Analysis of gene expression within the nervous system is particularly challenging because of the heterogeneity between cell types and the complex distributions patterns of specific mRNAs within single cells. Careful analysis of gene expression within the neurons must consider the fact that protein synthetic machinery is not restricted to the perikarya and that specific mRNAs can be localized to dendrites and subsynaptic locations. It is the contention of this proposal that conventional biochemical methods as well as micro-dissection approaches lack the ability to accurately measure gene expression within dendrites. This logic provides the rationale for this proposal and the need to develop technology capable of analysis of local changes in gene expression which are under regulatory control and vary during normal development, differentiation and disease processes. We have developed high resolution in situ hybridization methods to visualize the localization of mRNA within neurons. Further work is needed to develop methodology which is quantitative, highly sensitive and capable of analysis of changes in gene expression which are cell-type and/or cell- compartment specific. We propose to develop high-throughput technology in multi-color fluorescence in situ hybridization and non-isotopic methods at the ultrastructural level to permit quantitative analysis of gene transcription and localized gene expression within neurons. This proposal addresses four of the six areas of focus outlined in the RFA: (1) high throughput methods for quantifying the expression of multiple genes (2) methods for quantifying multiple spliced or edited variants of a given transcript (3) methods for comparing protein levels to corresponding mRNA levels within a cell (4) techniques for visualizing RNA distribution within cells. This approach has as its goal the assessment of the expression of many genes within a single cell. It relies on technology developed in the laboratory of the Co-Principal Investigator which allows the quantitative interrogation of sites of transcription within cells with a sensitivity sufficient to detect single molecules, or single nascent transcripts. This proposal describes a program of technological innovation based on the technical strengths of collaborations and existing expertise within the institution: that of FISH technology, primary neuronal culture, intracellular localization within neurons, slice cultures, long-term potentiation and in situ hybridization in brain sections.
| Muddashetty, Ravi S; Kelic, Sofija; Gross, Christina et al. (2007) Dysregulated metabotropic glutamate receptor-dependent translation of AMPA receptor and postsynaptic density-95 mRNAs at synapses in a mouse model of fragile X syndrome. J Neurosci 27:5338-48 |
| Eom, Taesun; Antar, Laura N; Singer, Robert H et al. (2003) Localization of a beta-actin messenger ribonucleoprotein complex with zipcode-binding protein modulates the density of dendritic filopodia and filopodial synapses. J Neurosci 23:10433-44 |
| Tiruchinapalli, Dhanrajan M; Oleynikov, Yuri; Kelic, Sofija et al. (2003) Activity-dependent trafficking and dynamic localization of zipcode binding protein 1 and beta-actin mRNA in dendrites and spines of hippocampal neurons. J Neurosci 23:3251-61 |
| Zhang, H L; Eom, T; Oleynikov, Y et al. (2001) Neurotrophin-induced transport of a beta-actin mRNP complex increases beta-actin levels and stimulates growth cone motility. Neuron 31:261-75 |
