Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause late-onset Parkinson’s disease, but its physiological function has remained largely unknown. Here we report that LRRK2 activates a calcium-dependent protein kinase kinase-β (CaMKK-β)/adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway which is followed by a persistent increase in autophagosome formation. Simultaneously, LRKR2 overexpression increases the levels of the autophagy receptor p62 in a protein synthesis-dependent manner, and decreases the number of acidic lysosomes. The LRRK2-mediated effects result in increased sensitivity of cells to stressors associated with abnormal protein degradation. These effects can be mimicked by the lysosomal Ca2+-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) and can be reverted by an NAADP receptor antagonist or expression of dominant-negative receptor constructs. Collectively, our data indicate a molecular mechanism for LRRK2 deregulation of autophagy and reveal previously unidentified therapeutic targets.
Pathogenic mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause autosomal-dominant and certain cases of sporadic Parkinson's disease (PD). The G2019S substitution in LRRK2 is the most common genetic determinant of PD identified so far, and maps to a specific region of the kinase domain called the activation segment. Here, we show that autophosphorylation of LRRK2 is an intermolecular reaction and targets two residues within the activation segment. The prominent pathogenic G2019S mutation in LRRK2 results in altered autophosphorylation, and increased autophosphorylation and substrate phosphorylation, through a process that seems to involve reorganization of the activation segment. Our results suggest a molecular mechanistic explanation for how the G2019S mutation enhances the catalytic activity of LRRK2, thereby leading to pathogenicity. These findings have important implications for therapeutic strategies in PD.
BackgroundHirschsprung disease (HSCR), which is congenital obstruction of the bowel, results from a failure of enteric nervous system (ENS) progenitors to migrate, proliferate, differentiate, or survive within the distal intestine. Previous studies that have searched for genes underlying HSCR have focused on ENS-related pathways and genes not fitting the current knowledge have thus often been ignored. We identify and validate novel HSCR genes using whole exome sequencing (WES), burden tests, in silico prediction, unbiased in vivo analyses of the mutated genes in zebrafish, and expression analyses in zebrafish, mouse, and human.ResultsWe performed de novo mutation (DNM) screening on 24 HSCR trios. We identify 28 DNMs in 21 different genes. Eight of the DNMs we identified occur in RET, the main HSCR gene, and the remaining 20 DNMs reside in genes not reported in the ENS. Knockdown of all 12 genes with missense or loss-of-function DNMs showed that the orthologs of four genes (DENND3, NCLN, NUP98, and TBATA) are indispensable for ENS development in zebrafish, and these results were confirmed by CRISPR knockout. These genes are also expressed in human and mouse gut and/or ENS progenitors. Importantly, the encoded proteins are linked to neuronal processes shared by the central nervous system and the ENS.ConclusionsOur data open new fields of investigation into HSCR pathology and provide novel insights into the development of the ENS. Moreover, the study demonstrates that functional analyses of genes carrying DNMs are warranted to delineate the full genetic architecture of rare complex diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-017-1174-6) contains supplementary material, which is available to authorized users.
Hirschsprung disease (HSCR, OMIM 142623) is a developmental disorder characterized by the absence of ganglion cells along variable lengths of the distal gastrointestinal tract, which results in tonic contraction of the aganglionic gut segment and functional intestinal obstruction. The RET proto-oncogene is the major gene for HSCR with differential contributions of its rare and common, coding and noncoding mutations to the multifactorial nature of this pathology. Many other genes have been described to be associated with the pathology, as NRG1 gene (8p12), encoding neuregulin 1, which is implicated in the development of the enteric nervous system (ENS), and seems to contribute by both common and rare variants. Here we present the results of a comprehensive analysis of the NRG1 gene in the context of the disease in a series of 207 Spanish HSCR patients, by both mutational screening of its coding sequence and evaluation of 3 common tag SNPs as low penetrance susceptibility factors, finding some potentially damaging variants which we have functionally characterized. All of them were found to be associated with a significant reduction of the normal NRG1 protein levels. The fact that those mutations analyzed alter NRG1 protein would suggest that they would be related with HSCR disease not only in Chinese but also in a Caucasian population, which reinforces the implication of NRG1 gene in this pathology.
Mutations in the parkin gene cause autosomal-recessive, juvenile-onset parkinsonism, and parkin dysfunction may also play a role in the pathogenesis of sporadic Parkinson disease (PD). Although its precise function remains largely unknown, parkin seems to play a neuroprotective role. Several studies indicate that changes in parkin solubility induced by post-translational modifications, such as S-nitrosylation or dopamine modification, comprise one mechanism of parkin inactivation associated with disease. Protein phosphorylation events have recently been linked to the molecular mechanism(s) underlying PD, but the role of this post-translational modification for parkin function has remained unclear. Here we report that compound phosphorylation of parkin by both casein kinase I and cyclin-dependent kinase 5 (cdk5) decreases parkin solubility, leading to its aggregation and inactivation. Combined kinase inhibition partially reverses the aggregative properties of several pathogenic point mutants in cultured cells. Enhanced parkin phosphorylation is detected in distinct brain areas of individuals with sporadic PD and correlates with increases in the levels of p25, the activator of cdk5. These findings indicate that casein kinase I and cdk5 may represent novel combinatorial therapeutic targets for treating PD.
