Faculty Bibliography

Development and implementation of microarray techniques to quantify expression levels of dozens to hundreds to thousands of transcripts simultaneously within select tissue samples from normal control subjects and neurodegenerative diseased brains has enabled scientists to create molecular fingerprints of vulnerable neuronal populations in Alzheimer's disease (AD) and related disorders. A goal is to sample gene expression from homogeneous cell types within a defined region without potential contamination by expression profiles of adjacent neuronal subpopulations and nonneuronal cells. The precise resolution afforded by single cell and population cell RNA analysis in combination with microarrays and real-time quantitative polymerase chain reaction (qPCR)-based analyses allows for relative gene expression level comparisons across cell types under different experimental conditions and disease progression. The ability to analyze single cells is an important distinction from global and regional assessments of mRNA expression and can be applied to optimally prepared tissues from animal models of neurodegeneration as well as postmortem human brain tissues. Gene expression analysis in postmortem AD brain regions including the hippocampal formation and neocortex reveals selectively vulnerable cell types share putative pathogenetic alterations in common classes of transcripts, for example, markers of glutamatergic neurotransmission, synaptic-related markers, protein phosphatases and kinases, and neurotrophins/neurotrophin receptors. Expression profiles of vulnerable regions and neurons may reveal important clues toward the understanding of the molecular pathogenesis of various neurological diseases and aid in identifying rational targets toward pharmacotherapeutic interventions for progressive, late-onset neurodegenerative disorders such as mild cognitive impairment (MCI) and AD

Dysfunction of cholinergic basal forebrain (CBF) neurons of the nucleus basalis (NB) is a cardinal feature of Alzheimer's disease (AD) and correlates with cognitive decline. Survival of CBF neurons depends upon binding of nerve growth factor (NGF) with high-affinity (trkA) and low-affinity (p75(NTR)) neurotrophin receptors produced within CBF neurons. Since trkA and p75(NTR) protein levels are reduced within CBF neurons of people with mild cognitive impairment (MCI) and mild AD, trkA and/or p75(NTR) gene expression deficits may drive NB degeneration. Using single cell expression profiling methods coupled with custom-designed cDNA arrays and validation with real-time quantitative PCR (qPCR) and in situ hybridization, individual cholinergic NB neurons displayed a significant down regulation of trkA, trkB, and trkC expression during the progression of AD. An intermediate reduction was observed in MCI, with the greatest decrement in mild to moderate AD as compared to controls. Importantly, trk down regulation is associated with cognitive decline measured by the Global Cognitive Score (GCS) and the Mini-Mental State Examination (MMSE). In contrast, there is a lack of regulation of p75(NTR) expression. Thus, trk defects may be a molecular marker for the transition from no cognitive impairment (NCI) to MCI, and from MCI to frank AD

Molecular mechanisms underlying tauopathy remain undetermined. In the current study, single cell gene expression profiling was coupled with custom-designed cDNA array analysis to evaluate tau expression and other cytoskeletal elements within individual neuronal populations in patients with no cognitive impairment (NCI), mild cognitive impairment (MCI), and Alzheimer's disease (AD). Results revealed a shift in the ratio of three-repeat tau (3Rtau) to four-repeat tau (4Rtau) mRNAs within individual human cholinergic basal forebrain (CBF) neurons within nucleus basalis (NB) and CA1 hippocampal neurons during the progression of AD, but not during normal aging. A shift in 3Rtau to 4Rtau may precipitate a cascade of events in the selective vulnerability of neurons, ultimately leading to frank neurofibrillary tangle (NFT) formation in tauopathies including AD.

The ability to form tenable hypotheses regarding the neurobiological basis of normative functions as well as mechanisms underlying neurodegenerative and neuropsychiatric disorders is often limited by the highly complex brain circuitry and the cellular and molecular mosaics therein. The brain is an intricate structure with heterogeneous neuronal and nonneuronal cell populations dispersed throughout the central nervous system. Varied and diverse brain functions are mediated through gene expression, and ultimately protein expression, within these cell types and interconnected circuits. Large-scale high-throughput analysis of gene expression in brain regions and individual cell populations using modern functional genomics technologies has enabled the simultaneous quantitative assessment of dozens to hundreds to thousands of genes. Technical and experimental advances in the accession of tissues, RNA amplification technologies, and the refinement of downstream genetic methodologies including microarray analysis and real-time quantitative PCR have generated a wellspring of informative studies pertinent to understanding brain structure and function. In this review, we outline the advantages as well as some of the potential challenges of applying high throughput functional genomics technologies toward a better understanding of brain tissues and diseases using animal models as well as human postmortem tissues

Intracellular management of cholesterol is a critical process in the brain. Deficits with cholesterol transport and storage are linked to neurodegenerative disorders such as Neimann-Pick disease type C and Alzheimer's disease. One protein putatively involved in cholesterol transport is metastatic lymph node 64 (MLN64). MLN64 localizes to late endosomes which are part of the cholesterol internalization pathway. However, a detailed pattern of MLN64 expression in the brain is unclear. Using immunocytochemical and immunoblot analyses, we demonstrated the presence of MLN64 in several tissue types and various regions within the brain. MLN64 immunostaining in the CNS was heterogeneous, indicating selective expression in discrete specific cell populations and regions. MLN64 immunoreactivity was detected in glia and neurons, which displayed intracellular labeling consistent with an endosomal localization. Although previous studies suggested that MLN64 may promote steroid production in the brain, MLN64 immunoreactivity did not colocalize with steroidogenic cells in the CNS. These results demonstrate that MLN64 is produced in the mouse and human CNS in a restricted pattern of expression, suggesting that MLN64 serves a cell-specific function in cholesterol transport.

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.