate critical molecular insights into mammalian CNS axon regeneration. Furthermore, we have shown that the Na+/K+-ATPase acts as a key regulator of regeneration of injured mature CNS axons and suggests that successful regeneration requires, in part, reactivation of embryonic signals. Identifying the specific role of DNA methylation in CNS regeneration promises novel therapeutic targets for CNS injury and disease.