Trafficking of the Menkes copper transporter ATP7A is regulated by clathrin-, AP-2-, AP-1-, and Rab22-dependent steps.

The transporter ATP7A mediates systemic copper absorption and provides cuproenzymes in the trans-Golgi network (TGN) with copper. To regulate metal homeostasis, ATP7A constitutively cycles between the TGN and plasma membrane (PM). ATP7A trafficking to the PM is elevated in response to increased copper load and is reversed when copper concentrations are lowered. Molecular mechanisms underlying this trafficking are poorly understood. We assess the role of clathrin, adaptor complexes, lipid rafts, and Rab22a in an attempt to decipher the regulatory proteins involved in ATP7A cycling. While RNA interference (RNAi)-mediated depletion of caveolin 1/2 or flotillin had no effect on ATP7A localization, clathrin heavy chain depletion or expression of AP180 dominant-negative mutant not only disrupted clathrin-regulated pathways, but also blocked PM-to-TGN internalization of ATP7A. Depletion of the µ subunits of either adaptor protein-2 (AP-2) or AP-1 using RNAi further provides evidence that both clathrin adaptors are important for trafficking of ATP7A from the PM to the TGN. Expression of the GTP-locked Rab22aQ64L mutant caused fragmentation of TGN membrane domains enriched for ATP7A. These appear to be a subdomain of the mammalian TGN, showing only partial overlap with the TGN marker golgin-97. Of importance, ATP7A remained in the Rab22aQ64L-generated structures after copper treatment and washout, suggesting that forward trafficking out of this compartment was blocked. This study provides evidence that multiple membrane-associated factors, including clathrin, AP-2, AP-1, and Rab22, are regulators of ATP7A trafficking.

The dyslexia-associated KIAA0319 protein undergoes proteolytic processing with {gamma}-secretase-independent intramembrane cleavage.

The KIAA0319 gene has been associated with reading disability in several studies. It encodes a plasma membrane protein with a large, highly glycosylated, extracellular domain. This protein is proposed to function in adhesion and attachment and thought to play an important role during neuronal migration in the developing brain. We have previously proposed that endocytosis of this protein could constitute an important mechanism to regulate its function. Here we show that KIAA0319 undergoes ectodomain shedding and intramembrane cleavage. At least five different cleavage events occur, four in the extracellular domain and one within the transmembrane domain. The ectodomain shedding processing cleaves the extracellular domain, generating several small fragments, including the N-terminal region with the Cys-rich MANEC domain. It is possible that these fragments are released to the extracellular medium and trigger cellular responses. The intramembrane cleavage releases the intracellular domain from its membrane attachment. Our results suggest that this cleavage event is not carried out by γ-secretase, the enzyme complex involved in similar processing in many other type I proteins. The soluble cytoplasmic domain of KIAA0319 is able to translocate to the nucleus, accumulating in nucleoli after overexpression. This fragment has an unknown role, although it could be involved in regulation of gene expression. The absence of DNA-interacting motifs indicates that such a function would most probably be mediated through interaction with other proteins, not by direct DNA binding. These results suggest that KIAA0319 not only has a direct role in neuronal migration but may also have additional signaling functions.

The dyslexia-associated protein KIAA0319 interacts with adaptor protein 2 and follows the classical clathrin-mediated endocytosis pathway.

Recently, genetic studies have implicated KIAA0319 in developmental dyslexia, the most common of the childhood learning disorders. The first functional data indicated that the KIAA0319 protein is expressed on the plasma membrane and may be involved in neuronal migration. Further analysis of the subcellular distribution of the overexpressed protein in mammalian cells indicates that KIAA0319 can colocalize with the early endosomal marker early endosome antigen 1 (EEA1) in large intracellular vesicles, suggesting that it is endocytosed. Antibody internalization assays with full-length KIAA0319 and deletion constructs confirmed that KIAA0319 is internalized and showed the importance of the cytoplasmic juxtamembranal region in this process. The present study has identified the medium subunit (mu2) of adaptor protein 2 (AP-2) as a binding partner of KIAA0319 in a yeast two-hybrid screen. Using Rab5 mutants or depletion of the mu-subunit of AP-2 or clathrin heavy chain by RNA interference, we demonstrate that KIAA0319 follows a clathrin-mediated endocytic pathway. We also identify tyrosine-995 of KIAA0319 as a critical amino acid required for the interaction with AP-2 and subsequent internalization. These results suggest the surface expression of KIAA0319 is regulated by endocytosis, supporting the idea that the internalization and recycling of the protein may be involved in fine tuning its role in neuronal migration.

