Rab7l1 plays a role in regulating surface expression of toll like receptors and downstream signaling in activated macrophages.

Rab GTPases are known for controlling intracellular membrane traffic in a GTP-dependent manner. Rab7l1, belonging to family of Rab GTPases, is important for both endosomal sorting and retrograde transport. In our previous study, we identified a novel role of Rab7l1 in phagosome maturation. However, its role in regulating macrophage innate-effector signaling and cytokine response is not clearly understood. In this study, we have demonstrated that upon treatment of Rab7l1-knocked-down (Rab7l1-KD) THP-1 macrophages with lipopolysaccharide (LPS) and Pam3CSK4 has led to higher induction levels of tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) as compared to the control cells that received scrambled shRNA. Similar results were observed in Rab7l1-KD RAW 264.7 and Balb/c peritoneal macrophages. The phospho-ERK 1/2 (extracellular signal-regulated kinase 1/2) and phospho-p38 MAPK (mitogen-activated protein kinase) levels, known to be responsible for higher induction of TNF-α and IL-10 respectively, were higher in Rab7l1-KD THP-1 macrophages which also displayed higher nuclear translocation of p50/p65 nuclear factor kappa B (NF-κB) upon stimulation with LPS. Surface expression levels of toll-like receptor 2 (TLR2), TLR4 and CD14 receptors were higher in Rab7l1-KD THP-1 macrophages as compared to the control cells. However, intracellular levels of these receptors were lower in Rab7l1-KD THP-1 macrophages as compared to the control group. Together, our study suggests that Rab7l1 has a role in regulating MAPK signaling and cytokine effector responses in macrophages by regulating the surface expression of membrane receptors.

Rabaptin5 acts as a key regulator for Rab7l1-mediated phagosome maturation process.

Phagosome maturation is an important innate defence mechanism of macrophages against pathogen infections. Phagosome-lysosome (P-L) fusion is a highly regulated process. Different RabGTPases are involved in P-L fusion. Rab7l1 is shown to regulate P-L fusion process. In this study, we demonstrate that Rabaptin5 is a guanine nucleotide exchange factor (GEF) for Rab7l1. We reveal that Rabaptin5 interacts with Rab7l1-GTP form and promotes its recruitment to phagosome. In the absence of Rabaptin5, localization of P-L markers like EEA1, Rab7, LAMP1 and LAMP2 was found to be poorer. Thus, our data suggest that Rabaptin5 works upstream to Rab7l1 and triggers Rab7l1 activation for further recruitment of P-L markers and downstream regulation of phagosomal maturation process.

Expression of RAB7L1 in Patients with Pituitary Adenomas.

Pituitary adenomas (PA) are neoplasms that arise predominantly in the adenohypophysis. They are generally divided into three categories depending on their biological behavior: benign adenomas, invasive adenomas, and carcinomas. They represent 10%-25% of all intracranial neoplasms, and their estimated prevalence in the general population is 17%. RAB7L1, located at the PARK16 locus, is a Rab GTPase key regulator in vesicle trafficking. Recent genome-wide association studies have linked variants in RAB7L1 to the risk of Parkinson's disease. However, the association between RAB7L1 and PAs is still unexplored. Thirty patients with pituitary adenomas who had undergone surgical resection at Jiangyin People's Hospital (Jiangsu, China) from 2014 to 2019 were selected. The RAB7L1 expression level was assessed by qPCR, Western blot, and immunohistochemical staining. The level of the RAB7L1 differential expression was closely related to the patients' age and size of the PAs. In contrast, the RAB7L1 expression level was found to be unrelated to gender, Knosp classification, or type of PA. Our study found that the RAB7L1 expression level was higher in adenoma tissues from older PA patients, and the RAB7L1 expression level was higher in adenoma tissues from patients with smaller adenomas (size≤2cm) than those with larger adenomas (size>2cm).

