Abstract
Aggregation of α-synuclein (αS) leads to the hallmark neuropathology of Parkinson’s disease (PD) and related synucleinopathies. αS has been described to exist in both cytosolic and membrane-associated forms, the relative abundance of which has remained unsettled. To study αS under the most relevant conditions by a quantitative method, we cultured and matured rodent primary cortical neurons for >17 days and determined αS cytosol:membrane distribution via centrifugation-free sequential extractions based on the weak ionic detergent digitonin. We noticed that at lower temperatures (4 °C or room temperature), αS was largely membrane-associated. At 37 °C, however, αS solubility was markedly increased. In contrast, the extraction of control proteins (GAPDH, cytosolic; calnexin, membrane) was not affected by temperature. When we compared the relative distribution of the synuclein homologs αS and β-synuclein (βS) under various conditions that differed in temperature and digitonin concentration (200–1200 μg/ml), we consistently found αS to be more membrane-associated than βS. Both proteins, however, exhibited temperature-dependent membrane binding. Under the most relevant conditions (37 °C and 800 μg/ml digitonin, i.e., the lowest digitonin concentration that extracted cytosolic GAPDH to near completion), cytosolic distribution was 49.8% ± 9.0% for αS and 63.6% ± 6.6% for βS. PD-linked αS A30P was found to be largely cytosolic, confirming previous studies that had used different methods. Our work highlights the dynamic nature of cellular synuclein behavior and has important implications for protein-biochemical and cell-biological studies of αS proteostasis, such as testing the effects of genetic and pharmacological manipulations.
Highlights
ΑS has been implicated as a key pathogenic protein in both sporadic and familial Parkinson’s disease (PD) since its discovery as the first PD-associated gene product [1] and the major constituent of Lewy bodies [2], the hallmark cytopathology of synucleinopathies
ACCELERATED COMMUNICATION: Temperature-dependent synuclein membrane interactions cortical neurons seem to exist in an equilibrium of soluble and membrane-associated forms. αS was found to be more strongly membrane-associated than βS, while both synucleins exhibit a pronounced temperature dependence of their membrane interactions: body temperature promotes cytosolic localization up to 50% or >60%, lower temperatures are associated with predominant membrane association of the synuclein proteins
To assess αS cytosol:membrane distribution in situ, i.e., without lifting cells off the culture dishes, we turned to digitonin-based sequential extraction of cellular proteins
Summary
ΑS has been implicated as a key pathogenic protein in both sporadic and familial PD (and related synucleinopathies) since its discovery as the first PD-associated gene product [1] and the major constituent of Lewy bodies [2], the hallmark cytopathology of synucleinopathies. Human wild-type (wt) αS expressed in rat neurons behaved to the endogenous protein while familial-PD (fPD)-linked αS A30P was found to be largely cytosolic, in line with previous studies that had used orthogonal methods [27]. Digitonin-based, centrifugation-free sequential extraction to achieve minimally disruptive cytosol:membrane protein separation αS has been demonstrated to exhibit several aspects of dynamic behavior in its natural cellular environment, as reviewed recently by Yeboah et al [28].
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