Добірка наукової літератури з теми "Endolysosomal system"

Endosomes, lysosomes and lysosome-related organelles are emerging as important Ca2+ storage cellular compartments with a central role in intracellular Ca2+ signalling. Endocytosis at the plasma membrane forms endosomal vesicles which mature to late endosomes and culminate in lysosomal biogenesis. During this process, acquisition of different ion channels and transporters progressively changes the endolysosomal luminal ionic environment (e.g. pH and Ca2+) to regulate enzyme activities, membrane fusion/fission and organellar ion fluxes, and defects in these can result in disease. In the present review we focus on the physiology of the inter-related transport mechanisms of Ca2+ and H+ across endolysosomal membranes. In particular, we discuss the role of the Ca2+-mobilizing messenger NAADP (nicotinic acid adenine dinucleotide phosphate) as a major regulator of Ca2+ release from endolysosomes, and the recent discovery of an endolysosomal channel family, the TPCs (two-pore channels), as its principal intracellular targets. Recent molecular studies of endolysosomal Ca2+ physiology and its regulation by NAADP-gated TPCs are providing exciting new insights into the mechanisms of Ca2+-signal initiation that control a wide range of cellular processes and play a role in disease. These developments underscore a new central role for the endolysosomal system in cellular Ca2+ regulation and signalling.

Nicotinic acid adenosine dinucleotide phosphate (NAADP) is a Ca2+-mobilizing second messenger that regulates a wide range of biological activities. However, the mechanism of its biogenesis remains controversial. CD38 is the only enzyme known to catalyze NAADP synthesis from NADP and nicotinic acid. CD38-mediated catalysis requires an acidic pH, suggesting that NAADP may be produced in acidic endolysosomes, but this hypothesis is untested. In this study, using human cell lines, we specifically directed CD38 to the endolysosomal system and assessed cellular NAADP production. First, we found that nanobodies targeting various epitopes on the C-terminal domain of CD38 could bind to cell surface–localized CD38 and induce its endocytosis. We also found that CD38 internalization occurred via a clathrin-dependent pathway, delivered CD38 to the endolysosome, and elevated intracellular NAADP levels. We also created a CD38 variant for lysosome-specific expression, which not only withstood the degradative environment in the lysosome, but was also much more active than WT CD38 in elevating cellular NAADP levels. Supplementing CD38-expressing cells with nicotinic acid substantially increased cellular NAADP levels. These results demonstrate that endolysosomal CD38 can produce NAADP in human cells. They further suggest that CD38's compartmentalization to the lysosome may allow for its regulation via substrate access, rather than enzyme activation, thereby providing a reliable mechanism for regulating cellular NAADP production.

Abnormal accumulation of the protein α- synuclein (α-syn) into proteinaceous inclusions called Lewy bodies (LB) is the neuropathological hallmark of Parkinson’s disease (PD) and related disorders. Interestingly, a growing body of evidence suggests that LB are also composed of other cellular components such as cellular membrane fragments and vesicular structures, suggesting that dysfunction of the endolysosomal system might also play a role in LB formation and neuronal degeneration. Yet the link between α-syn aggregation and the endolysosomal system disruption is not fully elucidated. In this review, we discuss the potential interaction between α-syn and the endolysosomal system and its impact on PD pathogenesis. We propose that the accumulation of monomeric and aggregated α-syn disrupt vesicles trafficking, docking, and recycling, leading to the impairment of the endolysosomal system, notably the autophagy-lysosomal degradation pathway. Reciprocally, PD-linked mutations in key endosomal/lysosomal machinery genes (LRRK2, GBA, ATP13A2) also contribute to increasing α-syn aggregation and LB formation. Altogether, these observations suggest a potential synergistic role of α-syn and the endolysosomal system in PD pathogenesis and represent a viable target for the development of disease-modifying treatment for PD and related disorders.

