Parkinson's VPS35[D620N] mutation induces LRRK2-mediated lysosomal association of RILPL1 and TMEM55B.

We demonstrate that the Parkinson's VPS35[D620N] mutation alters the expression of ~220 lysosomal proteins and stimulates recruitment and phosphorylation of Rab proteins at the lysosome. This recruits the phospho-Rab effector protein RILPL1 to the lysosome where it binds to the lysosomal integral membrane protein TMEM55B. We identify highly conserved regions of RILPL1 and TMEM55B that interact and design mutations that block binding. In mouse fibroblasts, brain, and lung, we demonstrate that the VPS35[D620N] mutation reduces RILPL1 levels, in a manner reversed by LRRK2 inhibition and proteasome inhibitors. Knockout of RILPL1 enhances phosphorylation of Rab substrates, and knockout of TMEM55B increases RILPL1 levels. The lysosomotropic agent LLOMe also induced LRRK2 kinase-mediated association of RILPL1 to the lysosome, but to a lower extent than the D620N mutation. Our study uncovers a pathway through which dysfunctional lysosomes resulting from the VPS35[D620N] mutation recruit and activate LRRK2 on the lysosomal surface, driving assembly of the RILPL1-TMEM55B complex.

Parkinson's disease-associated 18 bp promoter variant of DJ-1 alters REST binding and regulates its expression.

Parkinson's disease (PD) is a progressive neurodegenerative disorder with a complex etiology. Presence of autosomal mutations in PARK7/DJ-1 gene has been associated with early-onset PD. Growing evidence has suggested that DJ-1 acts as a putative sensor of oxidative stress. Reduced levels of DJ-1 protein have been reported in the cerebrospinal fluid of sporadic PD patients. Several case-control association studies have identified DJ-1 g.168_185del (rs200968609) variants conferring susceptibility towards PD pathogenesis. Similarly, among the PD patients in eastern India, the deletion allele (g.168_185) of this DJ-1 promoter polymorphism was found to be associated with PD. Hence, we aimed to find out the functional contribution of this promoter variant of DJ-1 in PD pathogenesis. The expression of DJ-1 was observed to be significantly reduced in the presence of both deletion and duplication sequences as identified from the luciferase promoter activity assay. The transcription factor binding prediction tool identified DJ-1 promoter 18 bp insertion polymorphism as the only binding partner of REST (RE1 Silencing Transcription Factor). Transient Chromatin Immuno-precipitation (ChIP) assay further confirmed this prediction. Previous reports have highlighted the role of REST in regulating the expression of stress-responsive genes. Our study has identified the functional involvement of DJ-1 promoter variants and REST-mediated regulation of DJ-1 expression in PD pathogenesis.

Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms.

Autosomal dominant mutations in LRRK2 that enhance kinase activity cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. Here, we explore whether LRRK1, a less studied homolog of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that in LRRK1 knock-out cells, phosphorylation of Rab7A at Ser72 was most impacted. This residue lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinson's mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. The PPM1H Rab phosphatase when overexpressed dephosphorylates Rab7A. Finally, the interaction of Rab7A with its effector RILP is not affected by LRRK1 phosphorylation and we observe that maximal stimulation of the TBK1 or PINK1 pathway does not elevate Rab7A phosphorylation. Altogether, these findings reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology with distinct substrate specificity.

Structural Basis for Rab8a Recruitment of RILPL2 via LRRK2 Phosphorylation of Switch 2.

Rab8a is associated with the dynamic regulation of membrane protrusions in polarized cells. Rab8a is one of several Rab GTPases that are substrates of leucine-rich repeat kinase 2 (LRRK2), a serine/threonine kinase that is linked to Parkinson's disease. Rab8a is phosphorylated at T72 (pT72) in its switch 2 helix and recruits the phospho-specific effector RILPL2, which subsequently regulates ciliogenesis. Here, we report the crystal structure of phospho-Rab8a (pRab8a) in complex with the RH2 (RILP homology) domain of RILPL2. The complex is a heterotetramer with RILPL2 forming a central α-helical dimer that bridges two pRab8a molecules. The N termini of the α helices cross over, forming an X-shaped cap (X-cap) that orients Arg residues from RILPL2 toward pT72. X-cap residues critical for pRab8a binding are conserved in JIP3 and JIP4, which also interact with LRRK2-phosphorylated Rab10. We propose a general mode of recognition for phosphorylated Rab GTPases by this family of phospho-specific effectors.

Role of Apolipoprotein E, Cathepsin D, and Brain-Derived Neurotrophic Factor in Parkinson's Disease: A Study from Eastern India.

