HIGHLIGHTS a-Syn normally binds RA and translocates to the nucleus to regulate transcription Nuclear translocation of a-Syn is regulated by calreticulin and Ca 2+ Nuclear translocation of a-Syn is linked to PD

α-Synuclein (α-Syn) is a protein implicated in the pathogenesis of Parkinson’s disease (PD). It is an intrinsically disordered protein that binds acidic phospholipids. Growing evidence supports a role for α-Syn in membrane trafficking, including, mechanisms of endocytosis and exocytosis, although the exact role of α-Syn in these mechanisms is currently unclear. Here we investigate the associations of α-Syn with the acidic phosphoinositides (PIPs), phosphatidylinositol 4,5-bisphosphate (PI4,5P2) and phosphatidylinositol 3,4-bisphosphate (PI3,4P2). Our results show that α-Syn colocalizes with PIP2 and the phosphorylated active form of the clathrin adaptor protein 2 (AP2) at clathrin-coated pits. Using endocytosis of transferrin as an indicator for clathrin mediated endocytosis (CME), we find that α-Syn involvement in endocytosis is specifically mediated through PI4,5P2 levels on the plasma membrane. In accord with their effects on PI4,5P2 levels, the PD associated A30P, E46K and A53T mutations in α-Syn further enhance CME in neuronal and non-neuronal cells. However, Lysine to Glutamic acid substitutions at the KTKEGV repeat domain of α-Syn, that interfere with phospholipid binding, are ineffective in enhancing CME. We further show that the rate of synaptic vesicle (SV) endocytosis is differentially affected by the α-Syn mutations and associates with their effects on PI4,5P2 levels, however, with the exception of the A30P mutation. This study provides evidence for a critical involvement of PIPs in α-Syn-mediated membrane trafficking.

α-Synuclein is a protein involved in the pathogenesis of synucleinopathies, including Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). We investigated the role of neuronal α-Syn in myelin composition and abnormalities. The phospholipid content of purified myelin was determined by 31P NMR in two mouse lines modeling PD, PrP-A53T α-Syn and Thy-1 wt-α-Syn. Significantly higher levels of phospholipids were detected in myelin purified from brains of these α-Syn transgenic mouse models than in control mice. Nevertheless, myelin ultrastructure appeared intact. To further investigate the effect of α-Syn on myelin abnormalities, we systematically analyzed the striatum, a brain region associated with neurodegeneration in PD. An age and disease-dependent loss of myelin basic protein (MBP) signal was detected by immunohistochemistry in striatal striosomes (patches). The age-dependent loss of MBP signal was associated with lower P25α levels in oligodendrocytes. In addition, we found that α-Syn inhibited oligodendrocyte maturation and the formation of membranous sheets in vitro. Based on these results we concluded that neuronal α-Syn is involved in the regulation and/or maintenance of myelin phospholipid. However, axonal hypomyelination in the PD models is evident only in progressive stages of the disease and associated with α-Syn toxicity.

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Abstractα-Synuclein (α-Syn) is a protein implicated in the pathogenesis of Parkinson’s disease (PD). It is an intrinsically disordered protein that binds acidic phospholipids. Growing evidence supports a role for α-Syn in membrane trafficking, including, mechanisms of endocytosis and exocytosis, although the exact role of α-Syn in these mechanisms is currently unclear. Here we have investigated the role of α-Syn in membrane trafficking through its association with acidic phosphoinositides (PIPs), such as phosphatidylinositol 4,5-bisphosphate (PI4,5P2) and phosphatidylinositol 3,4-bisphosphate (PI3,4P2). Our results show that α-Syn colocalizes with PIP2 and the phosphorylated active form of the clathrin adaptor AP2 at clathrin-coated pits. Using endocytosis of transferrin, an indicator of clathrin mediated endocytosis (CME), we find that α-Syn involvement in endocytosis is specifically mediated through PI4,5P2 levels. We further show that the rate of synaptic vesicle (SV) endocytosis is differentially affected by α-Syn mutations. In accord with their effects on PI4,5P2 levels at the plasma membrane, the PD associated E46K and A53T mutations further enhance SV endocytosis. However, neither A30P mutation, nor Lysine to Glutamic acid substitutions at the KTKEGV repeat domain of α-Syn, that interfere with phospholipid binding, affect SV endocytosis. This study provides evidence for a critical involvement of PIPs in α-Syn-mediated membrane trafficking.Significance Statementα-Synuclein (α-Syn) protein is known for its causative role in Parkinson’s disease. α-Syn is normally involved in mechanisms of membrane trafficking, including endocytosis, exocytosis and synaptic vesicles cycling. However, a certain degree of controversy regarding the exact role of α-Syn in these mechanisms persists. Here we show that α-Syn acts to increase plasma membrane levels PI4,5P2 and PI3,4P2 to facilitate clathrin mediated and synaptic vesicles endocytosis. Based on the results, we suggest that α-Syn interactions with the acidic phosphoinositides facilitate a shift in their homeostasis to support endocytosis.