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Lysoplex: An efficient toolkit to detect DNA sequence variations in the autophagy-lysosomal pathway.

ABSTRACT: The autophagy-lysosomal pathway (ALP) regulates cell homeostasis and plays a crucial role in human diseases, such as lysosomal storage disorders (LSDs) and common neurodegenerative diseases. Therefore, the identification of DNA sequence variations in genes involved in this pathway and their association with human diseases would have a significant impact on health. To this aim, we developed Lysoplex, a targeted next-generation sequencing (NGS) approach, which allowed us to obtain a uniform and accurate coding sequence coverage of a comprehensive set of 891 genes involved in lysosomal, endocytic, and autophagic pathways. Lysoplex was successfully validated on 14 different types of LSDs and then used to analyze 48 mutation-unknown patients with a clinical phenotype of neuronal ceroid lipofuscinosis (NCL), a genetically heterogeneous subtype of LSD. Lysoplex allowed us to identify pathogenic mutations in 67% of patients, most of whom had been unsuccessfully analyzed by several sequencing approaches. In addition, in 3 patients, we found potential disease-causing variants in novel NCL candidate genes. We then compared the variant detection power of Lysoplex with data derived from public whole exome sequencing (WES) efforts. On average, a 50% higher number of validated amino acid changes and truncating variations per gene were identified. Overall, we identified 61 truncating sequence variations and 488 missense variations with a high probability to cause loss of function in a total of 316 genes. Interestingly, some loss-of-function variations of genes involved in the ALP pathway were found in homozygosity in the normal population, suggesting that their role is not essential. Thus, Lysoplex provided a comprehensive catalog of sequence variants in ALP genes and allows the assessment of their relevance in cell biology as well as their contribution to human disease.

SUBMITTER: Di Fruscio G

PROVIDER: S-EPMC4502703 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Lysoplex: An efficient toolkit to detect DNA sequence variations in the autophagy-lysosomal pathway.

Di Fruscio Giuseppina G Schulz Angela A De Cegli Rossella R Savarese Marco M Mutarelli Margherita M Parenti Giancarlo G Banfi Sandro S Braulke Thomas T Nigro Vincenzo V Ballabio Andrea A

Autophagy 20150101 6

The autophagy-lysosomal pathway (ALP) regulates cell homeostasis and plays a crucial role in human diseases, such as lysosomal storage disorders (LSDs) and common neurodegenerative diseases. Therefore, the identification of DNA sequence variations in genes involved in this pathway and their association with human diseases would have a significant impact on health. To this aim, we developed Lysoplex, a targeted next-generation sequencing (NGS) approach, which allowed us to obtain a uniform and ac ...[more]

PMID: 26075876

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Project description:The mechanisms of the control and activity of the autophagy-lysosomal protein degradation machinery are emerging as an important theme for neurodevelopment and neurodegeneration. However, the underlying regulatory and functional networks of known genes controlling autophagy and lysosomal function and their role in disease are relatively unexplored. We performed a systems biology-based integrative computational analysis to study the interactions between molecular components and to develop models for regulation and function of genes involved in autophagy and lysosomal function. Specifically, we analyzed transcriptional and microRNA-based post-transcriptional regulation of these genes and performed functional enrichment analyses to understand their involvement in nervous system-related diseases and phenotypes. Transcriptional regulatory network analysis showed that binding sites for transcription factors, SREBP1, USF, AP-1 and NFE2, are common among autophagy and lysosomal genes. MicroRNA enrichment analysis revealed miR-130, 98, 124, 204 and 142 as the putative post-transcriptional regulators of the autophagy-lysosomal pathway genes. Pathway enrichment analyses revealed that the mTOR and insulin signaling pathways are important in the regulation of genes involved in autophagy. In addition, we found that glycosaminoglycan and glycosphingolipid pathways also make a major contribution to lysosomal gene regulation. The analysis confirmed the known contribution of the autophagy-lysosomal genes to Alzheimer and Parkinson diseases and also revealed potential involvement in tuberous sclerosis, neuronal ceroidlipofuscinoses, sepsis and lung, liver and prostatic neoplasms. To further probe the impact of autophagy-lysosomal gene deficits on neurologically-linked phenotypes, we also mined the mouse knockout phenotype data for the autophagylysosomal genes and found them to be highly predictive of nervous system dysfunction. Overall this study demonstrates the utility of systems biology-based approaches for understanding the autophagy-lysosomal pathways and gaining additional insights into the potential impact of defects in these complex biological processes.

