Alpha synuclein modulates mitochondrial Ca2+ uptake from ER during cell stimulation and under stress conditions

Abstract

Alpha synuclein (a-syn) is an intrinsically disordered protein prevalent in neurons, and aggregated forms are associated with synucleinopathies including Parkinson’s disease (PD). Despite the biomedical importance and extensive studies, the physiological role of a-syn and its participation in etiology of PD remain uncertain. We showed previously in model RBL cells that a-syn colocalizes with mitochondrial membranes, depending on formation of N-terminal helices and increasing with mitochondrial stress1. We have now characterized this colocalization and functional correlates in RBL, HEK293, and N2a cells. We find that expression of a-syn enhances stimulated mitochondrial uptake of Ca2+ from the ER, depending on formation of its N-terminal helices but not on its disordered C-terminal tail. Our results are consistent with a-syn acting as a tether between mitochondria and ER, and we show increased contacts between these two organelles using structured illumination microscopy. We tested mitochondrial stress caused by toxins related to PD, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP/MPP+) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and found that a-syn prevents recovery of stimulated mitochondrial Ca2+ uptake. The C-terminal tail, and not N-terminal helices, is involved in this inhibitory activity, which is abrogated when phosphorylation site serine-129 is mutated (S129A). Correspondingly, we find that MPTP/MPP+ and CCCP stress is accompanied by both phosphorylation (pS129) and aggregation of a-syn. Overall, our results indicate that a-syn can participate as a tethering protein to modulate Ca2+ flux between ER and mitochondria, with potential physiological significance. A-syn can also prevent cellular recovery from toxin-induced mitochondrial dysfunction, which may represent a pathological role of a-syn in the etiology of PD.

A-Syn(ful) MAM: A Fresh Perspective on a Converging Domain in Parkinson’s Disease

“…A comprehensive study led by Przedborski and Schon used several different techniques to demonstrate that wild-type monomeric αSyn is localized to ER–mitochondria contact sites at MAM domains and not within the mitochondria, as was previously thought [ 51 ]. Since then, several other studies from different groups have also demonstrated that αSyn can be found at MAM domains [ 52 , 53 , 54 , 55 ]. Yet, other studies have continued to associate αSyn within the mitochondria itself [ 56 , 57 , 58 ].…”

“…Lastly, since the ER is the primary site for intracellular Ca 2+ storage, Ca 2+ homeostasis at MAM domains is crucial in ER and mitochondrial communication, with the tight control of mitochondrial Ca 2+ import essential for cellular homeostasis. To investigate Ca 2+ homeostasis, independent measurements of Ca 2+ stores at the ER and Ca 2+ import into the mitochondria have been routinely performed, thereby indirectly assessing Ca 2+ homeostasis at MAMs [ 53 , 95 ]. Improved developments in genetically encoded calcium indicators (GECIs), such as fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET), have better enabled the steady-state assessment of Ca 2+ concentration [ 96 , 97 ].…”

“…αSyn has been found at the MAM in different studies [ 52 , 53 , 55 , 61 ]. Increasing levels of αSyn have been shown to increase the number of ER–mitochondria contact sites and consequently increase mitochondrial Ca 2+ uptake from the ER [ 53 ].…”

“…αSyn has been found at the MAM in different studies [ 52 , 53 , 55 , 61 ]. Increasing levels of αSyn have been shown to increase the number of ER–mitochondria contact sites and consequently increase mitochondrial Ca 2+ uptake from the ER [ 53 ]. In contrast, pathogenic point mutations in αSyn have been shown to reduce ER–mitochondria apposition, communication, and MAM activity [ 61 ].…”

“…A recent study by Ramezani and colleagues found that αSyn has a tight “dual-anchor” attachment to the ER and mitochondria and could itself regulate the MAM by tightening and loosening ER–mitochondria contact sites [ 53 ]. Using small unilamellar vesicles (SUVs) as a model to mimic the composition and curvature of synaptic vesicles, this work led by Eliezer and Baird proposed that αSyn can function as a double-anchor, binding two synaptic vesicles 50 nm across and thereby tightening their interaction [ 31 , 235 ].…”