The endolysosomal system comprises a unique environment for proteolysis, which is regulated in a manner that apparently does not involve protease inhibitors. The system comprises a series of membrane-bound intracellular compartments, within which endocytosed material and redundant cellular components are hydrolysed. Endocytosed material tends to flow vectorially through the system, proceeding through the early endosome, the endosome carrier vesicle, the late endosome and the lysosome. Phagocytosis and autophagy provide alternative entry points into the system. Late endosomes, lysosome/late endosome hybrid organelles, phagosomes and autophagosomes are the principal sites for proteolysis. In each case, hydrolytic competence is due to components of the endolysosomal system, i.e. proteases, lysosome-associated membrane proteins, H+-ATPases and possibly cysteine transporters. The view is emerging that lysosomes are organelles for the storage of hydrolases, perhaps in an inactivated form. Once a substrate has entered a proteolytically competent environment, the rate-limiting proteolytic steps are probably effected by cysteine endoproteinases. As these are affected by pH and possibly redox potential, they may be regulated by the organelle luminal environment. Regulation is probably also affected, among other factors, by organelle fusion reactions, whereby the meeting of enzyme and substrate may be controlled. Such systems would permit simultaneous regulation of a number of unrelated hydrolases.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant familial Parkinson’s disease (PD), with pathogenic mutations enhancing LRRK2 kinase activity. There is a growing body of evidence indicating that LRRK2 contributes to neuronal damage and pathology both in familial and sporadic PD, making it of particular interest for understanding the molecular pathways that underlie PD. Although LRRK2 has been extensively studied to date, our understanding of the seemingly diverse functions of LRRK2 throughout the cell remains incomplete. In this review, we discuss the functions of LRRK2 within the endolysosomal pathway. Endocytosis, vesicle trafficking pathways, and lysosomal degradation are commonly disrupted in many neurodegenerative diseases, including PD. Additionally, many PD-linked gene products function in these intersecting pathways, suggesting an important role for the endolysosomal system in maintaining protein homeostasis and neuronal health in PD. LRRK2 activity can regulate synaptic vesicle endocytosis, lysosomal function, Golgi network maintenance and sorting, vesicular trafficking and autophagy, with alterations in LRRK2 kinase activity serving to disrupt or regulate these pathways depending on the distinct cell type or model system. LRRK2 is critically regulated by at least two proteins in the endolysosomal pathway, Rab29 and VPS35, which may serve as master regulators of LRRK2 kinase activity. Investigating the function and regulation of LRRK2 in the endolysosomal pathway in diverse PD models, especially in vivo models, will provide critical insight into the cellular and molecular pathophysiological mechanisms driving PD and whether LRRK2 represents a viable drug target for disease-modification in familial and sporadic PD.

Until recently, the mechanisms that regulate endolysosomal calcium homoeostasis were poorly understood. The discovery of the molecular target of NAADP (nicotinic acid–adenine dinucleotide phosphate) as the two-pore channels resident in the endolysosomal system has highlighted this compartment as an important calcium store. The recent findings that dysfunctional NAADP release leads to defective endocytic function which in turn results in secondary lipid accumulation in the lysosomal storage disease Niemann–Pick type C, is the first evidence of a direct connection between a human disease and defective lysosomal calcium release. In the present review, we provide a summary of the current knowledge on mechanisms of calcium homoeostasis within the endolysosomal system and how these mechanisms may be affected in human metabolic disorders.

Hereditary spastic paraplegia (HSP) refers to a group of neurological disorders involving the degeneration of motor neurons. Due to their clinical and genetic heterogeneity, finding common effective therapeutics is difficult. Therefore, a better understanding of the common pathological mechanisms is necessary. The role of several HSP genes/proteins is linked to the endolysosomal and autophagic pathways, suggesting a functional convergence. Furthermore, impairment of these pathways is particularly interesting since it has been linked to other neurodegenerative diseases, which would suggest that the nervous system is particularly sensitive to the disruption of the endolysosomal and autophagic systems. In this review, we will summarize the involvement of HSP proteins in the endolysosomal and autophagic pathways in order to clarify their functioning and decipher some of the pathological mechanisms leading to HSP.

