Temperature is a key determinant of alpha- and beta-synuclein membrane interactions in neurons

Aggregation of alpha-synuclein (alpha S) leads to the hallmark neuropathology of Parkinson's disease (PD) and related synucleinopathies. alpha S has been described to exist in both cytosolic and membrane-associated forms, the relative abundance of which has remained unsettled. To study alpha S under the most relevant conditions by a quantitative method, we cultured and matured rodent primary cortical neurons for >17 days and determined alpha S cytosol:membrane distribution via centrifugation-free sequential extractions based on the weak ionic detergent digitonin. We noticed that at lower temperatures (4 degrees C or room temperature), alpha S was largely membrane-associated. At 37 degrees C, however, alpha S 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 alpha S and beta-synuclein (beta S) under various conditions that differed in temperature and digitonin concentration (200-1200 mu g/ml), we consistently found alpha S to be more membrane-associated than beta S. Both proteins, however, exhibited temperature-dependent membrane binding. Under the most relevant conditions (37 degrees C and 800 mu g/ml digitonin, i.e., the lowest digitonin concentration that extracted cytosolic GAPDH to near completion), cytosolic distribution was 49.8% +/- 9.0% for alpha S and 63.6% +/- 6.6% for beta S. PD-linked alpha S 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 alpha S proteostasis, such as testing the effects of genetic and pharmacological manipulations.