Astrocytes are the most abundant glial cell type in the central nervous system (CNS). Most astrocytes are born during the early postnatal period in the rodent brain and mature alongside neurons, demonstrating remarkable morphological structural complexity, and attaining maturity in the second postnatal month. We have shown that astrocyte morphogenesis is regulated in part by brain-derived neurotrophic factor (BDNF) via signaling through the truncated tropomyosin receptor kinase B (TrkB) receptor. TrkB is the primary receptor for BDNF which is broadly expressed and released by neurons in developing and mature brain. TrkB has two predominant isoforms expressed in central nervous system (CNS), the full length TrkB (TrkB.FL) receptor and truncated TrkB (TrkB.T1) receptor. We recently demonstrated in the adult rodent cortex that TrkB.T1 is largely specific to astrocytes and over 90% of all Ntrk2 expression in astrocytes attributed to TrkB.T1. In contrast TrkB.FL is the predominant isoform expressed by neurons. It is not known how astrocytes and neurons regulate their specific TrkB isoform expression, although previous studies in bulk frontal cortical tissue from human postmortem samples indicate that DNA methylation level in promoter region and 3' UTR region of NTRK2 is negatively correlated with TrkB.T1 expression levels, but not with TrkB.FL expression. The mechanism of TrkB.T1 isoform-specific expression and the role of TrkB.T1 in astrocyte developmental process are unknown.
In this dissertation, we aimed to determine in the DNA methylation contributes to isoform specific expression of TrkB.T1. We thus profiled the 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in neurons, astrocytes and microglia utilizing nanopore sequencing. We identified robust differences in cell-type specific TrkB isoform expression is associated with significantly different 5mC and 5hmC patterns in neurons and astrocytes. Further, we investigated the role of TrkB.T1 in cortical astrocyte developmental processes and astrocyte function during early postnatal development (postnatal day (P) 8, P14, P28 and P60). RNA sequencing of TrkB.T1 deficient astrocytes isolated at these timepoints revealed aberrant gene expression in astrocyte maturation, while pathway analysis indicated disruptions in synapse organization, neurotransmitter transport and exocytotic processes. Subsequent functional secretory proteomics highlighted disruptions in metabolism and lipid regulation, particularly cholesterol transport, suggesting potential implications for synapse formation. We observed dysregulated spine density in the motor and somatosensory cortices from TrkB.T1-deficient astrocytes relative to control astrocytes. These findings suggest that TrkB.T1 deficiency adversely affects normal astrocyte development, which in turn affects neuronal synapse development. This study provides new insights into the role of BDNF/TrkB.T1 signaling in CNS development and lays the groundwork for evaluating astrocyte BDNF/TrkB.T1 signaling in neurological diseases. / Doctor of Philosophy / Astrocytes are an abundant brain cell type that play crucial roles in maintaining brain health and supporting neuron functions. Astrocytes develop right after birth and reach full maturity by the second month in rodents. Throughout life, astrocytes play crucial roles in stabilizing the brain's environment and supporting most brain functions. the formation of astrocyte complex morphology is regulated by brain-derived neurotrophic factor (BDNF), which interacts with a specific receptor called TrkB.T1 found mainly in astrocytes. Another form of this receptor, TrkB.FL, is primarily found in neurons. Despite the importance of TrkB.T1 in astrocyte development and function, the reason for high expression and its role in astrocytes were not well understood. Previous studies in human postmortem samples indicated that DNA methylation level of the gene NTRK2 is negatively linked to TrkB.T1 expression but not TrkB.FL expression. However, the mechanisms behind TrkB.T1 isoform-specific expression and its role in astrocyte development are unknown.
In this study, we used advanced sequencing techniques to analyze DNA methylation patterns, a chemical modification that can control gene activity, in neurons, astrocytes, and microglia, which are three different cell types in the brain. Our study discovered distinct methylation patterns in all three cell types and supported previous research DNA methylation effect on gene regulation and alternative gene splicing. With the data obtain from this study, we also observed a significant difference in DNA methylation may cause that TrkB.T1 is mainly expressed in astrocytes and TrkB.FL is mainly expressed in neurons. The study further investigated TrkB.T1 deficient astrocytes at different developmental stages. By examining RNA from astrocytes, we found abnormal gene expression in astrocyte maturation. Further analysis showed changes in cholesterol transport, suggesting potential implications for synapse formation. Finally, we observed the dysregulated spine density in the animals with astrocyte specific TrkB.T1 deletion. These findings suggest that TrkB.T1 deficiency adversely affects normal astrocyte development, which in turn affects neuronal synapse development. These findings provide new insights into the expression mechanisms and functions of TrkB.T1 in astrocytes, enhancing our understanding of how these cells support brain health and function.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/120684 |
| Date | 23 July 2024 |
| Creators | Wei, Xiaoran |
| Contributors | Biomedical and Veterinary Sciences, Olsen, Michelle Lynne, Theus, Michelle Hedrick, Yu, Guoqiang, Morton, Paul D., Jarome, Timothy |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | Creative Commons Attribution-ShareAlike 4.0 International, http://creativecommons.org/licenses/by-sa/4.0/ |
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