AbstractLate-onset Alzheimer’s disease is the most common dementia type, yet no treatment exists to stop the neurodegeneration. Evidence from monogenic lysosomal diseases, neuronal pathology and experimental models suggest that autophagic and endolysosomal dysfunction may contribute to neurodegeneration by disrupting the degradation of potentially neurotoxic molecules such as amyloid-β and tau. However, it is uncertain how well the evidence from rare disorders and experimental models capture causal processes in common forms of dementia, including late-onset Alzheimer’s disease. For this reason, we set out to investigate if autophagic and endolysosomal genes were enriched for genetic variants that convey increased risk of Alzheimer’s disease; such a finding would provide population-based support for the endolysosomal hypothesis of neurodegeneration. We quantified the collective genetic associations between the endolysosomal system and Alzheimer’s disease in three genome-wide associations studies (combined n = 62 415). We used the Mergeomics pathway enrichment algorithm that incorporates permutations of the full hierarchical cascade of SNP-gene-pathway to estimate enrichment. We used a previously published collection of 891 autophagic and endolysosomal genes (denoted as AphagEndoLyso, and derived from the Lysoplex sequencing platform) as a proxy for cellular processes related to autophagy, endocytosis and lysosomal function. We also investigated a subset of 142 genes of the 891 that have been implicated in Mendelian diseases (MenDisLyso). We found that both gene sets were enriched for genetic Alzheimer’s associations: an enrichment score 3.67 standard deviations from the null model (P = 0.00012) was detected for AphagEndoLyso, and a score 3.36 standard deviations from the null model (P = 0.00039) was detected for MenDisLyso. The high enrichment score was specific to the AphagEndoLyso gene set (stronger than 99.7% of other tested pathways) and to Alzheimer’s disease (stronger than all other tested diseases). The APOE locus explained most of the MenDisLyso signal (1.16 standard deviations after APOE removal, P = 0.12), but the AphagEndoLyso signal was less affected (3.35 standard deviations after APOE removal, P = 0.00040). Additional sensitivity analyses further indicated that the AphagEndoLyso Gene Set contained an aggregate genetic association that comprised a combination of subtle genetic signals in multiple genes. We also observed an enrichment of Parkinson’s disease signals for MenDisLyso (3.25 standard deviations) and for AphagEndoLyso (3.95 standard deviations from the null model), and a brain-specific pattern of gene expression for AphagEndoLyso in the Gene Tissue Expression Project dataset. These results provide evidence that a diffuse aggregation of genetic perturbations to the autophagy and endolysosomal system may mediate late-onset Alzheimer’s risk in human populations.

Lysosomes are intracellular organelles that were considered for a long time to be simply an acidic and hydrolytically active end point of trafficking routes for degradation, in the last 20 years, light has been shed on their functional heterogeneity and striking role in signalling and nutrient homeostasis. While the dynamic nature and variety of lysosomal functions are now better appreciated, the mechanisms governing lysosomal fusion, reformation, signalling, and homeostasis remain to be fully elucidated, and are investigated here. In this study, endolysosomes which formed by fusion of late endosomes with lysosomes and are thought to be the predominant site of hydrolytic activity, were further characterised. Using live cell imaging and fluorescent labelling, the proportion of endolysosomes in the total pool of lysosomes was estimated using probes to their acidity and cathepsin activity, and their larger size compared to storage lysosomes was observed. The endolysosomal membrane was also shown to be marked by Rab7, Rab9, PI(3,5)P2 supporting the role of endolysosomes a highly active and dynamic principal site of hydrolase activity. The contributions of VAMP7 and VAMP8 to endolysosome fusion, measured by delivery of endocytosed cargo from late endosomes to endolysosomes, were analysed by CRISPR-Cas9 mediated knockout. Cells lacking VAMP7 and VAMP8 had no effect on delivery to endolysosomes, however at EM level, they displayed extensive tethering between late endocytic organelles, and accumulated small tethered vesicles. YKT6 knockdown impeded delivery to endolysosomes in VAMP7+VAMP8 knockout cells, which was rescued by VAMP7 expression, suggesting YKT6 substituted for VAMP7 in lysosome fusion. Following the hypothesis that reversible dissociation of V1 and Vo sectors of the V- ATPase may control the increase in pH of reforming storage lysosomes, cells expressing tagged V1G1 and Voa3 were generated. These markers of both sectors are present on endolysosomal membranes, and on the emerging endolysosomal tubules, suggesting the V1 and Vo sectors remain associated at this earliest stage of lysosome reformation, but these markers are still in development. IV Two assays were developed to give a readout of, and assess lysosomal stress. Firstly, an assay measuring TFEB-GFP translocation to the nucleus gave a robust and quantifiable readout of lysosomal perturbation. Secondly, a qPCR assay was developed to measure lysosomal gene upregulation as a downstream reporter of TFEB-activating lysosomal perturbations, however this assay, despite being more lysosome-specific, lacked the consistency and dynamic range of the TFEB translocation quantification. In summary, lysosomes are a heterogeneous collection of organelles, which have been better characterised primarily according to their acidity and hydrolytic capacity. Additionally, more SNAREs appear to be involved in lysosome fusion in cells than suggested by cell free assays, and I have developed tools to trace the V-ATPase during reformation of lysosomes after fusion to form endolysosomes. Lastly, I have developed a robust, reporter for a range of lysosomal stress-inducing conditions, providing a broad indication of their effects on lysosomal signalling and homeostasis.

