Faculty Bibliography

The development of gene array techniques to quantify expression levels of dozens to thousands of genes simultaneously within selected tissue samples from control and diseased brain has enabled researchers to generate expression profiles of vulnerable neuronal populations in several neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, schizophrenia, multiple sclerosis, and Creutzfeld-Jakob disease. Intriguingly, gene expression analysis reveals that vulnerable brain regions in many of these diseases share putative pathogenetic alterations in common classes of genes, including decrements in synaptic transcript levels and increments in immune response transcripts. Thus, gene expression profiles of diseased neuronal populations may reveal mechanistic clues to the molecular pathogenesis underlying various neurological diseases and aid in identifying potential therapeutic targets. This chapter will review how regional and single cell gene array technologies have advanced our understanding of the genetics of human neurological disease.

Galanin (GAL)-containing fibers enlarge and hyperinnervate remaining cholinergic basal forebrain (CBF) neurons within the anterior nucleus basalis (NB) in late-stage Alzheimer's disease (AD). Whether GAL hypertrophy occurs in the CBF in the prodromal or early stages of AD remains unknown. The present study used GAL immunohistochemistry and an unbiased semiquantitative scoring method to evaluate GAL innervation in the anterior NB of subjects clinically diagnosed as having no cognitive impairment, mild cognitive impairment or early-stage (mild/moderate) AD. There was no difference in GAL fiber staining within the anterior NB across the three clinical groups examined. Furthermore, GAL fiber innervation was not correlated with the number of NB neurons expressing the nerve growth factor receptors p75(NTR) or TrkA or with cortical choline acetyltransferase activity in the same cases. Single-cell gene expression analysis demonstrated that cholinergic NB neurons express mRNA for the GAL receptors GALR1, GALR2 and GALR3, yet the levels of these mRNAs were unchanged across the three diagnostic groups. These observations indicate that GAL hypertrophy within the anterior NB subfield is a late-stage AD response, which may play a role in regulating the cholinergic tone of remaining basocortical projection neurons.

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

Granule cell (GC) neurogenesis increases following seizures, and some newborn GCs develop in abnormal locations within the hilus. These ectopic GCs (EGCs) display robust spontaneous and evoked excitatory activity. However, the pattern of afferent input they receive has not been fully defined. This study used electron microscopic immunolabeling to quantitatively evaluate mossy fiber (MF) input to EGCs since MFs densely innervate the hilus normally and undergo sprouting in many animal models of epilepsy. EGC dendrites were examined in tissue from epileptic rats that had initially been treated with pilocarpine to induce status epilepticus and subsequently had spontaneous seizures. MF terminals were labeled with a zinc transporter-3 antibody, and calbindin immunoreactivity was used to label hilar EGCs and GC layer GCs. The pattern of input provided by sprouted MF terminals to EGC dendrites was then compared to the pattern of MF input to GC dendrites in the inner molecular layer (IML), where most sprouted fibers are thought to project. Analysis of EGC dendrites demonstrated that MF terminals represented their predominant source of afferent input: they comprised 63% of all terminals and, on average, occupied 40% and 29% of the dendritic surface in the dorsal and ventral dentate gyrus, respectively, forming frequent synapses. These measures of connectivity were significantly greater than comparable values for MF innervation of GC dendrites located in the IML of the same tissue sections. Thus, EGCs develop a pattern of synaptic connections that could help explain their previously identified predisposition to discharge in epileptiform bursts and suggest that they play an important role in the generation of seizure activity in the dentate gyrus

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

Despite numerous neuroendocrinological studies of seizures, the influence of estrogen and progesterone on seizures and epilepsy remains unclear. This may be due to the fact that previous studies have not systematically compared distinct endocrine conditions and included all relevant controls. The goal of the present study was to conduct such a study using pilocarpine as chemoconvulsant. Thus, age and weight-matched, intact or ovariectomized rats were tested to determine incidence of status epilepticus and to study events leading to status. Intact female rats were sampled at each cycle stage (proestrus, estrus, metestrus, or diestrus 2). Convulsant was administered at the same time of day, 10:00-10:30 a.m. Statistical analysis showed that there was a significantly lower incidence of status on the morning of estrus, but differences were attenuated in older animals. Ovariectomized rats were distinct in their rapid progression to status. These results show that the incidence of status in female rats following pilocarpine injection, and the progression to pilocarpine-induced status, are influenced by reproductive state as well as age. The hormonal milieu present specifically on the morning of estrus appears to decrease susceptibility to pilocarpine-induced status, particularly at young ages. In contrast, the chronic absence of reproductive steroids that characterizes the ovariectomized rat leads to a more rapid progression to status. This dissociation between incidence vs. progression provides new insight into the influence of estrogen and progesterone on seizures

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

Macroautophagy, which is a lysosomal pathway for the turnover of organelles and long-lived proteins, is a key determinant of cell survival and longevity. In this study, we show that neuronal macroautophagy is induced early in Alzheimer's disease (AD) and before beta-amyloid (Abeta) deposits extracellularly in the presenilin (PS) 1/Abeta precursor protein (APP) mouse model of beta-amyloidosis. Subsequently, autophagosomes and late autophagic vacuoles (AVs) accumulate markedly in dystrophic dendrites, implying an impaired maturation of AVs to lysosomes. Immunolabeling identifies AVs in the brain as a major reservoir of intracellular Abeta. Purified AVs contain APP and beta-cleaved APP and are highly enriched in PS1, nicastrin, and PS-dependent gamma-secretase activity. Inducing or inhibiting macroautophagy in neuronal and nonneuronal cells by modulating mammalian target of rapamycin kinase elicits parallel changes in AV proliferation and Abeta production. Our results, therefore, link beta-amyloidogenic and cell survival pathways through macroautophagy, which is activated and is abnormal in AD

In addition to its potent effects on vasculature, it has become clear that vascular endothelial growth factor (VEGF) has effects on both neurons and glia, and recent studies suggest that it can be neuroprotective. To determine potential mechanisms underlying this neuroprotection, recombinant human VEGF was bath applied to adult rat hippocampal slices, and both extracellular and intracellular recordings were used to examine intrinsic properties and synaptic responses of hippocampal principal neurons. Initial studies in area CA1 showed that VEGF significantly reduced the amplitude of responses elicited by Schaffer collateral stimulation, without influencing membrane properties. Similar effects occurred in CA3 pyramidal cells and dentate gyrus granule cells when their major glutamatergic afferents were stimulated. Because VEGF expression is increased after seizures, effects of VEGF were also examined in rats with recurrent spontaneous seizures. VEGF reduced spontaneous discharges in slices from these rats but had surprisingly little effect on epileptiform discharges produced by disinhibition of slices from control rats. These results demonstrate a previously unknown effect of VEGF on neuronal activity and also demonstrate a remarkable potency in the epileptic brain. Based on this, we suggest that VEGF or VEGF-related targets could provide useful endpoints to direct novel therapeutic strategies for epilepsy