Lysosomal Defects Research Articles

Two NPC1 homologous proteins are involved in asexual reproduction, pathogenicity, and lipid trafficking in Phytophthora sojae

Niemann-Pick type C (NPC) disease is characterized by lysosomal lipid storage disorders and defects in lipid trafficking, primarily due to mutations in the NPC1 protein. Two NPC1 homologous proteins are present in the genome of Phytophthora sojae, named as PsNPC1-1 and PsNPC1-2. Both proteins exhibit high sequence identity, consistent conserved functional domains, similar gene expression patterns, and comparable subcellular localization. Deletion of a single PsNPC1 gene did not result in significant phenotypic changes. However, simultaneous deletion of both PsNPC1 genes led to reduced mycelial growth, decreased sporangial production, impaired pathogenicity, and an inability to release normal zoospores in P. sojae. Furthermore, dysfunction of PsNPC1s did not completely block the absorption and utilization of exogenous sterols by P. sojae. While lipidome analysis revealed that the relative contents of fatty acyls, sphingolipids and saccharolipids were significantly elevated in the double-gene deletion mutant, alongside obvious alterations in glycerophospholipid and glycerolipid metabolism. Additionally, we observed a significant down-regulation of PsCDP-AP protein along with its interactions with both PsNPC1s. Deletion of PsCDP-AP also impaired asexual reproduction and virulence of P. sojae. These findings demonstrate that both PsNPC1 proteins may collaborate with other key regulators to modulate asexual reproduction, pathogenicity and lipid trafficking in P. sojae.

Glycolysis inhibition functionally reprograms T follicular helper cells and reverses lupus.

Systemic lupus erythematosus (SLE) is an autoimmune disease in which the production of pathogenic autoantibodies depends on T follicular helper (T FH ) cells. This study was designed to investigate the mechanisms by which inhibition of glycolysis with 2-deoxy-d-glucose (2DG) reduces the expansion of T FH cells and the associated autoantibody production in lupus-prone mice. Integrated cellular, transcriptomic, epigenetic and metabolic analyses showed that 2DG reversed the enhanced cell expansion and effector functions, as well as mitochondrial and lysosomal defects in lupus T FH cells, which include an increased chaperone-mediated autophagy induced by TLR7 activation. Importantly, adoptive transfer of 2DG-reprogrammed T FH cells protected lupus-prone mice from disease progression. Orthologs of genes responsive to 2DG in murine lupus T FH cells were overexpressed in the T FH cells of SLE patients, suggesting a therapeutic potential of targeting glycolysis to eliminate aberrant T FH cells and curb the production of autoantibodies inducing tissue damage.

Ceramide lowering rescues respiratory defects in a Drosophila model of acid sphingomyelinase deficiency.

Types A and B Niemann-Pick disease (NPD) are inherited multisystem lysosomal storage disorders due to mutations in the SMPD1 gene. Respiratory dysfunction is a key hallmark of NPD, yet the mechanism for this is underexplored. SMPD1 encodes acid sphingomyelinase (ASM), which hydrolyses sphingomyelin to ceramide and phosphocholine. Here, we present a Drosophila model of ASM loss-of-function, lacking the fly orthologue of SMPD1, dASM, modelling several aspects of the respiratory pathology of NPD. dASM is expressed in the late-embryonic fly respiratory network, the trachea, and is secreted into the tracheal lumen. Loss of dASM results in embryonic lethality, and the tracheal lumen fails to fill normally with gas prior to eclosion. We demonstrate that the endocytic clearance of luminal constituents prior to gas-filling is defective in dASM mutants, and is coincident with autophagic, but not lysosomal defects, in late stage embryonic trachea. Finally, we show that although bulk sphingolipids are unchanged, dietary loss of lipids in combination with genetic and pharmacological block of ceramide synthesis rescues the airway gas-filling defects. We highlight myriocin as a potential therapeutic drug for the treatment of the developmental respiratory defects associated with ASM deficiency, and present a new NPD model amenable to genetic and pharmacological screens.