La maladie d'Alzheimer (MA) est une pathologie complexe et multifactorielle pour laquelle il n'existe actuellement aucun traitement. Plusieurs hypothèses ont été proposées pour expliquer l'apparition et la progression de cette maladie, dont la cascade amyloïde, qui prédomine dans le domaine de la recherche depuis 30 ans. La voie amyloïdogène nécessite l'endocytose de la protéine APP dans les endosomes précoces où elle subit deux clivages protéolytiques, d'abord par la β-sécrétase pour produire le fragment C99, puis par la γ-sécrétase pour produire le peptide Aβ. L'une des hypothèses actuelles est que les anomalies de l'endocytose et le dysfonctionnement du système endolysosomal dans les neurones constitueraient un des mécanismes neuropathologiques précoces de la MA, bien avant la cascade neurotoxique générée par Aβ et les dépôts amyloïdes. Nous défendons l'hypothèse que des modifications de l'organisation membranaire, notamment au cours du vieillissement ou dues à des déséquilibres lipidiques, pourraient exacerber ou favoriser ces dysfonctionnements. Pour cette étude, nous avons utilisé un modèle de neuroblastome humain surexprimant la protéine mutante APPswe. Nous avons tout d'abord vérifié la présence d'anomalies endolysosomales typiques de la MA (endosomes hypertrophiés, trafic vésiculaire bloqué), auxquelles nous avons également associé une faible production d'exosomes, conditions de stress chronique que nous avons corrélées à la mort neuronale. Incriminant dans un premier temps une production continue d'Aβ dans ces cellules, nous avons cherché à réduire son impact en inhibant l'activité γ-sécrétase. Cela n'a pas amélioré le stress, mais l'a au contraire aggravé, ce qui nous a conduit à considérer que c'est le fragment C99 de l'APP, c'est-à-dire le substrat de la production d'Aβ, qui est le produit amyloïde central de la cascade neurotoxique observée dans les cellules surexprimant l'APP. Les effets délétères du C99 doivent se produire avant ceux de l'Aβ, expliquant la précocité connue des altérations endolysosomales. S'accumulant à la suite de l'inhibition de la γ-sécrétase, le fragment C99 interagit davantage avec la protéine Rab5, spécifique de l'endosome précoce. La maturation de cette dernière est ainsi empêchée, bloquant le trafic vésiculaire du système endolysosomal. Comme les interactions entre C99 et Rab5 se produisent au niveau de la membrane des endosomes, nous avons modifié la composition lipidique de la bicouche et exploré les conséquences sur ces interactions. À cette fin, nous avons traité des cellules SH-SY5Y-APPswe par de l'acide docosahexaénoïque (DHA, C22:6 n-3), le principal acide gras polyinsaturé des membranes neuronales et connu pour ses propriétés neuroprotectrices contre le stress amyloïde et la MA. L'effet bénéfique attendu sur la survie neuronale a bien été observé, en parallèle au déblocage du trafic endolysosomal et à la production d'exosomes. Tous ces changements ont été corrélés à une dispersion entre C99 et Rab5 dans la membrane, suggérant que le traitement par le DHA a pu initier un remodelage membranaire. Ce remodelage peut conduire à une relocalisation des protéines, les endosomes pouvant alors échanger Rab5 contre Rab7 et évoluer en endosomes tardifs, levant ainsi le blocage initial. À notre connaissance, il s'agit de la première preuve que le DHA peut corriger un phénotype directement lié à la MA, mais sa capacité à remodeler la membrane neuronale a déjà été démontrée par notre équipe pour préserver la signalisation par le facteur neurotrophique CNTF dans le cerveau de souris âgées. Nous ignorons quels principes mécanistiques pourraient régir ces effets bénéfiques, certainement non spécifiques, mais nous supposons qu'en préservant l'organisation des membranes des neurones âgés ou soumis à un stress chronique, ils pourraient prévenir ou restaurer une partie des dommages subis, augmenter les chances de survie des neurones et ainsi ralentir le développement de la MA
