Faculty Bibliography

Advances in high throughput cloning strategies have led to sequencing of the human genome as well as progress in the sequencing of the genome of several other species. Consequently, the field of molecular genetics is blossoming into a multidisciplinary entity that is revolutionizing the way researchers evaluate a myriad of critical concepts such as development, homeostasis, and disease pathogenesis. There is tremendous interest in the quantitative assessment of tissue-specific expression of both newly identified and well characterized specific genes and proteins. At present, an ideal approach is to assess gene expression in single elements recorded physiologically in living preparations or by immunocytochemical or histochemical methods in fixed cells in vitro or in vivo. The quantity of RNA harvested from individual cells is not sufficient for standard RNA extraction methods. Therefore, exponential polymerase-chain reaction based analyses, and linear RNA amplification including amplified antisense RNA amplification and a newly developed terminal continuation RNA amplification methodology have been developed for use in combination with microdissection procedures and cDNA/oligonucleotide microarray platforms. RNA amplification is a series of intricate procedures to amplify genetic signals from minute quantities of starting materials for microarray analysis and other downstream genetic methodologies. RNA amplification procedures effectively generate quantities of RNA through in vitro transcription. The present report illustrates practical usage of RNA amplification technologies within the context of regional, population cell, and single cell analyses in the brain

OBJECTIVE: The goal is to determine cellular and molecular mechanisms that regulate regenerative and neurodegenerative responses within the adult mouse dentate gyrus after axotomy of the principal glutamatergic input, the perforant path (PP). METHODS: A 'molecular fingerprint' of the dentate gyrus was generated to provide an extensive, concurrent representation of genes, with an emphasis on glutamate receptor subunits and related markers of glutamatergic neurotransmission. Reorganization of the hippocampal formation was evaluated by regional microdissection of the dentate gyrus, followed by terminal continuation RNA amplification and custom-designed cDNA microarray analysis after unilateral PP transections at five time-points (0, 1, 10, 14, and 30 days post-lesion). Gene-expression profiles garnered from the ipsilateral side of PP transected hippocampal formation (including the dentate gyrus) were compared and contrasted with those of naive subjects, sham surgical subjects, and mice subjected to unilateral occipital cortex lesions. Specific gene array observations were validated by immunoblot analysis. RESULTS: Results indicated selective regulation of specific transcripts, including AMPA and NMDA glutamate-receptor subunits, excitatory amino acid transporters, glutamate receptor interacting protein genes, and glial-associated markers across the time-course of the lesion study. CONCLUSION: The goal was to identify messenger RNAs from specific classes of relevant transcripts that change over time in relationship to the synaptic and cellular alterations to help understand mechanisms that underlie lesion-induced synaptic plasticity

Neurogenesis has been demonstrated in the adult mammalian hippocampus by the immunohistochemical identification of cells co-labeled with the neuronal marker NeuN and bromodeoxyuridine (BrdU), a marker for DNA synthesis. Whether these newly born neurons exhibit a genetic signature similar to that of existing hippocampal cells remains unknown. Recent advances in single cell RNA amplification techniques provide a unique method for profiling the mRNA complement of cells developed during adult neurogenesis. Standard protocols for identifying BrdU-positive cells requires an acid denaturation step that may preclude the amplification of cellular RNA for expression analysis. We first tested whether the BrdU reaction product was visible in monkey hippocampal tissue following treatment with dilutions of HCl (2-0.2 M) or citric acid (1.0-0.1 M). BrdU-labeled cells were visible only in tissue sections treated with 2 M HCl. RNA amplification was not compromised in cells dual-labeled for BrdU and NeuN using the 2 M HCl acid denaturation step. These cells express mRNAs encoding a wide variety of functional protein subclasses including glutamate receptors. The present study demonstrates for the first time that BrdU immunohistochemisty is compatable with gene array technology in the primate hippocampus to evaluate subclasses of genes in newborn neurons

To evaluate molecular events associated with the aging process in animal models and human tissues, microarray analysis is performed at the regional and cellular levels to define transcriptional patterns or mosaics that may lead to better understanding of the mechanism(s) that drive senescence. In this review, we outline the experimental and analytical issues associated with high-throughput genomic analyses in aging brain and other tissues for a comprehensive evaluation of the current state of microarray analysis in aging paradigms. Ultimately, the goal of these studies is to apply functional genomics and proteomics approaches to aging research to develop new tools to assess age in cell- and tissue-specific manners in order to develop aging biomarkers for pharmacotherapeutic interventions and disease prevention