Aggregation and cytotoxicity of misfolded ␣-synuclein is postulated to be crucial in the disease process of neurodegenerative disorders such as Parkinson's disease and DLB (dementia with Lewy bodies). In this study, we detected misfolded and aggregated ␣-synuclein in a Triton X-100 insoluble fraction as well as a high molecular weight product by gel electrophoresis of temporal neocortex from DLB patients but not from controls. We also found similar Triton X-100 insoluble forms of ␣-synuclein in an ␣-synuclein transgenic mouse model and in an in vitro model of ␣-synuclein aggregation. Introducing the molecular chaperone Hsp70 into the in vivo model by breeding ␣-synuclein transgenic mice with Hsp70-overexpressing mice led to a significant reduction in both the high molecular weight and detergent-insoluble ␣-synuclein species. Concomitantly, we found that Hsp70 overexpression in vitro similarly reduced detergent-insoluble ␣-synuclein species and protected cells from ␣-synuclein-induced cellular toxicity. Taken together, these data demonstrate that the molecular chaperone Hsp70 can reduce the amount of misfolded, aggregated ␣-synuclein species in vivo and in vitro and protect it from ␣-synuclein-dependent toxicity.␣-Synuclein is a natively unfolded molecule that can selfaggregate to form oligomers and fibrillar intermediates (1-5) that accumulate to form Lewy bodies (LBs) 1 and Lewy neurites in neurons at risk for degeneration in Parkinson's disease and dementia with Lewy bodies (DLB) (6 -14). For the most part, these ␣-synuclein aggregates are densely compact and can be immunostained for multiple additional components including ubiquitin, synphilin-1, and heat shock proteins (HSPs), which suggests that protein misfolding or degradation is altered in cells that develop LBs. Mouse models of ␣-synuclein aggregation exist that mimic the findings in human brains and show intracellular ␣-synuclein aggregates (15-17). Aggregated ␣-synuclein molecules are less detergent-soluble, and these detergent-insoluble species of ␣-synuclein can be detected in human brain, transgenic mouse models, and in vitro models (18 -21). Although it is known that the conformation of ␣-synuclein in LBs is significantly different from that in the neuropil (22), it is unclear which conformation of ␣-synuclein contributes to inclusion formation. In addition to the formation of intracellular aggregates, ␣-synuclein is also cytotoxic. It has been postulated that ␣-synuclein oligomers found in Parkinson's disease tissue by Western blot analysis represent the toxic species (5).HSPs belong to the family of chaperone proteins and are important in both refolding misfolded proteins and directing proteins toward proteasomal degradation (23-25). HSPs can be protective in several neurodegeneration models (26 -29), and recent data in the fly model suggest that overexpression of the molecular chaperone Hsp70 protects against ␣-synuclein-induced degeneration (26, 30). Hsp70 and its related co-chaperones may be important in ␣-synuclein misfolding. In fact, sever...

In the version of this caption initially published, the cover artwork was credited to Erin Dewalt, based on imagery from the author, rather than stating that it was created by Michael B. Battles and the design was by Erin Dewalt. The error has been corrected in the HTML and PDF versions of the caption. ERRATUM In the version of this article initially published, the genus name 'Mycoplasma' was incorrectly used in place of the correct 'Mycobacterium'. The error has been corrected in the HTML and PDF versions of the article. ERRATUM npg

␣-Synuclein is a major component of Lewy bodies, the pathological hallmark of Parkinson disease, dementia with Lewy bodies, and related disorders. Misfolding and aggregation of ␣-synuclein is thought to be a critical cofactor in the pathogenesis of certain neurodegenerative diseases. In the current study, we investigate the role of the carboxyl terminus of Hsp70-interacting protein (CHIP) in ␣-synuclein aggregation. We demonstrate that CHIP is a component of Lewy bodies in the human brain, where it colocalizes with ␣-synuclein and Hsp70. In a cell culture model, endogenous CHIP colocalizes with ␣-synuclein and Hsp70 in intracellular inclusions, and overexpression of CHIP inhibits ␣-synuclein inclusion formation and reduces ␣-synuclein protein levels. We demonstrate that CHIP can mediate ␣-synuclein degradation by two discrete mechanisms that can be dissected using deletion mutants; the tetratricopeptide repeat domain is critical for proteasomal degradation, whereas the U-box domain is sufficient to direct ␣-synuclein toward the lysosomal degradation pathway. Furthermore, ␣-synuclein, synphilin-1, and Hsp70 all coimmunoprecipitate with CHIP, raising the possibility of a direct ␣-synuclein-CHIP interaction. The fact that the tetratricopeptide repeat domain is required for the effects of CHIP on ␣-synuclein inclusion morphology, number of inclusions, and proteasomal degradation as well as the direct interaction of CHIP with Hsp70 implicates a cooperation of CHIP and Hsp70 in these processes. Taken together, these data suggest that CHIP acts a molecular switch between proteasomal and lysosomal degradation pathways.␣-Synuclein is a major component of Lewy bodies (LBs) 1 found in Parkinson disease, dementia with Lewy body disease (DLB), the Lewy body variant of Alzheimer disease, and glial cytoplasmic inclusions found in multiple system atrophy (1-3). In these neurodegenerative disorders, collectively referred to as synucleinopathies, LBs are characterized by abnormal fibrillar aggregates of ␣-synuclein protein in the cytoplasm of selective populations of neurons and glial cells (4 -7). Three different missense mutations in the ␣-synuclein gene (7-9) as well as triplication of the ␣-synuclein gene (10) have been shown to cause dominant inherited Parkinson disease in a small subset of human patients. Moreover, overexpression of ␣-synuclein in transgenic mice, flies, and viral vector-transduced rodents leads to ␣-synuclein pathology accompanied by neuronal dysfunction, the loss of synaptic terminals, and/or neuronal cell loss (11-16). These studies implicate a molecular pathological role of ␣-synuclein in Lewy body-related neurodegenerative diseases.In living cells, various stresses cause unfolded or misfolded proteins to accumulate. Heat shock proteins recognize misfolded proteins and aid refolding. In addition to chaperone activity, heat shock proteins have been shown to facilitate degradation of highly misfolded proteins by transferring them to the ubiquitin proteasome degradation system (17-21). Molecular chaperones...

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