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In vivo and crude extract analysis of polyQ aggregation intermediates

Award Number: R15NS081681
ORGANIZATION: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 09/15/2012
PERIOD OF PERFORMANCE END DATE: 10/31/2016

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In vivo and crude extract analysis of polyQ aggregation intermediates - DESCRIPTION (provided by applicant): The over-arching goal of this project is to understand the molecular and cellular mechanisms that drive glutamine-repeat (polyQ) protein aggregation. Such polyQ sequences are found in at least twelve human genetic diseases including Huntington's disease and Machado-Joseph disease. It is well established from in vitro experiments that stable intermediates, or oligomers, may be present during the assembly of polyQ proteins into fibrils. Such oligomers have been shown to have cytotoxic and pathogenic properties in cells. We propose that native sequences flanking the polyQ stretches in mutant proteins will greatly influence the distribution of aggregation species in the context of the cell, and that the activities of molecular machinery that mitigate protein aggregation may be influenced by such flanking sequences. A recent study, using crude extracts from a mouse neuroblastoma cell line (2), has identified the presence of oligomers using the newly developed analytical ultracentrifuge with fluorescence detection capability. This technique will be used, combined with FRAP (fluorescence recovery after photobleaching, carried out using a confocal microscope), to probe the aggregation states of polyQ-containing proteins both in vivo and in crude extracts in the worm, C. elegans. The effect of protein flanking sequences will be explored on the relative distribution of oligomers, and its correlation to cytotoxicity. To further assess biological significance for the role of various aggregation states in disease, D. melanogaster will be used to discover how two well-studied chaperones (Hsp104 and Hsp70) might mitigate the effects of intermediate aggregation states, using the same set of fluorescence techniques. Key reagents and fly lines are being developed while the principal investigator is on sabbatical leave in the laboratory of Dr. Nancy Bonini, an HHMI investigator at the University of Pennsylvania. The Bonini laboratory has expertise in the study of the role of several polyQ- containing proteins on fly development and aging (3-8). Preliminary work with C. elegans will be carried out in the laboratory of Dr. Christopher Link, at the University of Colorado, Boulder in the summer of 2012. He has expertise in the use of this animal model to study protein aggregative diseases (9-14). The products of this critical work will be brought back to Haverford, and will provide an important set of tools for undergraduate students to use in their senior research work. Two faculty in the Biology Department at Haverford College already use these animal models for their research, so there is adequate infrastructure support in place, which will allow for a sharing of resources, and provide new synergies in the existing research programs. By involving undergraduate researchers in all aspects of the project, this award will also contribute significantly to the education and training of future generations of biomedical scientists. PUBLIC HEALTH RELEVANCE: Twelve human diseases involve polyQ-driven aggregation, and the pathway to sequestration into inclusion bodies or nuclear inclusions has been shown to be complex, involving the accumulation of toxic oligomers. New biophysical and imaging techniques have been recently developed that allow scientists to probe aggregation both in vivo and in crude extracts, and the PI plans to use fluorescence techniques (using a confocal microscope, and characterization of molecular populations using an analytical ultracentrifuge) to probe polyQ aggregation using D. melanogaster and C. elegans model systems. The ability to mitigate potentially toxic intermediate populations will be explored using chaperones, proteins whose function is to prevent or reduce protein aggregation.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2017 ( Subtotal = -$844 )
2017	2012	CORPORATION OF HAVERFORD COLLEGE, THE	370 LANCASTER AVE	HAVERFORD	PA	19041	MONTGOMERY	USA	Extramural Research Programs in the Neurosciences and Neurological Disorders	000	1	1/27/2017	NEW	-$844
														Subtotal = -$844

Issue Date FY: 2016 ( Subtotal = $0 )
2016	2012	HAVERFORD COLLEGE		HAVERFORD	PA	19041	MONTGOMERY	USA	Extramural Research Programs in the Neurosciences and Neurological Disorders	000	1	8/31/2016	NEW	$0
														Subtotal = $0

Issue Date FY: 2012 ( Subtotal = $350,612 )
2012	2012	HAVERFORD COLLEGE	370 Lancaster Ave	HAVERFORD	PA	19041	MONTGOMERY	USA	Extramural Research Programs in the Neurosciences and Neurological Disorders	000	1	9/12/2012	NEW	$350,612
														Subtotal = $350,612

Grand Total All Awards = $349,768

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In vivo and crude extract analysis of polyQ aggregation intermediates

Award Number: R15NS081681

ORGANIZATION: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 09/15/2012

PERIOD OF PERFORMANCE END DATE: 10/31/2016

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