back to 1998 Society for Neuroscience Meeting

New Approaches and Technologies for Analysis of Neural Genes

Sponsored by:
The National Institute of Mental Health, NIH
(Division of Basic and Clinical Neuroscience Research)

Program

Saturday, November 7, 1998
1:00 - 500 p.m.
Los Angeles Convention Center, Room 301 A

Organizer: Hemin Chin, Ph.D., Genetics Research Branch, DBCNR, NIMH, NIH
	Speakers:
1:00 - 1:40 p.m.	Dan Geschwind, M.D., Ph.D. University of California at Los Angeles Neural Gene Discovery Using Genomic and Subtraction-Generated Libraries on DNA Microarrays: Progress and Pitfalls
1:50 - 2:30 p.m.	Karl K. Johe, Ph.D. NeuralSTEM Biopharmaceuticals Region-Specific CNS Stem Cells for Discovering Neural Differentiation Genes
2:40 - 3:20 p.m.	John L. Rubenstein, M.D., Ph.D. University of California at San Francisco Role of the Dlx Genes in Forebrain Development
3:30 - 4:10 p.m.	Donald B. Arnold, Ph.D. Harvard University Studying Transcription and Transport in Neurons using Biolistic Transfection of Brain Slices
4:20 - 5:00 p.m.	Robert M. Sapolsky, Ph.D. Stanford University Gene Therapy against Necrotic Neurological Insults

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  (Missing beginning and end portion. Audience questions inaudible.)

Abstract:

We have undertaken a multidisciplinary approach to studying genes that may contribute to cerebral lateralization (asymmetry) and the development of linguistic abilities. Two approaches that incorporate DNA microarray technology have been used. In the first, a powerful PCR-based technique, Representational Difference Analysis (RDA) has been used to identify genes that are differentially expressed by the developing left and right cerebral superior temporal gyrus (STG) and surrounding regions in the 19 week gestation human fetus, during the epoch of neurogenesis and neuronal migration, and antecedent to the development of gross cerebral asymmetries. Most cDNA subtraction techniques are inefficient in the CNS due to the complexity of low abundance messages present in the nervous system. RDA coupled with high throughput screening with 2-color fluorescent probes on cDNA microarrays increases the representation of rare-differentially expressed clones and improves screening throughput. Rather than completing the traditional 4 rounds of RDA, 400 clones from each of 4 rounds (total 1600) of an RDA subtracting L-R and R-L fetal STG were screened on microarrays. A significant proportion of genes in rounds two to four appear differentially expressed. Two to three rounds of RDA appear optimal for generating a significant percentage of clones that are differentially expressed, while preserving the complexity of the subtracted mixture in this system. Over half of the products in round 3 that were sequenced represent novel human genes. Despite the complexity of these tissues, the preliminary screen of 1600 RDA subtraction products has identified 2 candidate asymmetrically expressed genes. The RDA output was compared with the output of another powerful PCR-based subtraction technique, suppressive subtractive hybridization (SSH). No significant differential expression was detected in the screen of SSH, suggesting that RDA is a more powerful technique for comparing complex tissues. The results of quantitative analysis and secondary screens using RT-PCR will be presented and further directions discussed.

The second approach relies on the use of DNA microarrays for comparative genomic hybridization to aid in correlating a specific chromosomal region with cognitive and behavioral phenotypes in patients with Klinefelter's syndrome (KF; XXY). KF has a prevalence of 1/800 among males and typically presents as infertility. Many KF patients have verbal learning disabilities and other features that suggest that they have alterations in the typical pattern of cerebral dominance. Curiously, XXX females and XYY males, in addition to other unique features, both share developmental reading disability in common with XXY males. In comparison, Turner's syndrome patients have relative parity of verbal and performance measures. These data lead us to suspect that the specific cognitive defect seen in KF may be due to gene dosage, reflecting anomalous expression of a gene located in the non-inactivated pseudoautosomal regions (PAR) of the sex chromosomes. Although, alternative mechanisms such as parental imprinting or non-inactivation of a gene outside the PAR may be responsible, we hypothesize that some non-dyslexic KF subjects will carry deletions involving the responsible non-inactivated gene on the extra X chromosome. In order to detect these potential deletions in the Xp and Xq PARs, we have constructed an ordered microarray of inter-Alu amplified genomic fragments derived from mapped Cosmid and BAC clones. Thus far, we have been able to create an array with inter-Alu products spaced on average 150-200 Kb apart, allowing for the detection of deletions more than 10-fold above the resolution of routine cytogenetics. We demonstrate that 2-color fluorescent hybridization allows for quantitative determination of the 3:2 ratio of gene dosage necessary to identify deletions of one X PAR. Since no analysis of deletions in a size range below the level of routine cytogenetics has been done in KF patients, this work may provide a significant contribution to the understanding of the these patients even in the event that the culpable region lies outside the known PARs.

