Antony A. Cooper

Alpha-synuclein (alphaSyn) misfolding is associated with several devastating neurodegenerative disorders, including Parkinson's disease (PD). In yeast cells and in neurons alphaSyn accumulation is cytotoxic, but little is known about its normal function or pathobiology. The earliest defect following alphaSyn expression in yeast was a block in endoplasmic reticulum (ER)-to-Golgi vesicular trafficking. In a genomewide screen, the largest class of toxicity modifiers were proteins functioning at this same step, including the Rab guanosine triphosphatase Ypt1p, which associated with cytoplasmic alphaSyn inclusions. Elevated expression of Rab1, the mammalian YPT1 homolog, protected against alphaSyn-induced dopaminergic neuron loss in animal models of PD. Thus, synucleinopathies may result from disruptions in basic cellular functions that interface with the unique biology of particular neurons to make them especially vulnerable.

Heterozygous mutations in GBA1, the gene encoding lysosomal glucocerebrosidase, are the most frequent known genetic risk factor for Parkinson's disease. Reduced glucocerebrosidase and α-synuclein accumulation are directly related in cell models of Parkinson's disease. We investigated relationships between Parkinson's disease-specific glucocerebrosidase deficits, glucocerebrosidase-related pathways, and α-synuclein levels in brain tissue from subjects with sporadic Parkinson's disease without GBA1 mutations. Brain regions with and without a Parkinson's disease-related increase in α-synuclein levels were assessed in autopsy samples from subjects with sporadic Parkinson's disease (n = 19) and age- and post-mortem delay-matched controls (n = 10). Levels of glucocerebrosidase, α-synuclein and related lysosomal and autophagic proteins were assessed by western blotting. Glucocerebrosidase enzyme activity was measured using a fluorimetric assay, and glucocerebrosidase and α-synuclein messenger RNA expression determined by quantitative polymerase chain reaction. Related sphingolipids were analysed by mass spectrometry. Multivariate statistical analyses were performed to identify differences between disease groups and regions, with non-parametric correlations used to identify relationships between variables. Glucocerebrosidase protein levels and enzyme activity were selectively reduced in the early stages of Parkinson's disease in regions with increased α-synuclein levels although limited inclusion formation, whereas GBA1 messenger RNA expression was non-selectively reduced in Parkinson's disease. The selective loss of lysosomal glucocerebrosidase was directly related to reduced lysosomal chaperone-mediated autophagy, increased α-synuclein and decreased ceramide. Glucocerebrosidase deficits in sporadic Parkinson's disease are related to the abnormal accumulation of α-synuclein and are associated with substantial alterations in lysosomal chaperone-mediated autophagy pathways and lipid metabolism. Our data suggest that the early selective Parkinson's disease changes are likely a result of the redistribution of cellular membrane proteins leading to a chronic reduction in lysosome function in brain regions vulnerable to Parkinson's disease pathology.

Newly synthesized vacuolar hydrolases such as carboxypeptidase Y (CPY) are sorted from the secretory pathway in the late-Golgi compartment and reach the vacuole after a distinct set of membrane-trafficking steps. Endocytosed proteins are also delivered to the vacuole. It has been proposed that these pathways converge at a "prevacuolar" step before delivery to the vacuole. One group of genes has been described that appears to control both of these pathways. Cells carrying mutations in any one of the class E VPS (vacuolar protein sorting) genes accumulate vacuolar, Golgi, and endocytosed proteins in a novel compartment adjacent to the vacuole termed the "class E" compartment, which may represent an exaggerated version of the physiological prevacuolar compartment. We have characterized one of the class E VPS genes, VPS27, in detail to address this question. Using a temperature-sensitive allele of VPS27, we find that upon rapid inactivation of Vps27p function, the Golgi protein Vps10p (the CPY-sorting receptor) and endocytosed Ste3p rapidly accumulate in a class E compartment. Upon restoration of Vps27p function, the Vps10p that had accumulated in the class E compartment could return to the Golgi apparatus and restore correct sorting of CPY. Likewise, Ste3p that had accumulated in the class E compartment en route to the vacuole could progress to the vacuole upon restoration of Vps27p function indicating that the class E compartment can act as a functional intermediate. Because both recycling Golgi proteins and endocytosed proteins rapidly accumulate in a class E compartment upon inactivation of Vps27p, we propose that Vps27p controls membrane traffic through the prevacuolar/endosomal compartment in wild-type cells.