Over-expression and aggregation of α-synuclein (ASyn) are linked to the onset and pathology of Parkinson's disease and related synucleinopathies. Elevated levels of the stress induced chaperone, Hsp70, protects against ASyn misfolding and ASyn-driven neurodegeneration in cell and animal models, yet there is minimal mechanistic understanding of this important protective pathway. It is generally assumed that Hsp70 binds to ASyn using its canonical and promiscuous substrate-binding cleft to limit aggregation. Here we report that this activity is due to a novel and unexpected mode of Hsp70 action, involving neither ATP nor the typical substrate-binding cleft. We use novel ASyn oligomerization assays to show that Hsp70 directly blocks ASyn oligomerization, an early event in ASyn misfolding. Using truncations, mutations and inhibitors, we confirmed that Hsp70 interacts with ASyn via an as yet unidentified, non-canonical interaction site in the C-terminal domain. Finally, a biological role for a non-canonical interaction was observed in H4 neuroglioma cells. Together, these findings suggest that new chemical approaches will be required to target Hsp70-ASyn interaction in synucleinopathies. Such approaches are likely to be more specific than targeting Hsp70 canonical actions. Additionally, these results raise the question of whether other misfolded proteins might also engage via the same non-canonical mechanism.KEYWORDS: α-Synuclein, chaperone, Hsp70, protein misfolding, Parkinson's Disease, Lewy body dementia, synucleinopathy Neuropathological, biochemical and genetic evidence strongly implicates α-Synuclein (ASyn) in the onset and progression of Parkinson's disease (PD) and related syncucleinopathies including Lewy body dementias (LBD), multiple systems atrophy and Alzheimer's disease. Aggregated ASyn inclusions are a hallmark of these diseases [1], and ASyn gene mutations or multiplications cause early onset PD or LBD [2,3,4]. The sequential misfolding of ASyn into oligomers and fibrils is central to the pathogenesis of the synucleinopathies. ASyn gene multiplications and mutations causing PD and LBD are associated with enhanced oligomer formation [5,6,7]. Significant evidence supports the hypothesis that prion-like transmission of misfolded ASyn underlies disease progression [8,9]. The severity of disease correlates with the progressive spread of aggregated ASyn in patients [10], and misfolding is associated with toxicity in cell and animal models [11]. As shown for other amyloid misfolding proteins, compelling evidence supports pre-fibrillar ASyn oligomers and not fibrillar deposits as the pathogenic species in disease [5,12,13,14,15,16]. ASyn oligomers are directly toxic to cells [16], and mutations enhancing ASyn oligomer formation increase ASyn toxicity in neurons and rodents [12,13,14].The constitutive (Hsc70) and inducible (Hsp70) forms of the 70-KDa cytosolic heat shock molecular chaperones (Hsp70s) assist a wide variety of folding processes and provide broad protection against protein misfolding in the cell. ...