Region-Specific CNS Stem Cells for Discovering Neural Differentiation Genes
Karl K. Johe, Ph.D., NeuralSTEM Biopharmaceuticals

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Abstract:

CNS stem cells are the multipotential precursor cells of fetal and adult mammalian CNS that can differentiate into neurons, astrocytes, and oligodendrocytes. NeuralStem Biopharmaceuticals has developed the important technology to isolate, expand, and differentiate mammalian, including human, CNS stem cells from most regions of the brain and spinal cord. One immediate application of this technology is to produce functionally active human neurons in commercially useful quantities for discovering novel therapeutic genes and compounds for CNS disorders.

The CNS stem cell technology provides a controlled human neuronal culture system where the complex processes of neuronal birth, differentiation, and maturation occur in a predictable time schedule with a predictable outcome. This situation is rarely found during the normal CNS development since it is a continuous process. Thus, for discovering novel genes with therapeutic value by means of comparison and contrast, the CNS stem cell culture system is a cleaner, better defined, and more accurate source of genes than the developing brain and spinal cord.

The CNS stem cell cultures are not a single population of identical stem cells. Rather, they are a mixture of distinct stem cell populations with distinct intrinsic information for generating neuronal and perhaps even glial subtypes. Just what the degree of heterogeneity is within a given stem cell culture is yet to be determined. However, we know for certain that stem cells from different regions of fetal CNS generate different sets of region-specific neurons, such as dopaminergic neurons from ventral midbrain but not dorsal midbrain, cholinergic neurons from septum and spinal cord but not from hippocampus or cortex. The CNS stem cell culture system, therefore, recapitulates a significant period of CNS development in vivo. And this is precisely the period where genes of highest therapeutic potential such as neurotrophic factors, neuronal differentiation factors, neuronal phenotype-determining factors, and other potential drug targets are at play.

Role of the Dlx Genes in Forebrain Development
John Rubenstein M.D., Ph.D, Nina Ireland Laboratory of Developmental Neuroscience, UCSF

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Abstract:

The striatum has a central role in many neurobiological processes, yet little is know about the molecular control of its development. Inroads to this subject have been made due to the discovery of transcription factors, such as the Dlx genes, whose expression patterns suggest that they have a role in striatal development. There are four known Dlx genes (Dlx-1, -2, -5 and -6) that are expressed in the developing striatum; these genes are expressed at different stages of differentiation within the striatal anlage. Mice lacking Dlx-1, -2 and -5 have been generated, and the effects of these mutations on striatal development have been characterized. While the single mutants have only subtle abnormalities, the Dlx-1 & -2 double mutant has a severe defect in the development of the basal telencephalon. The Dlx-1 & -2 double mutants have a time-dependent block in striatal differentiation. In these mutants, early-born neurons migrate into a striatum-like region, which is enriched for markers of the striosome (patch) compartment. However, later-born neurons accumulate within the proliferative zone due to a block in their migration. Several lines of evidence suggest that mutations in Dlx-1 and Dlx-2 produce abnormalities in the development of the striatal subventricular zone, and in the differentiation of striatal matrix neurons. The Dlx-1 & -2 mutants not only affect differentiation of striatal projection neurons; the production of neocortical and olfactory bulb interneurons, which migrate from the proliferative zones of the basal ganglia, is also inhibited. Thus, the Dlx genes are required in the basal ganglia primordia for the production of a subventricular zone that produces the late-born striatal projection neurons and interneuron precursors that migrate into the olfactory bulb and neocortex.

Studying Transcription and Transport in Neurons using Biolistic Transfection of Brain Slices
Donald B. Arnold, Ph.D., Clapham Laboratory, HHMI, Children's Hospital, Boston

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Abstract:

The study of basic mechanisms of cellular specification in neurons has been hampered by the resistance of non mitotic neurons to conventional DNA transfection methods. I have adapted the biolistic transfection technique, which was originally developed for use in plant cells, to transfect cultured brain slices. Biolistics involves loading DNA onto 1 mm diameter gold beads and then "shooting" them into cells with a shock wave of high pressure helium. When used in combination with organotypic brain slices, it enables the transfection of isolated neurons in an environment where their morphology and connectivity is preserved. To study the developmental and dynamic regulation of gene expression, I examined the control of transcription of the Calbindin gene, which encodes a calcium binding protein that is highly and specifically expressed in Purkinje cells of the cerebellum at a very early stage in development. I found that the expression of calbindin and calmodulin II are under the control of a novel calcium sensitive promoter element. This element consists of a repeated 10 bp GA rich motif and is capable of mediating Purkinje cell-specific expression in cerebellar slices.

To understand mechanisms of synaptic targeting and transport, I used biolistic transfection to study localization of ion channels and PSD-95 in cortical pyramidal cells. I found that a single PDZ domain and the SH3 domain are necessary for targeting of PSD-95 to postsynaptic sites. In addition, the shaker potassium channel Kv1.4 was able to target PSD-95 to axons. These experiments provide evidence that the location of subcellular targeting for both PSD-95 and ion channels to which it binds is determined by signals on the channel.

Gene Therapy against Necrotic Neurological Insults
Robert M. Sapolsky, Ph.D., Stanford University

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