RAB7L1 Participates in Secondary Brain Injury Induced by Experimental Intracerebral Hemorrhage in Rats.

RAB7, a member of RAS oncogene family-like 1 (RAB7L1), is a GTPase belonging to the Rab family and acts as an upstream regulator to regulate the kinase activity of leucine-rich repeat kinase 2 (LRRK2). Although LRRK2 has been shown to aggravate secondary brain injury (SBI) after intracerebral hemorrhage (ICH), it is unknown whether RAB7L1 is also involved in this process. The purpose of the present study was to investigate the role of RAB7L1 in ICH-induced SBI in vivo. Autologous blood was injected into adult male Sprague-Dawley rats to induce an ICH model in vivo. The results showed that the protein levels of RAB7L1 increased after ICH. Overexpression of RAB7L1 induced neuronal apoptosis and damage, as demonstrated by TUNEL-positive and FJB-positive cells, and exacerbated ICH-induced learning and cognitive dysfunctions; in contrast, downregulation of RAB7L1 via RNA interference yielded comparatively opposite changes in these parameters. In summary, this study demonstrates that RAB7L1 promotes SBI after ICH and may represent a potential target for ICH therapy.

The Parkinson's Disease Protein LRRK2 Interacts with the GARP Complex to Promote Retrograde Transport to the trans-Golgi Network.

Mutations in Leucine-rich repeat kinase 2 (LRRK2) cause Parkinson's disease (PD). However, the precise function of LRRK2 remains unclear. We report an interaction between LRRK2 and VPS52, a subunit of the Golgi-associated retrograde protein (GARP) complex that identifies a function of LRRK2 in regulating membrane fusion at the trans-Golgi network (TGN). At the TGN, LRRK2 further interacts with the Golgi SNAREs VAMP4 and Syntaxin-6 and acts as a scaffolding platform that stabilizes the GARP-SNAREs complex formation. Therefore, LRRK2 influences both retrograde and post-Golgi trafficking pathways in a manner dependent on its GTP binding and kinase activity. This action is exaggerated by mutations associated with Parkinson's disease and can be blocked by kinase inhibitors. Disruption of GARP sensitizes dopamine neurons to mutant LRRK2 toxicity in C. elegans, showing that these pathways are interlinked in vivo and suggesting a link in PD.

Comprehensive analysis of PM20D1 QTL in Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a complex disorder caused by a combination of genetic and non-genetic risk factors. In addition, an increasing evidence suggests that epigenetic mechanisms also accompany AD. Genetic and epigenetic factors are not independent, but multiple loci show genetic-epigenetic interactions, the so-called quantitative trait loci (QTLs). Recently, we identified the first QTL association with AD, namely Peptidase M20 Domain Containing 1 (PM20D1). We observed that PM20D1 DNA methylation, RNA expression, and genetic background are correlated and, in turn, associated with AD. We provided mechanistic insights for these correlations and had shown that by genetically increasing and decreasing PM20D1 levels, AD-related pathologies were decreased and accelerated, respectively. However, since the PM20D1 QTL region encompasses also other genes, namely Nuclear Casein Kinase and Cyclin Dependent Kinase Substrate 1 (NUCKS1); RAB7, member RAS oncogene family-like 1 (RAB7L1); and Solute Carrier Family 41 Member 1 (SLC41A1), we investigated whether these genes might also contribute to the described AD association. RESULTS: Here, we report a comprehensive analysis of these QTL genes using a repertoire of in silico methods as well as in vivo and in vitro experimental approaches. First, we analyzed publicly available databases to pinpoint the major QTL correlations. Then, we validated these correlations using a well-characterized set of samples and locus-specific approaches-i.e., Sanger sequencing for the genotype, cloning/sequencing and pyrosequencing for the DNA methylation, and allele-specific and real-time PCR for the RNA expression. Finally, we defined the functional relevance of the observed alterations in the context of AD in vitro. Using this approach, we show that only PM20D1 DNA methylation and expression are significantly correlated with the AD-risk associated background. We find that the expression of SLC41A1 and PM20D1-but not NUCKS1 and RAB7L1-is increased in mouse models and human samples of AD, respectively. However, SLC41A1 and PM20D1 are differentially regulated by AD-related stressors, with only PM20D1 being upregulated by amyloid-ß and reactive oxygen species, and with only PM20D1 being neuroprotective when overexpressed in cell and primary cultures. CONCLUSIONS: Our findings reinforce PM20D1 as the most likely gene responsible of the previously reported PM20D1 QTL association with AD.

