Restorative Neurology and Neuroscience

Meffimphetamine (METH) is a drug of abuse with well-deseribed neurodegenerative effects. Some of the METH-induced degenerative manifestations are thought to be due to increased dopamine (DA) release in the cytoplasm of nerve terminals and subsequent extravasation in the synaptic cleft. Using an immortalized neural cell line, we have made use of the comprehensive cDNA array technology in order to compare and contrast the molecular effects of DA and METH. We found that the two compounds do have many similar but also different effects. Since these neural cells produce no DA, these results demonstrate that many of the METH-induced responses attributed to DA might, in fact, be intrinsic to METH itself. More biochmical studies are needed to investigate DA-independent METH deleterious events in the central nervous system.

Purpose: The human SH-SY5Y cell line is an established model for retinoic acid (RA)-induced neural differentiation. We employed a broad human 15K microarray (15,000 genes) and focused Neuroarray (1152 genes) to examine changes in gene expression early in the process of differentiation (6 hr), before morphology or growth changes are observed. Methods: 33 P-labeled CDNA probes prepared from RNA extracts of RA-treated and control cultures were hybridized to array membranes, and levels of expression were quantified and compared. Results: In the 15K array, 19 % of the genes were decreased (0.4 % were named genes and the remainder were expressed sequence tags (ESTs) or unknowns), and 9 % were increased (4.2 % named genes). In the Neuroarray, 3 % were decreased and 8 % were inereased. Conclusions: Summary gene profiles are presented, which include transcription factors, genes associated with cell cycle, cell shape, neurotransmission, intermediary filaments, and others. The prevalence of down-regulated genes in the broad 15K array and up-regulated genes in the neuro-focused array suggests a pattem shift in gene expression associated with differentiation. The predominance of ESTs among the down-regulated genes indicates a great number of as-yet-unidentified genes are repressed in early stage neural differentiation.

Embryonic stem (ES) cells have the ability to differentiatie into a variety of cell lineages. We are examining ES cell differentiation in vitro by using cDNA microarrays to generate a molecular phenotype for each cell type. El4 ES cells induced by retinoic acid after forming embryoid bodies differentiatie almost exclusively to neurons. We obtained expression pattems for about 8500 gene sequenees by comparing mRNAs from undifferentiated ES cells and their differentiated derivatives in a competitive hybridization. Our results indicate that the genes expressed by ES cells change dramatically as they differentiatie (58 gene sequences up-regulated, 34 down-regulated). Most notably, totipotent ES cells expressed high levels of a repressor of Hox expression (the polycomb homolog Mphl) and a co-repressor (CTBP2). Expression of these genes was undetectable in differentiated cells; the ES cell-derived neurons expressed a different set of transcriptional regulators, as weil as markers of neurogenesis. The gene expression profiles indicate that ES cells actively suppress differentiation by transcriptional repression; cell-cell contact in embryoid bodies and retinoic acid treatment may overcome this suppression, allowing expression of Hox genes and inducing a suite of neuronal genes. Gene expression profiles will be a useful outcome measure for comparing in vitro treatrnents of differentiating ES cells and other stem cells. Also, knowing the molecule phenotype of transplantable cells will allow correlation of phenotype with the success of the transplant.

Genetic subtraction studies may be useful tools for neural repair research by identifying genes expressed under one condition, but not under another. However, these studies suffer from some limitations, including a lack of heterogeneity of subtracted cDNA pools and the difficulty of screening out false positives in the subtracted pools. Our strategy to overcome these difficulties was to combine one subtractive method - representational difference analysis - with screening of the subtracted products using a custom CDNA microarray. Using the neurosphere culture system, we have used this stepwise approach in order to identify genes that are selectiveley expressed by CNS progenitor cells, but not by more differentiated cells. Following microarray screening, we confirmed the localization of putatively differentially expressed clones by in situ hybridization analysis. These genes, both novel and previously identified, now become candidate therapeutic targets for CNS repair strategies