Hirschsprung disease (HSCR) is the most common cause of neonatal intestinal obstruction. It is characterized by the absence of ganglia in the nerve plexuses of the lower gastrointestinal tract. So far, three common disease-susceptibility variants at the RET, SEMA3 and NRG1 loci have been detected through genome-wide association studies (GWAS) in Europeans and Asians to understand its genetic etiologies. Here we present a trans-ethnic meta-analysis of 507 HSCR cases and 1191 controls, combining all published GWAS results on HSCR to fine-map these loci and narrow down the putatively causal variants to 99% credible sets. We also demonstrate that the effects of RET and NRG1 are universal across European and Asian ancestries. In contrast, we detected a European-specific association of a low-frequency variant, rs80227144, in SEMA3 [odds ratio (OR) = 5.2, P = 4.7 × 10-10]. Conditional analyses on the lead SNPs revealed a secondary association signal, corresponding to an Asian-specific, low-frequency missense variant encoding RET p.Asp489Asn (rs9282834, conditional OR = 20.3, conditional P = 4.1 × 10-14). When in trans with the RET intron 1 enhancer risk allele, rs9282834 increases the risk of HSCR from 1.1 to 26.7. Overall, our study provides further insights into the genetic architecture of HSCR and has profound implications for future study designs.
Hirschsprung disease (HSCR, OMIM 142623) is a developmental disorder characterized by the absence of ganglion cells along variable lengths of the distal gastrointestinal tract, which results in tonic contraction of the aganglionic colon segment and functional intestinal obstruction. The RET proto-oncogene is the major gene associated to HSCR with differential contributions of its rare and common, coding and noncoding mutations to the multifactorial nature of this pathology. In addition, many other genes have been described to be associated with this pathology, including the semaphorins class III genes SEMA3A (7p12.1) and SEMA3D (7q21.11) through SNP array analyses and by next-generation sequencing technologies. Semaphorins are guidance cues for developing neurons implicated in the axonal projections and in the determination of the migratory pathway for neural-crest derived neural precursors during enteric nervous system development. In addition, it has been described that increased SEMA3A expression may be a risk factor for HSCR through the upregulation of the gene in the aganglionic smooth muscle layer of the colon in HSCR patients. Here we present the results of a comprehensive analysis of SEMA3A and SEMA3D in a series of 200 Spanish HSCR patients by the mutational screening of its coding sequence, which has led to find a number of potentially deleterious variants. RET mutations have been also detected in some of those patients carrying SEMAs variants. We have evaluated the A131T-SEMA3A, S598G-SEMA3A and E198K-SEMA3D mutations using colon tissue sections of these patients by immunohistochemistry. All mutants presented increased protein expression in smooth muscle layer of ganglionic segments. Moreover, A131T-SEMA3A also maintained higher protein levels in the aganglionic muscle layers. These findings strongly suggest that these mutants have a pathogenic effect on the disease. Furthermore, because of their coexistence with RET mutations, our data substantiate the additive genetic model proposed for this rare disorder and further support the association of SEMAs genes with HSCR.
In recent years, long non-coding RNAs have emerged as a novel class of regulators of cancer biological processes. While they are dysregulated in many cancer types, little is known about their expression and functional profiles. This study has been focused on the determination of the role of a specific lncRNA in papillary thyroid cancer. Quantitative reverse transcription PCR was performed to detect the expression levels of 84 lncRNAs in 61 papillary thyroid carcinoma tissues and their adjacent non-tumor tissues. The highest fold-change was obtained for lung cancer associated transcript 1 LUCAT1, and thus, this study determines the expression and biological implication of lncRNA LUCAT1 through different in vitro and ex vivo approaches in this tumor. LUCAT1 was specifically located at the cell nucleus in tumoral regions of patient tissues. Furthermore, LUCAT1 knockdown significantly reduced both cell proliferation and invasion ex vivo and induced cell-cycle arrest and apoptosis. These facts were corroborated by an enhanced expression of P21, P57, P53 and BAX, and a reduced expression of EZH2 and HDAC1. In addition, a significant decrease was observed on DNMT1 and NRF2 genes, helping to clarify the role of LUCAT1 on PTC. Our study reveals the involvement of LUCAT1 in PTC development, through acting in cell-cycle regulation, proliferation, epigenetic modifications through LUCAT1/ CDK1/ EZH2/ P57/ P21/ HDAC1/ DNMT1/ P53/ BAX axis and apoptosis, via extrinsic pathway activating caspases. These findings indicate that LUCAT1 is maybe a potential therapeutic target and molecular biomarker for PTC.
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