Association of RIT2 and RAB7L1 with Parkinson's disease: a case-control study in a Taiwanese cohort and a meta-analysis in Asian populations.

Several genome-wide association studies and meta-analyses on Parkinson's disease (PD)-related genes have identified several risk foci in Ras-related genes, particularly among Caucasian individuals. However, the corresponding results have been controversial among Asian individuals. We investigated whether 2 single-nucleotide polymorphisms of Ras-related genes, RIT2 (rs12456492) and RAB7L1 (rs823118), are associated with PD risk in Taiwanese individuals. In addition, we conducted a meta-analysis of all studies related to rs12456492 in Asian populations to resolve inconsistency in this locus. In total, 1103 Taiwanese individuals (588 patients with PD and 515 controls) and 1111 Taiwanese individuals (594 patients with PD and 517 controls) were genotyped for rs12456492 and rs823118. However, we could not confirm the association of rs12456492 and rs823118 with PD. Our current meta-analysis involving the rs12456492(A/G) variant demonstrated that the GG + GA genotypes, GG genotypes, and G allele may be risk factors for PD. RIT2 may increase PD risk in Asian individuals. The discrepancies between Caucasian and Asian populations may be due to differences in geographic region-specific genetic backgrounds and gene-environmental interactions.

RAB7L1-Mediated Relocalization of LRRK2 to the Golgi Complex Causes Centrosomal Deficits via RAB8A.

Mutations in the LRRK2 gene cause autosomal-dominant Parkinson's disease (PD), and both LRRK2 as well as RAB7L1 have been implicated in increased susceptibility to idiopathic PD. RAB7L1 has been shown to increase membrane-association and kinase activity of LRRK2, and both seem to be mechanistically implicated in the same pathway. Another RAB protein, RAB8A, has been identified as a prominent LRRK2 kinase substrate, and our recent work demonstrates that aberrant LRRK2-mediated phosphorylation of RAB8A leads to centrosomal alterations. Here, we show that RAB7L1 recruits LRRK2 to the Golgi complex, which causes accumulation of phosphorylated RAB8A in a pericentrosomal/centrosomal location as well as centrosomal deficits identical to those observed with pathogenic LRRK2. The centrosomal alterations induced by wildtype LRRK2 in the presence of RAB7L1 depend on Golgi integrity. This is in contrast to pathogenic LRRK2 mutants, which cause centrosomal deficits independent of Golgi integrity or largely independent on RAB7L1 expression. Furthermore, centrosomal alterations in the presence of wildtype LRRK2 and RAB7L1 are at least in part mediated by aberrant LRRK2-mediated RAB8A phosphorylation, as abolished by kinase inhibitors and reduced upon knockdown of RAB8A. These results indicate that pathogenic LRRK2, as well as increased levels of RAB7L1, cause centrosomal deficits in a manner dependent on aberrant RAB8A phosphorylation and centrosomal/pericentrosomal accumulation, suggesting that centrosomal cohesion deficits may comprise a useful cellular readout for a broader spectrum of the disease.

