Autosomal recessive juvenile parkinsonism (AR-JP), one of the most common familial forms of Parkinson disease, is characterized by selective dopaminergic neural cell death and the absence of the Lewy body, a cytoplasmic inclusion body consisting of aggregates of abnormally accumulated proteins. We previously cloned PARK2, mutations of which cause AR-JP (ref. 2), but the function of the gene product, parkin, remains unknown. We report here that parkin is involved in protein degradation as a ubiquitin-protein ligase collaborating with the ubiquitin-conjugating enzyme UbcH7, and that mutant parkins from AR-JP patients show loss of the ubiquitin-protein ligase activity. Our findings indicate that accumulation of proteins that have yet to be identified causes a selective neural cell death without formation of Lewy bodies. Our findings should enhance the exploration of the molecular mechanisms of neurodegeneration in Parkinson disease as well as in other neurodegenerative diseases that are characterized by involvement of abnormal protein ubiquitination, including Alzheimer disease, other tauopathies, CAG triplet repeat disorders and amyotrophic lateral sclerosis.

␣-Synuclein contributes to the pathogenesis of Parkinson's disease (PD), but its precise role in the disorder and its normal function remain poorly understood. Consistent with a presumed role in neurotransmitter release and its prominent deposition in the dystrophic neurites of PD, ␣-synuclein localizes almost exclusively to the nerve terminal. In brain extracts, however, ␣-synuclein behaves as a soluble, monomeric protein. Using a binding assay to characterize the association of ␣-synuclein with cell membranes, we find that ␣-synuclein binds saturably and with high affinity to characteristic intracellular structures that double label for components of lipid rafts. Biochemical analysis demonstrates the interaction of ␣-synuclein with detergent-resistant membranes and reveals a shift in electrophoretic mobility of the raft-associated protein. In addition, the A30P mutation associated with PD disrupts the interaction of ␣-synuclein with lipid rafts. Furthermore, we find that both the A30P mutation and raft disruption redistribute ␣-synuclein away from synapses, indicating an important role for raft association in the normal function of ␣-synuclein and its role in the pathogenesis of PD.

Autosomal recessive juvenile parkinsonism (AR‐JP) is a distinct clinical entity characterized by a selective degeneration of nigral neurons. Recently, the parkin gene responsible for AR‐JP has been identified. Now, we report the subcellular localization of Parkin protein in patients with AR‐JP or Parkinson's disease (PD) and in controls by immunoblotting and immunohistochemistry using antibodies raised against the Parkin molecule. Parkin protein was absent in all regions of the brains of patients with AR‐JP. Parkin protein was not decreased in the brains of sporadic PD patients. Immunoreactivity was detected in a few Lewy bodies. Parkin protein was located in both the Golgi complex and cytosol. Ann Neurol 1999;45:668–672

Considerable genetic and pathological evidence has implicated the small, soluble protein ␣-synuclein in the pathogenesis of familial and sporadic forms of Parkinsons disease (PD). However, the precise role of ␣-synuclein in the disease process as well as its normal function remain poorly understood. We recently found that an interaction with lipid rafts is crucial for the normal, pre-synaptic localization of ␣-synuclein. To understand how ␣-synuclein interacts with lipid rafts, we have now developed an in vitro binding assay to rafts purified from native membranes. Recapitulating the specificity observed in vivo, recombinant wild type but not PD-associated A30P mutant ␣-synuclein binds to lipid rafts isolated from cultured cells and purified synaptic vesicles. Proteolytic digestion of the rafts does not disrupt the binding of ␣-synuclein, indicating an interaction with lipid rather than protein components of these membranes. We have also found that ␣-synuclein binds directly to artificial membranes whose lipid composition mimics that of lipid rafts. The binding of ␣-synuclein to these raft-like liposomes requires acidic phospholipids, with a preference for phosphatidylserine (PS). Interestingly, a variety of synthetic PS with defined acyl chains do not support binding when used individually. Rather, the interaction with ␣-synuclein requires a combination of PS with oleic (18:1) and polyunsaturated (either 20:4 or 22:6) fatty acyl chains, suggesting a role for phase separation within the membrane. Furthermore, ␣-synuclein binds with higher affinity to artificial membranes with the PS head group on the polyunsaturated fatty acyl chain rather than on the oleoyl side chain, indicating a stringent combinatorial code for the interaction of ␣-synuclein with membranes.

Leucine-Rich Repeat Kinase 2 (LRRK2) is a causative gene for the autosomal dominant form of Parkinson's disease (PD). The gene encodes the approximately 280 kDa LRRK2 protein composed of domains such as leucine-rich repeats, Ras in complex proteins (Roc) followed by C-terminal of Roc (COR), mitogen-activated protein kinase kinase kinase (MAPKKK) and WD40. However, the normal function of the protein as well as its contribution to the pathogenesis of PD remains largely unknown. Here we describe the localization of LRRK2 in Golgi apparatus, plasma membrane and synaptic vesicles in cultured cells including mouse primary neurons. The membrane association of LRRK2 resists solubilization by ice-cold 1% Triton X-100, indicating its association through lipid rafts. To investigate whether mutations found in PD patients affect the localization of LRRK2, we transfected various LRRK2 mutants into cultured cells and performed fractionation experiments. Unexpectedly, the mutants are collected in both membrane and soluble fractions in a manner similar to wild type (WT). I2020T mutant LRRK2 associates with lipid rafts, similar to the WT. The lipid raft association of LRRK2 mutants as well as WT LRRK2 suggests that alteration of LRRK2 function on lipid rafts contributes to the pathogenesis of PD.