EGF-responsive striatal progenitor cells from rat brain have been maintained in culture in the form of neurospheres for six years without exhausting their renewal capacity. The events surrounding differentiation of stem cells in the brain after a long progenitorship remain a mystery. Using DNA microarray analysis we investigated differential gene expression, comparing progenitor cells in their neurosphere state with the cells 24 hours after induction of differentiation. Eighty-one genes showed increased expression in the differentiated condition. Genes associated with cellular growth, neurite outgrowth, and synaptogenesis were activated, including both anti-apoptotic and pro-apoptotic genes. Two transmitter- related genes, acetylcholine receptor-ß and glutamate receptor-ß-unit were also elevated-, these genes not only fit the profile of early neural development, but also reflect the characteristics of striatal neurons. In addition, there are approximately 30 expressed sequence tags (ES7) increased during neural differentiation. Forty-seven genes showed decreased expression; half of them are known genes related to the cell cycle, cell adhesion, transcription, and signaling. Tbe signaling and cell cycle genes may be responsible for the life-long self-renewal. These data demonstrate for the first time that life-long quiescent stem cells retain the potential to become activated and develop into specific types of brain cells. The six-year long-term neural stem cells are an excellent model for studying developmental neurobiological processes and aging.

Recent advances in DNA microarray technology have enabled the simultaneous evaluation of thousands of genes and the subsequent generation of massive amounts of biological data relevant to injury or diseases of the central nervous system (CNS). This technology has the potential to bridge the gap between molecular and systems neuroscience by efficiently revealing the discrete molecular aspects underlying the perturbations of complex systemic insults such as those resulting from traumatic brain injury (TBI). One of the more intriguing and as of yet not understood aspects of TBI that can be efficiently explored with DNA microarrays, is the sequence of molecular events that results in pronounced cell death in specific areas of the brain. The elucidation of these changes in gene expression underlying the mechanism of cell death following brain injury is of central importance in the design of future therapeutic agents. This review focuses on the technical aspects of microarray manufacture (photolithography, microspotting, and ink jet technology) and their utility in elucidating the molecular sequelae of brain injury.

Cluster analysis is a computational method that groups together similarly-shaped patterns. It may be applied to large-scale gene expression data to form new hypotheses regarding gene function. In the present study, we clustered the temporal expression patterns of genes expressed in the rat hippocampus during normal development and after a kainate-induced seizure injury at postnatal day 25. We found that two different methods, Euclidean hierarchical and K-means clustering, produced slightly different results, and concluded that different clustering methods may he used to complement one another. We also found that certain genes cluster together both during development and after seizure injury, consistent with the idea of sets of genes that act in concert under various conditions.

EDNA microarrays provide an efficient method to analyze gene expression patterns in thousands of genes in parallel. In some cases, large unfocused collections of cDNAs have been used in hybridization studies, in others small logically defined collections of tissue specific arrays have been used. Here we describe the bioinformatic selection of 1152 named human cDNAs specifically designed for neuroscience applications, arrayed on nylon membranes at high density. cDNAs were chosen which represent all the major cellular types of the brain including; neurons, astrocytes, microglia, and oligodendrocytes. Gene families chosen include cell type specific markers, ion-channels, transporters, receptors, and cell adhesion molecules among many others. These arrays were used with region specific samples from human brain to determine MRNA expression profiles for each region. Used with 33_p labeled complex probes, this is a low cost, highly sensitive approach for tbc investigator to focus on tissue specifie genes of interest where sampies of limiting arnounts of RNA are used. This selected set of brain-rele vant cDNAs should be widely useful in the analysis of gene expression patterns from brain tissues as well as neural cell lines.

Alzheimer's disease (AD) is the most common form of dementia, affecting as many as four million elderly people. lt results from abnormal changes in the brain that most likely begin long before cognitive impairment and other clinical symptoms become apparent. Little is known about the changes preceding or accompanying initiation of the disease. Using cDNA microarray, we previously reported candidate gene products whose expression is altered in the cerebral cortex of cases at risk for AD dementia. However, it is possible that the cDNA microarray evidence might have underestimated post-transcriptional modifications, and as a result, provided only a partial view of the biological problem of interest. Based on this hypothesis, we initiated a series of parallel high-throughput proteomic studies. We found that, consistent with the EDNA microarray evidence, the expression of proteins involved in synaptic activities was also altered in the brains of early AD cases. These studies support the feasibility and usefulness of high-throughput CDNA and protein microarray techniques to examine the sequential changes of distinctive gene expression patterns in the brain as a function of the progression of AD dementia. Our preliminary results also support the utility of high-throughput proteomic methodologies as a means to identify novel AD biomarkers from cerebral spinal fluid and/or from serum.