Parkinson's disease-associated mutant LRRK2 phosphorylates Rab7L1 and modifies trans-Golgi morphology.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the major genetic cause of autosomal-dominantly inherited Parkinson's disease. LRRK2 is implicated in the regulation of intracellular trafficking, neurite outgrowth and PD risk in connection with Rab7L1, a putative interactor of LRRK2. Recently, a subset of Rab GTPases have been reported as substrates of LRRK2. Here we examine the kinase activity of LRRK2 on Rab7L1 in situ in cells. Phos-tag analyses and metabolic labeling assays revealed that LRRK2 readily phosphorylates Golgi-localized wild-type Rab7L1 but not mutant forms that are distributed in the cytoplasm. In vitro assays demonstrated direct phosphorylation of Rab7L1 by LRRK2. Subsequent screening using Rab7L1 mutants harboring alanine-substitution for every single Ser/Thr residue revealed that Ser72 is a major phosphorylation site, which was confirmed by using a phospho-Ser72-specific antibody. Moreover, LRRK2 pathogenic Parkinson mutants altogether markedly enhanced the phosphorylation at Ser72. The modulation of Ser72 phosphorylation in Rab7L1 resulted in an alteration of the morphology and distribution of the trans-Golgi network. These data collectively support the involvement of Rab7L1 phosphorylation in the LRRK2-mediated cellular and pathogenetic mechanisms.

Association between PARK16 and Parkinson's disease: A meta-analysis.

Recent years, several case-control studies reported that two polymorphisms (rs947211 and 1572913) within the PARK16 locus were associated with the Parkinson's disease (PD). However, the results were still controversial. Herein, we conducted a comprehensive meta-analysis to estimate the associations between two polymorphisms and PD. Seven databases (PubMed, Google Scholar, EMBASE, Web of Science, CNKI (China National Knowledge Infrastructure), VIP and Wanfang) were searched to identify the eligible studies. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to evaluate the associations of two polymorphisms with PD susceptibility. Totally, 15 studies with 6637 cases and 6774 controls were included in our meta-analysis. The results showed that rs947211 variants were associated with a decreased risk of PD in overall population. Stratified analysis found that rs947211 variants were associated with a significantly decreased risk of PD in Northeast Asian population, but a slightly decreased risk of PD in Southeast Asian and Caucasian population. With regard to rs1572913 polymorphism, the results suggested that rs1572913 variants contribute to decrease the risk of PD. Therefore, our meta-analysis suggested that rs947211 variants (A allele, AG and GG genotypes) may decrease the risk of PD in overall population, particularly in Northeast Asian population; and T allele, TC and TT genotypes of rs1572913 variants contributed to decrease the risk of PD.

C9orf72 and RAB7L1 regulate vesicle trafficking in amyotrophic lateral sclerosis and frontotemporal dementia.

A non-coding hexanucleotide repeat expansion in intron 1 of the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), however, the precise molecular mechanism by which the C9orf72 hexanucleotide repeat expansion directs C9ALS/FTD pathogenesis remains unclear. Here, we report a novel disease mechanism arising due to the interaction of C9ORF72 with the RAB7L1 GTPase to regulate vesicle trafficking. Endogenous interaction between C9ORF72 and RAB7L1 was confirmed in human SH-SY5Y neuroblastoma cells. The C9orf72 hexanucleotide repeat expansion led to haploinsufficiency resulting in severely defective intracellular and extracellular vesicle trafficking and a dysfunctional trans-Golgi network phenotype in patient-derived fibroblasts and induced pluripotent stem cell-derived motor neurons. Genetic ablation of RAB7L1or C9orf72 in SH-SY5Y cells recapitulated the findings in C9ALS/FTD fibroblasts and induced pluripotent stem cell neurons. When C9ORF72 was overexpressed or antisense oligonucleotides were targeted to the C9orf72 hexanucleotide repeat expansion to upregulate normal variant 1 transcript levels, the defective vesicle trafficking and dysfunctional trans-Golgi network phenotypes were reversed, suggesting that both loss- and gain-of-function mechanisms play a role in disease pathogenesis. In conclusion, we have identified a novel mechanism for C9ALS/FTD pathogenesis highlighting the molecular regulation of intracellular and extracellular vesicle trafficking as an important pathway in C9ALS/FTD pathogenesis.

