Type of Document Dissertation Author Darcy, Michael URN etd-12082009-143750 Title Characterization of HDAC4's Role in Brain Degree Doctor of Philosophy Department Neuroscience Advisory Committee
Advisor Name Title Charles Ouimet Committee Chair Carlos Bolanos Committee Member Laura Keller Committee Member Mohamed Kabbaj Committee Member Colleen Kelley University Representative Keywords
- Morris Water Maze
- Traumatic Brain Injury
- Postsynaptic Density
- Dendritic Spines
Date of Defense 2009-12-02 Availability unrestricted AbstractEpigenetic regulation of gene expression involves a steady-state balance of acetylation carried about by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs act as transcriptional co-activators and HDACs interact with large multi-protein complexes to promote transcriptional repression. HDACs have only recently been characterized in mammalian cells, and most work has focused on the function of HDACs in vitro using biochemical analysis, inhibitors, and cultured cell types. HDAC4, a class II HDAC, displays the ability to shuttle between the cytoplasm and the nucleus where it can regulate transcriptional programs. HDAC4 plays a key role in calcium-dependent transcriptional regulation of many non-neuronal cell processes including cardiac hypertrophy and bone formation. HDAC4 mRNA is also highly expressed in brain; however protein expression and its underlying biological role in brain is still unclear.
HDAC4 localization in cultured neurons is dependent on neural activity and calcium-dependent signaling pathways. Mechanisms governing long-term changes in synaptic plasticity and learning and memory take place on dendritic spines, a site affected by many cognitive disorders. Dendritic spines act to compartmentalize calcium signaling and second messenger cascades leading to activation of enzymes and proteins associated with transcriptional regulation. Inhibition of HDACs has become a prevalent tool in exploring the role of HDACs in brain and has proven useful in many models of psychiatric and neurodegenerative disorders with more recent implication in the recovery or enhancement of synaptic plasticity and learning and memory. HDAC inhibition, however, is non-specific, and the localization of specific HDACs in brain and their role in these neuronal functions needs to be addressed. The similarity between HDAC4 regulation in non-neuronal cells and the processes initiated within a dendritic spine led to the hypothesis that HDAC4 may be present at the dendritic spine, where it can relay alterations of synaptic activity to the nucleus in order to regulate transcriptional programs affecting synaptic plasticity or other cell function.
For this dissertation, I report findings which establish the regional and novel subcellular localization pattern of HDAC4 expression in brain, identify a mechanism specific to synaptic activity at the dendritic spine which results in HDAC4 trafficking, and attempt to establish a direct interaction of HDAC4 to a key member of the scaffolding network within a dendritic spine. In additional studies, I report the effects of HDAC inhibition on learning and memory and lesion size using a model of traumatic brain injury (TBI) as well as the effects of amyloid plaque level on the localization pattern of HDAC4 in the hippocampus. These studies failed to illicit a significant change in the conditions tested and are not discussed in the main text, however, useful information regarding the role of HDAC inhibition and HDAC4 was obtained.
In brief, I report the localization of HDAC4 across brain regions germane to many pathological conditions such as Huntingtonís, Parkinsonís, and Alzheimerís disease. HDAC4 was found to be present in dendritic spines, enriched at the level of the post-synaptic density (PSD), and partially colocalized with post-synaptic density protein 95 (PSD-95), a key scaffolding protein for the formation and maintenance of dendritic spines. Furthermore, using hippocampal slice cultures to more closely represent in vivo synaptic connections, exogenous overexpression of HDAC4 localized to the cytoplasm and in dendritic spines. Dendritic spines, synaptic activity, and the ability to form memories are tightly regulated through the activation of N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Blockade of both NMDA and AMPA receptors together was necessary to induce the nuclear localization of HDAC4 in these cultures, a shift that was reversed upon removal of the antagonists or reduced by HDAC inhibition. Finally, HDAC4 was expressed along with PSD-95 in vitro as well as extracted from hippocampal tissue to explore whether HDAC4 was a direct member of the PSD-95 scaffolding network in vivo. HDAC4 failed to show a complex with PSD-95, however, indirect interactions may still exist which anchor HDAC4 to the PSD. Together, these results suggest HDAC4 can act as a synaptic monitor, translocating to the nucleus during synaptic blockade where it can alter transcriptional programs and gene expression. Isolating the biological role for individual HDAC isoforms remains a critical step in understanding the mechanisms behind therapeutic candidates such as HDAC inhibitors, which have been used clinically in non-neuronal disruption of cancerous cells, and show much promise in the alleviation of many symptoms resulting from various psychiatric and neurodegenerative disorders.
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