Novel Small Molecules Targeting the Intrinsically Disordered Structural Ensemble of α-Synuclein Protect Against Diverse α-Synuclein Mediated Dysfunctions

Tóth, Gergely; Neumann, Thomas; Berthet, Amandine; Masliah, Eliezer; Spencer, Brian; Tao, Jiahui; Jobling, Michael F.; Gardai, Shyra J.; Bertoncini, Carlos W.; Cremades, Nunilo; Bova, Michael P.; Ballaron, Stephen J.; Chen, Xiao Hua; Mao, Wenxian; Nguyen, Phuong; Tabios, Mariano; Tambe, Mitali A.; Rochet, Jean‐Christophe; Junker, Hans Dieter; Schwizer, Daniel; Sekul, Renate; Ott, Inge; Anderson, John P.; Szőke, Balázs; Hoffman, Wherly; Christodoulou, John; Yednock, Ted; Agard, David A.; McConlogue, Lisa

doi:10.1038/s41598-019-52598-4

Cited by 22 publications

(34 citation statements)

References 57 publications

(78 reference statements)

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“…In particular, Hsp70's impact on ASyn oligomerization, the initial step of fibrillization, has not been directly determined [25,26,27,28,30]. Using both previously reported assays [33] and a novel, biochemical ASyn oligomerization approach, we show that Hsp70 directly blocks ASyn oligomerization in a nucleotide-independent manner. Surprisingly, we show that a competitive inhibitor of the Hsp70 canonical substrate binding site, the NR peptide, does not block this action, indicating that a non-canonical site must be responsible for retarding ASyn oligomerization.…”

Section: Discussionmentioning

confidence: 88%

“…This scenario might be expected to result in untoward effects complicating therapeutic application for neurodegenerative diseases. One possible therapeutic approach targeting Hsp70's noncanonical action on ASyn would be the development of targeted ASyn pharmacological chaperones [33,47] which are small molecules that bind to and stabilize select ASyn conformations. Such compounds could impact ASyn's mode of interaction with Hsp70 allowing for enhanced blocking of ASyn oligomer formation.…”

Section: Discussionmentioning

confidence: 99%

“…Because fibrillization assays are complex and oligomers are the likely pathologic species, we reasoned that biochemical investigation of Hsp70's action on the earliest stages of ASyn oligomer formation would be most informative. In prior studies, we established biochemical ASyn oligomerization assays that are based on either the complementation of split Gaussia luciferase (gLuc) or FRET between fluorophores on separate ASyn molecules [33]. Here we develop an improved biochemical complementation assay using split NanoBit luciferase (nLuc)[34] tags placed on separate ASyn molecules (Fig 1).…”

Section: Novel Asyn Oligomerization Assaysmentioning

confidence: 99%

“…We next tested Hsp70 for impact on ASyn oligomerization. In three ASyn oligomerization assays, split Gaussia luciferase (gLuc) [33] (Fig 3A), nLuc ( Fig 3B), and FRET[33] (Fig 3C), Hsp70 impairs oligomer formation at similar IC50 concentrations (11.6 μM gLuc, 9.1 μM nLuc, 19.3 μM FRET). Hsp70 is unable to disassemble preformed oligomers (Fig 3D), which is consistent with previous findings [29].…”

Section: Hsp70 Blocks Asyn Oligomerization In An Atp Independent Mannermentioning

confidence: 99%

“…Note however, that nLuc signal developed at lower ASyn concentrations than the FRET signal, indicating the inherent avidity expected with any complementation system (Fig 1E vs 1F). The increased sensitivity is expected to be beneficial when exploring the effects of oligomerization modulators.Combined size exclusion chromatography and multi angle light scattering (SEC-MALS) analyses of either nLuc tagged (Fig 2A) or untagged ASyn (Fig 2B) species indicated that ASyn converts from a largely monomeric to a largely trimeric state over the incubation time course of the assays (24 hours for nLuc tagged species, 48 hours for untagged FRET assay species [33]), indicating that we are monitoring the early stages of oligomerization. Previously, it was shown that oligomers capable of seeding fibril formation can be formed in in vitro biochemical assays [36].…”

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confidence: 99%

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Hsp70 chaperone blocks α-synuclein oligomer formation via a novel engagement mechanism

Tao

Berthet

Citron

et al. 2020

Preprint

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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. ...

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Section: Discussionmentioning

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Section: Novel Asyn Oligomerization Assaysmentioning

confidence: 99%

Section: Hsp70 Blocks Asyn Oligomerization In An Atp Independent Mannermentioning

confidence: 99%

mentioning

confidence: 99%

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Hsp70 chaperone blocks α-synuclein oligomer formation via a novel engagement mechanism

Tao

Berthet

Citron

et al. 2020

Preprint

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The dopamine receptor agonist apomorphine stabilizes neurotoxic α‐synuclein oligomers

et al. 2021

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The misfolding and aggregation of the protein α-synuclein (aSyn) into potentially neurotoxic oligomers is believed to play a pivotal role in the neuropathogenesis of Parkinson's disease (PD). Herein, we explore how apomorphine (Apo), a nonselective dopamine D1 and D2 receptor agonist utilized in the therapy for PD, affects the aggregation and toxicity of aSyn in vitro. Our data indicated that Apo inhibits aSyn fibrillation leading to the formation of large oligomeric species (Apo-aSyn-O), which exhibit remarkable toxicity in mesencephalic dopaminergic neurons in primary cultures. Interestingly, purified Apo-aSyn-O, even at very low concentrations, seems to be capable of converting unmodified aSyn monomer into neurotoxic species. Collectively, our findings warn for a possible dangerous effect of Apo on aSyn misfolding/aggregation pathway.

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Cancer and Parkinson's Disease: Common Targets, Emerging Hopes

Filippou

Outeiro

2020

Movement Disorders

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Cancer and neurodegeneration are two major leading causes of morbidity and death worldwide. At first sight, the two fields do not seem to share much in common and, if anything, might be placed on opposite ends of a spectrum. Although neurodegeneration results in excessive neuronal cell death, cancer emerges from increased proliferation and resistance to cell death. Therefore, one might expect significant differences in the underlying pathophysiological mechanisms. However, the more we deepen our understanding of these two types of diseases, the more we appreciate the unexpected overlap between them. Although most epidemiological studies support an inverse association between the risk for development of neurodegenerative diseases and cancer, increasing evidence points to a positive correlation between specific types of cancer, like melanoma, and neurodegenerative diseases, like Parkinson's disease (PD). We believe that deciphering the molecular processes and pathways underlying one of these diseases may significantly increase our understanding about the other. Therefore, the identification of novel biomarkers and therapeutic approaches in cancer, may lead to improved diagnosis and treatment of neurodegeneration, and vice versa. In this Viewpoint, we summarize recent findings connecting both diseases and speculate that insights from one disease may inform on mechanisms, and help identify novel biomarkers and targets for intervention, possibly leading to improved management of both diseases. © 2020 International Parkinson and Movement Disorder Society

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Novel Small Molecules Targeting the Intrinsically Disordered Structural Ensemble of α-Synuclein Protect Against Diverse α-Synuclein Mediated Dysfunctions

Cited by 22 publications

References 57 publications

Hsp70 chaperone blocks α-synuclein oligomer formation via a novel engagement mechanism

Hsp70 chaperone blocks α-synuclein oligomer formation via a novel engagement mechanism

The dopamine receptor agonist apomorphine stabilizes neurotoxic α‐synuclein oligomers

Cancer and Parkinson's Disease: Common Targets, Emerging Hopes

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