Mechanisms of Mutant LRRK2 Neurodegeneration.

LRRK2 mutations are associated with the loss of neurons, that is to say toxicity, in patients and in experimental model systems. However, the mechanisms by which mutations can be linked to neurodegeneration are not fully defined. Here I will argue that mechanism in this context encompasses a variety of levels of information. Mutations or alterations in gene expression at a genetic level are one set of mechanisms that are reflected at the biochemical level likely in enhanced or persistent function of LRRK2. By impacting cellular pathways, prominently including changes in autophagy but also microtubule function, mitochondria and protein synthesis, in neurons and immune cells, the LRRK2 brain is primed for neurodegeneration in an age-dependent manner. These concepts have implications for not only modeling LRRK2 disease but also perhaps for Parkinson's disease more generally, including the development of therapeutic modalities.

RAB7L1 promoter polymorphism and risk of Parkinson's disease; a case-control study.

INTRODUCTION: Recent genome-wide association studies have explored some new loci in association with Parkinson's disease (PD). RAB7L1 is an important gene involved in one of the important neurological pathways, located in PARK16 locus. We performed a case-control study to examine the association between rs823144 SNP located in the promoter region of the RAB7L1 gene and PD risk in Iranian population. METHODS: A total of 960 samples including 480 PD patients and 480 healthy controls were collected for analysis of the RAB7L1 rs823144 polymorphism using polymerase chain reaction-restriction fragment length polymorphism (PCR - RFLP) method. RESULTS: We found significant differences in genotypic and allelic frequencies between patients and controls. Significant association was found between presence of minor allele (C) and decreased risk of PD development (p = 0.008, OR = 0.74 (0.605-0.924)). Also another significant association was observed between the CC genotype and PD (p = 0.004, OR = 0.441 (0.252-0.772)). CONCLUSION: Our data support the association between rs823144 and decreased risk of PD.

LRRK2 and Parkinson's Disease: From Lack of Structure to Gain of Function.

Mutations in LRRK2 comprise the most common cause for familial Parkinson's disease (PD), and variations increase risk for sporadic disease, implicating LRRK2 in the entire disease spectrum. LRRK2 is a large protein harbouring both GTPase and kinase domains which display measurable catalytic activity. Most pathogenic mutations increase the kinase activity, with increased activity being cytotoxic under certain conditions. These findings have spurred great interest in drug development approaches, and various specific LRRK2 kinase inhibitors have been developed. However, LRRK2 is a largely ubiquitously expressed protein, and inhibiting its function in some non-neuronal tissues has raised safety liability issues for kinase inhibitor approaches. Therefore, understanding the cellular and cell type-specific role(s) of LRRK2 has become of paramount importance. This review will highlight current knowledge on the precise biochemical activities of normal and pathogenic LRRK2, and highlight the most common proposed cellular roles so as to gain a better understanding of the cell type-specific effects of LRRK2 modulators.

Association of three candidate genetic variants in RAB7L1/NUCKS1, MCCC1 and STK39 with sporadic Parkinson's disease in Han Chinese.

