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Department of Pharmacology
James H. Eberwine, Ph.D.

Education:

1978	B.S. (Biochemistry)	Yale University
1979	M.A. (Biochemistry)	Columbia University
1984	Ph.D. (Biochemistry)	Columbia University

Research Summary:
The research efforts of my laboratory are directed towards understanding the molecular basis of neuronal functioning. Our experimental approach is reductionist in nature and involves analysis of gene expression in individual cells dispersed in culture, in the live slice preparation or from fixed pathological tissue specimens. We have developed various procedures that have enabled the analysis of cellular functioning using single cells as the experimental model. These procedures include those that permit an analysis of the mRNA complement, the protein complement and an assessment of mRNA movement and translation within single cells. This level of analysis is important since the biochemical composition of an individual cell may be diluted by that of surrounding cells. We are currently generating molecular fingerprints of various cell types and disease states. When this is complete, we hope that it will be possible to alter the cellular response to various challenges by altering the levels of these biological processes in a predictable manner. As part of these studies, we are examining the role of subcellular localization of mRNAs in regulating cellular function. We have shown that multiple mRNAs are localized in neuronal dendrites and have provided a formal proof of local mRNA translation in dendrites. Further, we have recently shown that the intracellular sites of localization and translation of these mRNAs can be altered by synaptic stimulation highlighting for the first time that in vivo translation of a mRNA can occur at different rates in distinct regions of a single cell (translation is primarily exponential in dendrites and linear in the cell soma). These multigenic biological processes are modulated by the activity or RNA binding proteins (RBPs). To assess and quantitate the involvement of RBPs we have developed the first methodologies that permit analysis of their functioning in live cells. These insights into the cell biology of neuronal function highlight the complexities that remain to be understood.

 

Selected Key Publications:

Single Cell and Subcellular Genomics, Pharmacology Newsletter, Vol. 17, No. 1, 2009.
Figures from Single Cell and Subcellular Genomics, Pharmacology Newsletter, Vol. 17, No. 1, 2009.

Figure 1. Morphology Rat Hippocampal Neuron. Rat hippocampal neurons were visualized using fluorescence microscopy after immunocytochemical localization of NeuN (nuclear and somatic localization, Yellow) and MAP2 (somato-dendritic localization, Red) with nucleus visualization (DAPI-Blue) and dendritic spines localization (Phalloidin, Green). It is on the tip of dendritic spines where synapses form and is the site where cellular aspects of learning and memory are initiated. Axons are not visible under these staining conditions (Courtesy of Peter Buckley).

Figure 2.. Cell viability/cytotoxicity assay. Neurons bathed in Elk-1 GFP mRNA (left-hand image) vs. neurons bathed in Elk-1 GFP mRNA plus phototransfection (right-hand image) are shown. Note the presence of nuclear dye accumulation in the photoporated neuron (right panel, white arrow), and the absence of nuclear dye accumulation in the non-photoporated neuron (left panel, white arrow). DIC images were taken immediately after phototransfection, and overlayed with images showing nuclear dye accumulation taken 3 hours after phototransfection. Black arrow shows nuclear dye accumulation in a neighboring astrocyte.

Zhang H, Kacharmina J, Miyashiro K, Greene M & Eberwine J: Protein quantification from complex protein mixtures using a novel proteomics methodology with single cell resolution. Proc Natl Acad Sci 98: 5497-5502, 2001.

Job C & Eberwine J: Identification of sites for exponential translation in living dendrites. Proc Natl Acad Sci 10.1073/pnas23148569 (early edition).

Job C & Eberwine J: Localization and translation of mRNA in dendrites and axons. Nat Rev Neurosci 2: 889-898, 2001.

Miyashiro K, Beckel-Mitchener A, Purk TP, Belt B, Kelly A, Becker K, Barret T, Weiler IJ, Greenough W & Eberwine J: Deficits in cellular functioning associated with the loss of FMRP in the CNS of knockout mice as revealed by antibody positioned RNA amplification. Neuron 37: 417-431, 2003.

Marciano P, Brettschneider J, Davis J, Manduchi E, Eastman S, Raghupathi R, Saatman K, Speed T, Stoechert C, Eberwine J* & McIntosh T*: Neuron-Specific mRNA Complexity Responses during Hippocampal Apoptosis after TBI. J Neurosci 24: 2866-2876, 2004. (*Co-Senior Authors)