The Sanfilippo syndrome type B (MPS IIIB) is a rare neurodegenerative disorder of children, caused by mutations in the gene encoding the enzyme alpha-N-acetylglucosaminidase and the resulting accumulation of heparan sulfate. The disease, which is chronic and progressive, is characterized by profound mental retardation and eventual dementia, with death usually in the late teens. There currently is no effective treatment. We previously developed a mouse model by targeted disruption of the Naglu gene in order to study this catastrophic disease. Activated microglia and astrocytes are present throughout the brain, starting at 1 month of age. However, immunohistochemistry has shown that certain neuronal changes are focal, with neurons in a few areas showing a constellation of secondary abnormalities in addition to the expected storage of glycosaminoglycan. One such area (designated as """"""""vulnerable"""""""") is the medial entorhinal cortex.
In Specific Aim 1, we will use microarray technology to identify changes in gene expression that are specific to neurons in the """"""""vulnerable"""""""" area of the mutant mouse brain. Using laser capture microdissection, we will obtain 500 -700 neurons from the medial entorhinal cortex of the mutant mice at 1, 3 and 6 months of age, and process total RNA for hybridization to oligonucleotide arrays representing the entire mouse genome. The feasibility of this procedure has been demonstrated in a preliminary experiment. Neurons will also be dissected from a """"""""less vulnerable"""""""" area of the brain, which does not show the secondary abnormalities (e.g., the lateral entorhinal cortex). Neurons from the two areas in each of 4 mutant mice of each age will be compared to each other as well as to neurons from corresponding areas of brain from control mice. Transcripts specific to neurons in the """"""""vulnerable"""""""" area of the mutant mouse brain will be confirmed by independent methods such as in situ hybridization, quantitative RT-PCR and immunohistochemistry for the corresponding proteins. This phase of the study is expected to uncover changes in gene expression that may underlie the selective vulnerability of certain neurons.
In specific aim 2, we will explore the significance and consequences of some of these changes. The changes of interest would be those involved in, e.g., signal transduction, organelle traffic, cell adhesion, and other important cellular processes. While the generation of hypotheses for this Specific Aim must await data obtained from microarray analyses, we used a finding in the feasibility experiment (a 7-fold increase of lysozyme transcript) as an example of how we would proceed from microarray data to a hypothesis for the significance of the elevated transcript. The purpose of these studies is to better understand the role of the secondary abnormalities in altering molecular and biochemical pathways in the Sanfilippo type B brain.
The Sanfilippo Syndrome type B is a rare but catastrophic neurodegenerative disorder of children, which can be studied in a mouse model. While all cells in this mouse share the same primary problem (storage of heparan sulfate), there is a set of neurons with additional pathological changes. Using microarray technology, we will identify genes that are expressed abnormally in these neurons, in order to discover pathways that might be involved in the development of the disease in the brain.
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