Molecular Chaperones and Huntington’s Disease

Donald B. DeFranco, Ph.D.
Department of Pharmacology
University of Pittsburgh School of Medicine

Abstract

Huntington disease (HD) is a genetic disorder of the central nervous system and is inherited as an autosomal dominant condition. In affected individuals, one gene of a gene pair s not functioning correctly and dominates the other working gene. HD belongs to a family of dominantly inherited neurodegenerative diseases that are characterized by the presence of a greater number of polyglutamine (polyQ) repeats in the disease gene product than would be found in the general population. The expanded number of polyQ repeats within huntingtin, the HD gene product, leads primarily to selective degeneration of striatal and cortical neurons.

Degenerating neurons in polyQ diseases accumulate neuronal inclusions within the cell’s nucleus that contain aggregate polyQ protein. Expression of polyQ-expanded proteins activates a molecular chaperone response leading to induction of various heat shock proteins (HSPs), such as HSP 70 and recruitment of these HSPs into inclusions. Overexpressoin of an HSP 70 partner, the “DnaJ protein,” reduces aggregation of some polyQ-expanded disease proteins and toxicity in vitro. The mechanism of this in vitro rescue is unknown and it is unclear why differences in chaperone rescue exist for distinct polyQ-expanded disease proteins.

Dr. DeFranco and his associates will examine the mechanism of molecular chaperone rescue of polyQ-expanded huntingtin aggregation and toxicity using various in vitro models. An understanding of the cellular factors and conditions that limit chaperone rescue of polyQ protein aggregation and toxicity in vitro could aid in the design of therapeutic approaches to exploit molecular chaperones for the management of diseases associated with protein misfolding.

Progress Report (as of 8/2002)

Huntington's Disease (HD) is a neurodegenerative disease that is caused by expanded CAG repeats in the coding sequence of the disease gene-huntingtin (htt). Expansion of CAG repeats in the htt gene leads to the expression of mutant protein with an expanded number of glutamine (Q) residues. The most affected area in HD is the striatum and the cortex. The most important pathological feature of HD is the formation of intracellular inclusions (aggregates) that contain polyglutamine-expanded htt protein. Ubiquitin, proteasome subunits and molecular chaperones have also been found to be recruited into these aggregates indicating that aggregation is likely to be caused by protein misfolding. Recently, it has been found that several proteins involved in transcriptional regulation are recruited into the polyQ-expanded htt aggregates, which leads to a loss of their function. For example, CBP (CREB binding protein) is found to be recruited into the nuclear and cytoplasmic aggregates in cell culture and transgenic mouse models of HD and in postmortem brain of HD patients. CBP's affect on transcription is compromised when it is recruited into polyQ-expanded htt aggregates.

We have used HT22 cells, a hippocampal cell line, to investigate the role of CBP in HD. We found that CBP behaves differently in cells expressing htt-polyQ (htt-99Q) aggregates. In cells that express htt-99Q that localizes to nuclear aggregates, CBP is either recruited into the aggregates or is degraded. In contrast, in cells that express htt-99Q in cytoplasmic aggregates, CBP remains diffusely distributed within nuclei. Nuclear localization of the Sp1 transcription factor is not affected by the presence of either nuclear or cytoplasmic htt-99Q aggregates. Ubiquitin and the molecular chaperone Hdj2 are both recruited into nuclear and cytoplasmic htt-99Q aggregates. Since CBP behaves differently between nuclear and cytoplamic aggregates expressing cells, and nuclear localization of htt-polyQ is more often associated with toxicity than cytoplasmic localization, we investigated whether the localization of aggregates affect cell toxicity within individual HT22 cells. We used a co-immunofluorescence method in which an antibody is used to detect htt-polyQ, and Tunnel staining to indicate DNA damage and loss of cell viability. We found that approximately 80% of the cells that contain nuclear aggregates also exhibit DNA damage (i.e. positive for Tunnel staining), while only 20% of cells which contain cytoplasmic aggregates are Tunnel positive.

Our data suggests that besides being recruited into polyQ aggregates, CBP can also be degraded. This suggests that the Ubiquitin-Proteasome Pathway (UPP) is still active, or perhaps hyperactive, towards specific substrates in cells expressing htt-polyQ aggregates. The fact that inhibition of the UPP blocks CBP degradation in cells expressing htt-99Q confirms the enhanced action of the proteasome on CBP.

Publications

Haibing Jiang, Fred C. Nucifora, Christopher A. Ross and Donald B. DeFranco. Cell death triggered by polyglutamine-expanded huntingtin in a neuronal cell line is associated with both the recruitment and degradation of CREB-Binding Protein (CBP). Hum. Mol. Genet., submitted 7/02.

Funding

Submitted in 2001:

“Molecular Chaperones and Huntington’s Disease”
Hereditary Disease Foundation
Not recommended for funding on first submission.

Planned Submission in 2002
“Alterations in the Ubiquitin-Proteasome Degradation System by Polyglutamine-expanded Huntingtin.
Hereditary Disease Foundation


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