Date of Award

August 2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

John Watt

Abstract

Axon regeneration is essential for effective repair following injury in the CNS. One of the burdens of aging is the lack of recovery following injury in the CNS. Our previous studies show that in 35-day-old rats, axon regeneration occurs in response to a unilateral hypothalamic lesion in the hypothalamus neurohypophyseal system. However, this regenerative ability is lost in 125-day-old rats. Furthermore, treatment of hypothalamic organotypic culture with ciliary neurotrophic factor (CNTF) promotes axonal outgrowth and survival. Our previous studies suggest that astrocytes may be playing critical roles in this maturational loss of regenerative capacity because only astrocytes expressed all the three receptors, necessary for CNTF activity. Additionally, CNTF immunoreactivity and P-STAT3 are localized in the ventral glial limitans, where astrocytes are also located. DNA methylation and microRNA are two epigenetic mechanisms that regulate transcriptional programming including axonal regeneration. These epigenetic regulations vary in different brain regions and with age. Although astrocytes are well known to play a generally supportive role for neurons, the specific roles that astrocytes play in the maturational loss of axonal regeneration in the hypothalamus are not known: the age-related changes in the transcriptome of astrocytes associated with maturational loss of axon regenerative ability in the hypothalamus is now known. Furthermore, how epigenetic factors regulate transcriptional changes to affect axonal regeneration in the hypothalamus is not known. Our central hypothesis is that maturational loss in axonal regeneration is due to age-related changes in the epigenetic regulation of gene expression programs in astrocytes. The overall aim of this study is to investigate the relationship between maturational alteration in the epigenome and transcriptome of hypothalamic astrocytes in the context of axonal regeneration. Our objectives are to determine age-related changes in the transcriptome of hypothalamic astrocytes associated with axonal regeneration; determine age-related changes in DNA methylation of hypothalamic astrocytes associated with axonal regeneration; and determine age-related changes in microRNA expression of hypothalamic astrocytes associated with axonal regeneration. With little modification to the traditional shaking method, astrocytes were isolated from the hypothalamus of 35 days and 125-day-old rats using enzymatic digestion and mechanical dissociation. Purification was done by shaking overnight. Astrocyte purification was confirmed using western blot and immunocytochemistry. Protein, mRNA, microRNA, and DNA were isolated using standard procedures and were used for western blot, RNA sequencing, microRNA sequencing, and DNA Methylation sequencing respectively. For bioinformatic analysis, FastQC was used for the initial quality check, and trimming of adapter sequences was done with trimmomatic. Differential expression analysis of both RNA sequencing and microRNA sequencing data was done using DESeq2. Functional analysis to determine biological processes and pathway enriched was done with clusterProfiler in all analyses. Determination of differential methylation was done with methylKit and genomation. RNA-seq validation was done with qPCR. For all analyses, we used Adjusted P-value of < 0.05 and > 1.5-fold change. Our RNA-seq analysis identified 1783 and 1770 differentially upregulated and downregulated genes respectively. Functionally analysis revealed the enrichment of axonogenesis and other related processes in the downregulated genes, while upregulated genes are enriched in immune response-related processes. Pathway analysis showed that axon guidance was inhibited. Furthermore, there was downregulation of many genes related to axon regeneration, such as semaphorins, including Sema3a, Sema4d, and Sema7a. Upon analyzing our microRNA sequencing, we identified 20 and 4 downregulated and upregulated microRNAs respectively. Some of these 24 microRNAs (e.g. miR-124-3p, miR-9a-5p, miR-1247-5p, and miR-1247-3p) have been previously reported to be involved in promoting axon regeneration. These 24 microRNAs are known to target 395 mRNAs based on three databases. Surprisingly, neither these targets nor their overlap with RNA-seq DEGs are enriched in axonal regeneration and other related processes. On the other hand, DNA methylation analysis reveals a strong enrichment of axonogenesis and related biological processes similar to what was seen in transcriptome analysis. Also, the overlap of genes of DMR and RNA seq are enriched in axonogenesis and other related processes. Specifically, genes in hypomethylated regions are enriched in axonogenesis and other related processes. Furthermore, the Kyoto Encyclopedia of Genes and Genome (KEGG) analysis revealed the enrichment of axon guidance, PI3K-Akt, and other similar pathways known to be related to axonal regeneration. Putting all these together, we conclude that there is an association between maturational loss of axonal regeneration and transcriptome changes in hypothalamic astrocytes. And that Methylation (but not MicroRNA) is the likely epigenetic mechanism used for the transcriptional regulation of axon regeneration by astrocytes in the hypothalamus. Further studies that overexpressed or knockdown specific genes of interest identified in this study, in an astrocytes-neuron co-culture or in vivo will shed more light to the specific biological relevance of specific genes in this context.

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