Project description:ATM (ataxia telangiectasia mutated) protein is found associated with multiple organelles including synaptic vesicles, endosomes and lysosomes, often in cooperation with ATR (ataxia telangiectasia and Rad3 related). Mutation of the ATM gene results in ataxia-telangiectasia (A-T), an autosomal recessive disorder with defects in multiple organs including the nervous system. Precisely how ATM deficiency leads to the complex phenotypes of A-T, however, remains elusive. Here, we reported that part of the connection may lie in autophagy and lysosomal abnormalities. We found that ATM was degraded through the autophagy pathway, while ATR was processed by the proteasome. Autophagy and lysosomal trafficking were both abnormal in atm-/- neurons and the deficits impacted cellular functions such as synapse maintenance, neuronal survival and glucose uptake. Upregulated autophagic flux was observed in atm-/- lysosomes, associated with a more acidic pH. Significantly, we found that the ATP6V1A (ATPase, H+ transporting, lysosomal V1 subunit A) proton pump was an ATM kinase target. In atm-/- neurons, lysosomes showed enhanced retrograde transport and accumulated in the perinuclear regions. We attributed this change to an unexpected physical interaction between ATM and the retrograde transport motor protein, dynein. As a consequence, SLC2A4/GLUT4 (solute carrier family 4 [facilitated glucose transporter], member 4) translocation to the plasma membrane was inhibited and trafficking to the lysosomes was increased, leading to impaired glucose uptake capacity. Together, these data underscored the involvement of ATM in a variety of neuronal vesicular trafficking processes, offering new and therapeutically useful insights into the pathogenesis of A-T.Abbreviations: 3-MA: 3-methyladenine; A-T: ataxia-telangiectasia; ALG2: asparagine-linked glycosylation 2 (alpha-1,3-mannosyltransferase); AMPK: adenosine 5'-monophosphate (AMP)-activated protein kinase; ATG5: autophagy related 5; ATM: ataxia telangiectasia mutated; ATP6V1A: ATPase, H+ transporting, lysosomal V1 subunit A; ATR: ataxia-telangiectasia and Rad3 related; BFA1: bafilomycin A1; CC3: cleaved-CASP3; CGN: cerebellar granule neuron; CLQ: chloroquine; CN: neocortical neuron; CTSB: cathepsin B; CTSD: cathepsin D; DYNLL1: the light chain1 of dynein; EIF4EBP1/4E-BP1: eukaryotic translation initiation factor 4E binding protein 1; Etop: etoposide; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HBS: HEPES-buffered saline; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HOMER1: homer protein homolog 1; KU: KU-60019; LAMP1: lysosomal-associated membrane protein 1; LC3B-II: LC3-phosphatidylethanolamine conjugate; Lyso: lysosome; LysopH-GFP: lysopHluorin-GFP; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAP2: microtubule associated protein 2; MAPK14: mitogen-activated protein kinase 14; MAPK8/JNK1: mitogen-activated protein kinase 8; MCOLN1/TRPML1: mucolipin 1; OSBPL1A: oxysterol binding protein like 1A; PIKK: phosphatidylinositol 3 kinase related kinase; Rapa: rapamycin; RILP: rab interacting lysosomal protein; ROS: reactive oxygen species; SEM: standard error of mean; SLC2A4/GLUT4: solute carrier family 2 (facilitated glucose transporter), member 4; TSC2/tuberin: TSC complex subunit 2; ULK1: unc-51 like kinase 1; UPS: ubiquitin-proteasome system; VE: VE-822; WCL: whole-cell lysate; WT: wild type.

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Dataset Information

Lysoplex: An efficient toolkit to detect DNA sequence variations in the autophagy-lysosomal pathway.

Publications

Lysoplex: An efficient toolkit to detect DNA sequence variations in the autophagy-lysosomal pathway.

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