Type of Document Dissertation Author Dietz, Karen Christine URN etd-02282008-160200 Title Dendritic Spines and Deacetylases Degree Doctor of Philosophy Department Biological Science, Department of Advisory Committee
Advisor Name Title Charles C Ouimet Committee Chair Mohamed Kabbaj Committee Member Paul Q Trombley Committee Member Thomas C.S. Keller III Committee Member Zuoxin Wang Committee Member Keywords
- Dendritic Spines
- Down's Syndrome
- Histone Deacetylases
Date of Defense 2008-02-18 Availability unrestricted AbstractThe work presented in this dissertation represents investigations into the mechanism of two neurological disorders. The first set of experiments was aimed at examining the morphology of post-synaptic structures called dendritic spines in a mouse model of Down’s syndrome. The second set of experiments was aimed at examining the regional pattern and cellular distribution of possible therapeutic targets, histone deacetylases, in treating the symptoms of Huntington’s disease.
In the first set of experiments, we examined a mouse model of Down’s syndrome, the Ts65Dn mouse, to determine if it mimics the dendritic spine abnormalities in area CA1 of hippocampus that have been documented in human individuals with the disorder. The Ts65Dn mouse represents a partial trisomy of the murine chromosome homologous to a large portion of human chromosome 21, which is present in 3 copies rather than 2 in Down’s syndrome. These mice show behavioral abnormalities and learning deficits that are thought to replicate the mental retardation that is a prominent characteristic of Down’s syndrome. As many of the behavioral and learning paradigms used to test these mice require hippocampal function, and neurons from hippocampal tissue taken from Down’s syndrome individuals show a reduction in the density of dendritic spines, we sought to determine if Ts65Dn mice exhibit the same morphological abnormality.
Dendritic spine densities on the apical branches of CA1 hippocampal neurons in Ts65Dn animals were not significantly different when compared to those from euploid (normal chromosome number) littermates. In addition, morphological analysis of dendritic spine shape demonstrated that the proportion of dendritic spines in each of the four major spine shape categories (stubby, thin, filopodia and mushroom) was not different between the two conditions. As the environment for cells in a cultured slice are most likely very different from those experienced in the intact animal, we examined if neurons in the intact brain exhibited signs of abnormal dendritic spine density in the Ts65Dn mouse. Analysis of dendritic spine densities on apical branches of CA1 hippocampal neurons from mice sacrificed at 2 weeks, 3 months or 6 months of age showed no significant differences between the euploid and trisomic conditions. Furthermore, Western blot analysis showed that Ts65Dn do not have reduced expression of a dendritic spine protein, drebrin, as has been reported in Down’s syndrome.
The second set of experiments that are described concern the immunohistochemical localization of two enzymes involved in the transcriptional regulation of genes. These enzymes, called histone deacetylases, or HDACs, aid in the regulation of histone acetylation levels as a mechanism to control access of transcription factors to gene sequences. Recently, it was found that compounds that inhibit enzymes that remove acetyl groups demonstrate therapeutic effects in animal models of Huntington’s disease, a neurodegenerative disorder that targets brain regions important for movement control. The work presented here describes the immunohistochemical localization of a HDAC2 and HDAC7.
HDAC7 immunohistochemistry was consistent with biochemical studies demonstrating that HDAC7 can be present in both the cytoplasm and the nucleus. A finding of interest is that not all neurons of the murine brain express HDAC7, nor is it localized to the same subcellular compartment in all cell types. Granule cells of the hippocampal dentate gyrus and of the cerebellum showed very little immunoreactivity for HDAC7. Apical dendrites of the CA1 and CA3 regions of hippocampus showed very heavy cytoplasmic staining. Deeper cortical layers showed pyramidal neurons with heavier staining than superficial pyramidal layers in almost all cortical regions, except for the orbital, insular and piriform cortices which showed heavy staining in superficial layers as well. In general, staining of the olfactory system appeared more intense than other sensory system regions. Fibers along the striatonigral bundle, from the caudate to the substantia nigra reticulata showed heavy immunoreactivity. These data suggest that targeting therapeutics to HDAC7 activity may indeed be useful, as the striatonigral pathway is a key component of movement control.
HDAC2 immunoreactivity confirmed that HDAC2 is strictly a nuclear localized protein. In addition, it appeared that HDAC2 is ubiquitously expressed throughout the murine brain in all brain regions. Interestingly, HDAC2 staining occurred only in neurons that showed NeuN staining, demonstrating that it is a neuron-specific protein. Therefore, therapeutic inhibition of HDACs that include inhibition of HDAC2 may affect many aspects of neurologic functioning in multiple brain regions.
It is our hope that this work will provide some added detail and knowledge to the greater neuroscience community, and will aid in the greater understanding of the organization and function of the brain and ultimately aid in development of treatments for and ultimately the cure of neurological diseases and disorders.
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