Alpha-synuclein locus duplication as a cause of familial Parkinson's disease.

Genomic triplication of the alpha-synuclein gene (SNCA) has been reported to cause hereditary early-onset parkinsonism with dementia. These findings prompted us to screen for multiplication of the SNCA locus in nine families in whom parkinsonism segregates as an autosomal dominant trait. One kindred was identified with SNCA duplication by semiquantitative PCR and confirmed by fluorescent in-situ hybridisation analysis in peripheral leucocytes. By contrast with SNCA triplication families, the clinical phenotype of SNCA duplication closely resembles idiopathic Parkinson's disease, which has a late age-of-onset, progresses slowly, and in which neither cognitive decline nor dementia are prominent. These findings suggest a direct relation between SNCA gene dosage and disease progression.

CYP2D6 polymorphism, pesticide exposure, and Parkinson's disease.

We performed a case-control study of Parkinson's disease (PD) in a population characterized by a high prevalence of pesticide exposure and studied the joint effect of pesticide exposure and CYP2D6. Although they are based on a small group of subjects with the joint exposure, our findings are consistent with a gene-environment interaction disease model according to which (1) pesticides have a modest effect in subjects who are not CYP2D6 poor metabolizers, (2) pesticides' effect is increased in poor metabolizers (approximately twofold), and (3) poor metabolizers are not at increased PD risk in the absence of pesticide exposure.

S18Y polymorphism in the UCH-L1 gene and Parkinson's disease: evidence for an age-dependent relationship.

We studied the relationship between Parkinson's disease (PD) and the S18Y polymorphism in the UCH-L1 gene and the effect on this relationship of age at onset, smoking, and pesticides. Patients requested free health coverage for PD to the Mutualité Sociale Agricole (MSA), the French health insurance organization for people whose work is related to agriculture. Controls requested reimbursement of health expenses to the MSA. A maximum of three controls were matched to each case. Analyses included participants with both parents born in Europe. There were no differences in S18Y genotypes between patients (n = 209; 67% SS, 32% SY, 1% YY) and controls (n = 488; 66% SS, 30% SY, 4% YY). The relationship between PD and S18Y was modified by age at onset (P = 0.03). The Y allele was inversely associated with PD for patients with onset before 61 years (odds ratio [OR] = 0.53; 95% confidence interval [CI], 0.29-0.99); there was no association for older patients (62-68 years: OR = 1.21; 95% CI, 0.67-2.20; >68 years: OR = 1.24; 95% CI, 0.67-2.31). Among patients, Y carriers had a later onset than noncarriers (P = 0.04). These findings were not modified or confounded by smoking and pesticides. In this community-based case-control study, carriers of the Y allele were at decreased risk of developing PD at a young age, independently of pesticides and smoking.

Association between Parkinson's disease and polymorphisms in the nNOS and iNOS genes in a community-based case-control study.

Excess of nitric oxide (NO) has been shown to exert neurotoxic impacts in the brain. Moreover, inhibition of two NO-synthesizing enzymes, neuronal NOS (nNOS) and inducible NOS (iNOS), displays neuroprotective effects in the MPTP model of Parkinson's disease (PD). These data suggest a possible involvement of NOS as factors controlling the resistance of the nigral dopaminergic neurons to environmental insults. Therefore, we investigated whether polymorphisms present in these genes could contribute to the risk of developing PD. We carried out a community-based case-control study among subjects enrolled in the Mutualité Sociale Agricole, the French health insurance organization for workers connected to agriculture. Two-hundred and nine PD patients and 488 controls of European (mostly French) ancestry and matched for age, sex and region of residency were included in this study. Associations were observed with polymorphisms present in exon 22 of iNOS (OR for AA carriers=0.50, 95% CI=0.29-0.86, P=0.01) and in exon 29 of nNOS (OR for carriers of the T allele=1.53, 95% CI=1.08-2.16, P=0.02); no association was observed with a polymorphism in exon 18 of nNOS (OR for carriers of the T allele=1.20, 95% CI=0.85-1.69, P=0.30). Moreover, a significant interaction of the nNOS polymorphisms with current and ever cigarette smoking was found (nNOS 18, P=0.05; nNOS 29, P=0.04). All together, these data favour an involvement of these two genes as new modifier genes in PD.