Neurodegeneration in Parkinson's disease is correlated with the occurrence of Lewy bodies, intracellular inclusions containing aggregates of the intrinsically disordered protein (IDP) α-Synuclein 1 . The aggregation propensity of α-Synuclein in cells is modulated by specific factors including posttranslational modifications 2,3 , Abelson-kinase-mediated phosphorylation 4,5 and interactions with intracellular machineries such as molecular chaperones, although the underlying mechanisms are unclear [6][7][8] . Here, we systematically characterize the interaction of molecular chaperones with α-Synuclein in vitro as well as in cells at the atomic level. We find that six vastly different molecular chaperones commonly recognize a canonical motif in α-Synuclein, consisting Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms *

Mutations in glucocerebrosidase (GBA) are the most prevalent genetic risk factor for Lewy body disorders (LBD)—collectively Parkinson’s disease, Parkinson’s disease dementia and dementia with Lewy bodies. Despite this genetic association, it remains unclear how GBA mutations increase susceptibility to develop LBD. We investigated relationships between LBD-specific glucocerebrosidase deficits, GBA-related pathways, and α-synuclein levels in brain tissue from LBD and controls, with and without GBA mutations. We show that LBD is characterised by altered sphingolipid metabolism with prominent elevation of ceramide species, regardless of GBA mutations. Since extracellular vesicles (EV) could be involved in LBD pathogenesis by spreading disease-linked lipids and proteins, we investigated EV derived from post-mortem cerebrospinal fluid (CSF) and brain tissue from GBA mutation carriers and non-carriers. EV purified from LBD CSF and frontal cortex were heavily loaded with ceramides and neurodegeneration-linked proteins including alpha-synuclein and tau. Our in vitro studies demonstrate that LBD EV constitute a “pathological package” capable of inducing aggregation of wild-type alpha-synuclein, mediated through a combination of alpha-synuclein–ceramide interaction and the presence of pathological forms of alpha-synuclein. Together, our findings indicate that abnormalities in ceramide metabolism are a feature of LBD, constituting a promising source of biomarkers, and that GBA mutations likely accelerate the pathological process occurring in sporadic LBD through endolysosomal deficiency.

The property of molecular chaperones to dissolve protein aggregates of Parkinson-related α-synuclein has been known for some time. Recent findings point to an even more active role of molecular chaperones preventing the transformation of α-synuclein into pathological states subsequently leading to the formation of Lewy bodies, intracellular inclusions containing protein aggregates as well as broken organelles found in the brains of Parkinson’s patients. In parallel, a short motif around Tyr39 was identified as being crucial for the aggregation of α-synuclein. Interestingly, this region is also one of the main segments in contact with a diverse pool of molecular chaperones. Further, it could be shown that the inhibition of the chaperone:α-synuclein interaction leads to a binding of α-synuclein to mitochondria, which could also be shown to lead to mitochondrial membrane disruption as well as the possible proteolytic processing of α-synuclein by mitochondrial proteases. Here, we will review the current knowledge on the role of molecular chaperones in the regulation of physiological functions as well as the direct consequences of impairing these interactions—i.e., leading to enhanced mitochondrial interaction and consequential mitochondrial breakage, which might mark the initial stages of the structural transition of α-synuclein towards its pathological states.

NMR spectroscopy is one of the basic tools for molecular structure elucidation. Unfortunately, resolution of the spectra is often limited by inter-nuclear couplings. The existing workarounds often alleviate the problem...

BACKGROUND: Tau aggregation in neurons and glial cells characterizes tauopathies as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). Tau proteolysis has been proposed as a trigger for tau aggregation and tau fragments have been observed in brain and cerebrospinal fluid (CSF). Our group identified a major tau cleavage at amino acid (aa) 224 in CSF; N-terminal tau fragments ending at aa 224 (N-224) were significantly increased in AD and lacked correlation to total tau (t-tau) and phosphorylated tau (p-tau) in PSP and CBD. OBJECTIVE: Previous studies have shown cleavage from calpain proteases at sites adjacent to aa 224. Our aim was to investigate if calpain-1 or-2 could be responsible for cleavage at aa 224. METHODS: Proteolytic activity of calpain-1, calpain-2, and brain protein extract was assessed on a custom tau peptide (aa 220-228), engineered with fluorescence resonance energy transfer (FRET) technology. Findings were confirmed with in-gel trypsination and mass spectrometry (MS) analysis of brain-derived bands with proteolytic activity on the FRET substrate. Finally, knockdown of the calpain-2 catalytic subunit gene (CAPN2) was performed in a neuroblastoma cell line (SH-SY5Y). RESULTS: Calpain-2 and brain protein extract, but not calpain-1, showed proteolytic activity on the FRET substrate. MS analysis of active gel bands revealed presence of calpain-2 subunits, but not calpain-1. Calpain-2 depletion and chemical inhibition suppressed proteolysis of the FRET substrate. CAPN2 knock-down caused a 76.4% reduction of N-224 tau in the cell-conditio ned media. CONCLUSIONS: Further investigation of the calpain-2 pathway in the pathogenesis of tauopathies is encouraged.

Dysregulation of post‐translational modifications (PTMs) like phosphorylation is often involved in disease. NMR may elucidate exact loci and time courses of PTMs at atomic resolution and near‐physiological conditions but requires signal assignment to individual atoms. Conventional NMR methods for this base on tedious global signal assignment that may often fail, as for large intrinsically disordered proteins (IDPs). We present a sensitive, robust alternative to rapidly obtain only the local assignment near affected signals, based on FOcused SpectroscopY (FOSY) experiments using selective polarisation transfer (SPT). We prove its efficiency by identifying two phosphorylation sites of glycogen synthase kinase 3 beta (GSK3β) in human Tau40, an IDP of 441 residues, where the extreme spectral dispersion in FOSY revealed unprimed phosphorylation also of Ser409. FOSY may broadly benefit NMR studies of PTMs and other hotspots in IDPs, including sites involved in molecular interactions.

Dysregulation of post-translational modifications (PTMs) like phosphorylation is often involved in disease. NMR may elucidate exact loci and time courses of PTMs at atomic resolution and near-physiological conditions but requires signal assignment to individual atoms. Conventional NMR methods for this base on tedious global signal assignment that may often fail, as for large intrinsically disordered proteins (IDPs). We present a sensitive, robust alternative to rapidly obtain only the local assignment near affected signals, based on FOcused SpectroscopY (FOSY) experiments using selective polarisation transfer (SPT). We prove its efficiency by identifying two phosphorylation sites of glycogen synthase kinase 3 beta (GSK3b) in human Tau40, an IDP of 441 residues, where the extreme spectral dispersion in FOSY revealed unprimed phosphorylation also of Ser409. FOSY may broadly benefit NMR studies of PTMs and other hotspots in IDPs, including sites involved in molecular interactions.

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