Previous studies identified that polymorphisms RAB7L1/NUCKS1 rs823118, MCCC1 rs12637471 and STK39 rs1955337 to be the risk loci for Parkinson's disease (PD) in a Caucasian population. However, the characteristics of these three polymorphisms in a Han Chinese population from mainland China were unknown. We examined genetic associations of rs823118, rs12637471 and rs1955337 with PD susceptibility in a Han Chinese population of 1016 sporadic PD patients and 1069 controls. We also conducted further stratified analysis according to age at onset and compared the clinical characteristics between minor allele carriers and non-carriers for each locus. In this study, the minor allele frequency (MAF) was significantly different of RAB7L1/NUCKS1 rs823118 (P = 0.003) and MCCC1 rs12637471 (P = 0.008) between cases and controls. Subjects of RAB7L1/NUCKS1 rs823118 with CC+CT genotypes had a decreased risk compared to those with TT genotype (P = 0.001) and this association also can be seen among younger population (<50 years, P = 0.011). For the MCCC1 rs12637471, subjects with GA+GG genotypes had an increased risk compared to those with AA genotype (P = 0.017). However, we did not observe any significant difference in allele and genotype distribution between PD patients and controls for rs1955337 in STK39. In addition, minor allele carriers cannot be distinguished from non-carriers based on their clinical features of the three loci. Our study provides strong support for the susceptibility role of rs823118 and rs12637471 in sporadic PD in a Han Chinese population.

The rs1572931 polymorphism of the RAB7L1 gene promoter is associated with reduced risk of Parkinson's disease.

INTRODUCTION: Parkinson's disease (PD) is one of the most common neurologic disorders affecting about 2% of people over 65 years old and both genetic and environmental factors are involved in its aetiology. The genetic part includes several genes and polymorphisms that are the direct cause of disease or its susceptibility factor. The rs1572931 polymorphism of RAB7L1 gene, located in the promoter region, has been recently studied and shown to be strongly associated with reducing risk of PD. In this study, we aim to investigate its association with PD in Iranian population. METHODS: We examined the association of rs1572931 polymorphism with PD in 490 unrelated Patients and 490 normal controls by PCR-RFLP method in Iranian subjects. RESULTS: A significant difference in genotype and allele frequencies was observed between patients and controls (p value = 0.003, OR (95% CI) = 0.71(0.56-0.90)). The TT genotype and the T allele were both significantly less frequent in PD cases. CONCLUSION: Our results confirmed the protective effect of the rs1572931 SNP on PD and replicated the results of previous studies, in Iranian subjects. We suggest further studies in other populations.

An association analysis of the rs1572931 polymorphism of the RAB7L1 gene in Parkinson's disease, amyotrophic lateral sclerosis and multiple system atrophy in China.

BACKGROUND AND PURPOSE: Recently, the rs1572931 single-nucleotide polymorphism (SNP) of the putative promoter of the member RAS oncogene family-like 1 (RAB7L1) gene was reported to be associated with reduced risk for Parkinson's disease (PD) in the Ashkenazi Jewish population. Ethnic-specific effects are an important consideration in genome-wide association studies. Considering that the clinical manifestations and pathological characteristics overlap between PD, amyotrophic lateral sclerosis (ALS) and multiple system atrophy (MSA), the possible associations between the rs1572931 SNP and these three diseases were studied in the Chinese population. METHODS: A total of 1011 PD patients, 778 sporadic ALS (SALS) patients, 264 MSA patients and 516 healthy controls (HC) were included in this study. All subjects were genotyped for the rs1572931 SNP by using polymerase chain reaction and direct sequencing. RESULTS: Significant differences were observed in the genotype and minor allele frequency (MAF) of rs1572931 between PD and HC (P = 0.0001 and P = 1.08E-04, respectively) and between late-onset PD and matched controls (P = 0.0011 and P = 0.0002, respectively). However, no differences were observed between early-onset PD and HC. The number of minor allele carriers was significantly lower in PD patients than in HC (P = 2.96E-05, odds ratio 0.63, 95% confidence interval 0.51-0.78). No differences were observed between groups with respect to sex, onset symptoms, absence or presence of cognition impairment, anxiety or depression. In addition, no differences were found in the genotype and MAF of rs1572931 between SALS and HC or between MSA and HC. CONCLUSION: Our results suggest that rs1572931 decreases the risk for PD but not for ALS and MSA in the Chinese population. However, the polymorphism is unlikely to be a common cause of SALS and MSA in the Chinese population.