Alzheimer's disease (AD) is a complex and multifactorial pathology for which there is no current treatment. Several hypotheses have been proposed to explain the onset and progression of this disease, including the amyloid cascade, which predominates the field of research for the past 30 years. The amyloidogenic pathway requires the endocytosis of the APP protein in early endosomes where it undergoes two proteolytic cleavages, first by β-secretase to produce the C99 fragment, and then by γ-secretase to produce the Aβ peptide. One of the current hypotheses is that abnormalities of endocytosis and dysfunction of the endolysosomal system in neurons would constitute one of the early neuropathological mechanisms of AD, well before the neurotoxic cascade generated by Aβ and amyloid deposits. We advocate the hypothesis that changes in membrane organization, particularly during aging or due to lipid imbalances, may exacerbate or promote these dysfunctions. For this study, we used a human neuroblastoma model overexpressing the mutant protein APPswe. We first verified the presence of typical AD endolysosomal abnormalities (enlarged endosomes, blocked vesicular trafficking), to which we also associated low exosome production, chronic stress conditions that we correlated with neuronal death. Initially incriminating continuously produced Aβ in these cells, we sought to reduce its impact by inhibiting γ-secretase activity. This did not ameliorate the stress, but instead aggravated it, leading us to consider that it is the C99 fragment of APP, i.e. the substrate of Aβ production, that is the central amyloid product in the neurotoxic cascade seen in APP-overexpressing cells. The deleterious effects of C99 must occur before those of Aβ, explaining the known precocity of endolysosomal alterations. Accumulating as a result of γ-secretase inhibition, the C99 fragment interacts further with the early endosome-specific Rab5 protein. Maturation of the latter is thus prevented, blocking vesicular trafficking of the endolysosomal system. As the interactions between C99 and Rab5 occur at the membrane level of endosomes, we have modified the lipid composition of the bilayer and explored the consequences on these interactions. For this purpose, we treated SH-SY5Y-APPswe cells with docosahexaenoic acid (DHA, C22:6 n-3), the major polyunsaturated fatty acid in neuronal membranes and known for its neuroprotective properties against Aβ toxicity and AD. The expected beneficial effect on neuronal survival was indeed observed, in parallel with the unblocking of endolysosomal trafficking and exosomal production. All these changes were correlated with a dispersion between C99 and Rab5 in the membrane, suggesting that DHA treatment may initiate membrane remodeling. This remodeling may lead to protein relocalization, whereby endosomes may exchange Rab5 for Rab7 to evolve into late endosomes, thereby overcoming the initial blockage. To our knowledge, this is the first evidence that DHA can correct a phenotype directly related to AD, but its ability to remodel the neuronal membrane was previously demonstrated by our team to preserve the neurotrophic CNTF signaling in the brain of aged mice. We do not know what mechanistic principles might govern these beneficial effects, which are certainly non-specific, but we assume that by preserving the organization of the membranes of aged or chronically stressed neurons, they may prevent or restore some of the damage suffered, increase the chances of neuronal survival and thus slow AD development

Manipulation of host phosphoinositide lipids has emerged as a key survival strategy utilized by pathogenic bacteria to establish and maintain a replication-permissive compartment within eukaryotic host cells. The human pathogen, Legionella pneumophila, infects and proliferates within the lung’s innate immune cells causing severe pneumonia termed Legionnaires’ disease. This pathogen has evolved strategies to manipulate specific host components to construct its intracellular niche termed the Legionella-containing vacuole (LCV). Paramount to LCV biogenesis and maintenance is the spatiotemporal regulation of phosphoinositides, important eukaryotic lipids involved in cell signaling and membrane trafficking. Through a specialized secretion system, L. pneumophila translocates multiple proteins that target phosphoinositides in order to escape endolysosomal degradation. By specifically binding phosphoinositides, these proteins can anchor to the cytosolic surface of the LCV or onto specific host membrane compartments, to ultimately stimulate or inhibit encounters with host organelles. Here, we describe the bacterial proteins involved in binding and/or altering host phosphoinositide dynamics to support intracellular survival of L. pneumophila.