A NAC domain mutation (E83Q) unlocks the pathogenicity of human alpha-synuclein and recapitulates its pathological diversity

Abstract

The alpha-synuclein mutation E83Q, the first in the NAC domain of the protein, was recently identified in a patient with dementia with Lewy bodies. We investigated the effects of this mutation on the aggregation of aSyn monomers and the structure, morphology, dynamic, and seeding activity of the aSyn fibrils in neurons. We found that it markedly accelerates aSyn fibrillization and results in the formation of fibrils with distinct structural and dynamic properties. In cells, this mutation is associated with higher levels of aSyn, accumulation of pS129, and increased toxicity. In a neuronal seeding model of Lewy body (LB) formation, the E83Q mutation significantly enhances the internalization of fibrils into neurons, induces higher seeding activity, and results in the formation of diverse aSyn pathologies, including the formation of LB-like inclusions that recapitulate the immunohistochemical and morphological features of brainstem LBs observed in brains of patients with Parkinson’s disease.

Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity

“…The pattern of pS129 non-specific immunoreactivity detected by WB could be mistaken for pS129 species seen in the aSyn neuronal seeding models. It is noteworthy that under conditions where significant pS129 immunoreactive aSyn aggregates are formed, these non-specific bands become less prominent 37 . These findings underscore the importance of not only validating the pS129 antibodies but also using the appropriate (1) control samples (e.g., recombinant WT and pS129 and lysates from aSyn KO neurons or cells that do not Forty nanograms of recombinant aSyn WT or phosphorylated at residue S129 (pS129, indicated by the red arrow) were used as positive controls.…”

“…We have recently shown that the nature of the PFF seeds is a strong determinant of the morphological diversity of aSyn aggregates we detect in our seeding neuronal model of LB-like inclusion formation. When hippocampal or cortical primary neurons are treated with mouse WT PFF, newly formed fibrils appear mainly as filamentousand ribbon-like aggregates or LBlike inclusions at D21 35,37 (Supplementary Fig. 7A).…”

“…The mouse aSyn PFFs used in this study are from the same batch prepared and characterized in Mahul-Mellier et al 32 . The E83Q aSyn PFFs used in this study are from the same batch prepared, characterized and used in Kumar et al 37 . All procedures were approved by the Swiss Federal Veterinary Office (authorization numbers VD 3392 and VD 3694).…”

“…WT and aSyn KO primary hippocampal neurons untreated or treated with WT aSyn mouse 32 or E83Q human PFFs 37 were washed twice with PBS, fixed in 4% PFA for 20 min at RT, and then immunostained as previously described 32,35,95 . The antibodies used are indicated in the corresponding legend section of each figure.…”

Opportunities and challenges of alpha-synuclein as a potential biomarker for Parkinson’s disease and other synucleinopathies

“…Moreover, other aSyn point mutations, such as the G51D mutation, were reported to phenotypically display common neuropathological features of PD and MSA 31 , 32 . More recently, an E83Q mutation in aSyn was identified in a patient suffering from DLB and atypical frontotemporal lobar degeneration 27 , 33 . Third, the level of aSyn aggregates in the cerebrospinal fluid (CSF) and skin biopsies distinguishes PD patients from controls with high accuracy 34 – 36 .…”