Parkinson's disease (PD) is a progressive neurodegenerative disease with complex etiology. Both genetic and environmental factors play significant role. Apart from candidate genes, some modifier genes have been reported to be associated with the altered risk of PD. Previous studies have identified Apolipoprotein E (APOE), Cathepsin D (CTSD), and Brain-Derived Neurotrophic Factor (BDNF) as key players of neurodegenerative pathways with their variants associated with different neurodegenerative diseases. Hence, this study aims to identify the potential role of these modifier genes in the pathogenesis of PD among Eastern Indian PD patients. A case-control study was performed using 302 clinically diagnosed PD patients and 304 ethnically matched controls. Promoter SNPs of APOE (rs449647, rs405509) and BDNF (rs56164415), and coding SNPs of APOE (rs429358, rs7412 resulting in ε2, ε3, and ε4 alleles), CTSD (rs17571), and BDNF (rs6265) were analyzed by PCR-RFLP and bidirectional sequencing. The effect of rs56164415 on BDNF expression was characterized by Luciferase assay. APOEε4 allele was significantly overrepresented (p value = 0.0003) among PD patients, whereas ε3 allele was predominant in the control population. The promoter haplotype (A-rs449647, G-rs405509) of APOE was preponderant among female PD patients posing risk. No association was found for CTSD polymorphism. The 'T/T' genotype of BDNF rs56164415 was overrepresented (p-value = 0.02) among early onset PD patients. Expression of BDNF for the 'T/T' variant was significantly lower (p-value = 0.012) than the 'C/C' variant, suggesting a possible role in PD pathogenesis. This study suggests that APOE and BDNF may serve as modifier loci among eastern Indian PD patients.

Potential Role of Brain-Derived Neurotrophic Factor and Dopamine Receptor D2 Gene Variants as Modifiers for the Susceptibility and Clinical Course of Wilson's Disease.

Wilson's disease (WD), an inborn error of copper metabolism caused by mutations in the ATPase copper transporting beta (ATP7B) gene, manifests variable age of onset and different degrees of hepatic and neurological disturbances. This complex phenotypical outcome of a classical monogenic disease can possibly be explained by modifier loci regulating the clinical course of the disease. The brain-derived neurotropic factor (BDNF), critical for the survival, morphogenesis, and plasticity of the neurons, and the dopamine receptor D2 (DRD2), one of the most abundant dopamine receptors in the brain, have been highlighted in the pathophysiology of various neuropsychiatric diseases. This study aims to identify the potential association between BDNF and DRD2 gene polymorphisms and WD and its clinical characteristics. A total of 164 WD patients and 270 controls from India were included in this study. Two BDNF polymorphisms [p.Val66Met (c.G196A) and c.C270T] and the DRD2 Taq1A (A2/A1 or C/T) polymorphism were examined for their association with WD and some of its clinical attributes, using polymerase chain reaction, restriction fragment length digestion, and bidirectional sequencing. The C allele and CC genotype of BDNF C270T were significantly overrepresented among controls compared to WD patients. In addition, a significantly higher proportion of the allele coding for Val and the corresponding homozygous genotype of BDNF Val66Met polymorphism was found among WD patients with age of onset later than 10 years. Furthermore, the A1A1 genotype of DRD2 Taq1A polymorphism was significantly more common among WD patients with rigidity. Our data suggest that both BDNF and DRD2 may act as potential modifiers of WD phenotype in the Indian context.

Influence of Apolipoprotein E polymorphism on susceptibility of Wilson disease.

Wilson disease (WD) is an autosomal-recessive disorder caused by mutations in the ATP7B gene leading to abnormal copper deposition in liver and brain. WD manifests diverse neurological and hepatic phenotypes and different age of onset, even among the siblings, with same mutational background suggesting complex nature of the disease and involvement of other candidate genes. In that context, Apolipoprotein E (APOE) and Prion Protein (PRNP) have been proposed to be potential candidates for modifying the WD phenotype and age of onset. This study aims to identify the contribution of APOE and PRNP polymorphisms on the variable phenotypic expression of Indian WD patients. A total of 171 WD patients and 291 controls from Indian population were included in this study. Two APOE cSNPs (rs429358 and rs7412) resulting in three isoforms and M129V (rs1799990) polymorphism of PRNP were examined for their association with WD and its clinical phenotypes. The APOE ԑ4 allele was found to be significantly overrepresented in WD patients compared to controls. However, the frequency of the APOE ԑ3 allele and ԑ3/ԑ3 genotype was significantly higher in WD patients without cognitive behavior impairment compared to the ones with the impairment. On the contrary, the PRNP allele representing Val129 was found to be present in higher proportion in WD patients with cognitive behavioral decline. Our data suggest that the APOE ԑ4 allele could act as a potential risk for the pathogenesis of WD. Also, APOE and PRNP might contribute toward the cognitive behavioral decline in a section of WD patients.