9317 Diabetogenic Stress-Mediated Beta Cell Extracellular Vesicle:Autophagy Pathway Crosstalk

Abstract Disclosure: A. Roy: None. G. Ariyaratne: None. A. Matveyenko: None. N. Javeed: None. Diabetogenic stressors including elevated free fatty acids (lipotoxicity), low-grade systemic inflammation, and proteotoxicity are central to the development of Type 2 Diabetes (T2D). Due to their high protein synthetic burden, β-cells employ autophagy and endo/lysosomal pathways to reduce protein aggregates, and protect β-cell mass and function. Recently, extracellular vesicles (EVs; 30-150 nm sized nanoparticles) have been shown to work in tandem with autophagy to facilitate cellular adaptation and intercellular communication. As diabetogenic stressors have been shown to perturb autophagy via lysosomal defects, our group has noted enhanced EV secretion. Thus, we hypothesize that diabetogenic factors enhance EV secretion through activation of an EV-based secretory autophagy pathway and that re-balancing of both pathways could improve β-cell function in T2D. To test this, MIN6 cells were exposed to free fatty acid palmitate (PAL; 24h), or pro-inflammatory cytokines IL-1β, TNFα, and IFNγ (CYTO; 48h). Expression of autophagic markers, LC3 and p62 were increased in CYTO and PAL conditions suggesting defects in auto/lysosomal function. Upon PAL/CYTO treatment in MIN6 cells, conditioned media EVs were isolated using differential ultracentrifugation. We noted enhanced protein expression of LC3-II and p62 in PAL- and cytoEVs, suggesting a diversion of immature autophagosome components into EVs due to lysosomal inhibition. To restore balance between autophagy:EV flux, we used EV biogenesis inhibitor, GW4869 (GW; 24h) or autophagy inducer, rapamycin (Rapa; 24h) on MIN6 cells exposed to PAL or CYTO. Isolated EVs were subjected to Nanoparticle Tracking Analysis (NTA) which showed a significant reduction in EV concentration with GW or Rapa addition (p<.05). Isolated mouse islet exposed to CYTO+GW/Rapa or PAL+GW/Rapa showed significant improvements in glucose stimulated insulin secretion (vs. CYTO or PAL; p<.05). Taken together, these data implicate activation of EV-based secretory autophagy in response to diabetogenic stressors and thus, potential targets to improve functional β-cell mass. Presentation: 6/2/2024

6681 Age and Sex Disparity in NASH and the Potential Role of Estradiol Treatment for NASH

Abstract Disclosure: M. Tripathi: None. S. Sakthivel: None. A. Suzuki: None. B.K. Singh: None. P.M. Yen: None. Introduction: NAFLD (nonalcoholic fatty liver disease) and its more severe form, NASH (nonalcoholic steatohepatitis) are sexually dimorphic conditions that exhibit higher prevalence in men than women during reproductive ages but have similar prevalence after menopause. Reproductive age women are protected from hepatic fibrosis relative to men, although they lose this protection after menopause. These sex differences in NASH have been attributed, at least partly, to different circulating estrogen levels. However, the underlying mechanism(s) is not fully understood. We thus conducted animal experiments to investigate: 1) sex differences in the histologic severity of NASH, necroinflammatory and fibrogenic activities, and other relevant pathways in young and old male and female mice and 2) the effects of estradiol administration on these parameters in old male and female mice to evaluate the therapeutic potential of estradiol in NASH. Methodology: Young/reproductive age (12-weeks old) and old/post-reproductive age female and male (48∼50-weeks weeks old) C57BL/6J mice were fed control chow diet (NCD) or high-fat methionine/choline-deficient (MCD) diet for six weeks to induce NASH. The old mice were administered 200nM 17β-estradiol (physiological) in drinking water. We analyzed serum and hepatic NASH parameters, ER stress, and lysosome-autophagy defects. Results and Conclusion: Among reproductive age mice with NASH, females developed less severe histologic inflammation and fibrosis and decreased gene expression of Il1b, Il6, Tgfb, Col1a1, Ccl2, and Ccl5, and lower hepatic collagen than males. Females with NASH also had less inhibition of hepatic autophagy and lysosomal protein expression than males with NASH. Among old mice, post-reproductive age females fed NCD or MCD exhibited more severe hepatic inflammation and fibrosis and had higher gene expression of inflammatory and fibrogenic markers with comparable autophagic inhibition and lysosomal defects as similar age males. Physiological doses of estradiol significantly reduced hepatic gene expression of inflammatory and fibrogenic markers, ER stress and lysosomal-autophagic defects in post-reproductive age females. In conclusion, similar to clinical NASH, we observed marked sex and age-related differences in the severity of NASH. Furthermore, physiological doses of estradiol treatment improved the hepatic histology, lysosome-autophagy, inflammation, and fibrosis in post-reproductive age female mice with NASH and suggests that estradiol supplementation may decrease or reverse NASH progression in post-menopausal women with NASH. Presentation: 6/2/2024

Functional Analysis of Human GBA1 Missense Mutations in Drosophila: Insights into Gaucher Disease Pathogenesis and Phenotypic Consequences.

The human GBA1 gene encodes lysosomal acid β-glucocerebrosidase, whose activity is deficient in Gaucher disease (GD). In Drosophila, there are two GBA1 orthologs, Gba1a and Gba1b, and Gba1b is the bona fide GCase encoding gene. Several fly lines with different deletions in the Gba1b were studied in the past. However, since most GD-associated GBA1 mutations are point mutations, we created missense mutations homologous to the two most common GD mutations: the mild N370S mutation (D415S in Drosophila) and the severe L444P mutation (L494P in Drosophila), using the CRISPR-Cas9 technology. Flies homozygous for the D415S mutation (dubbed D370S hereafter) presented low GCase activity and substrate accumulation, which led to lysosomal defects, activation of the Unfolded Protein Response (UPR), inflammation/neuroinflammation, and neurodegeneration along with earlier death compared to control flies. Surprisingly, the L494P (called L444P hereafter) flies presented higher GCase activity with fewer lysosomal defects and milder disease in comparison to that presented by the D370S homozygous flies. Treatment with ambroxol had a limited effect on all homozygous fly lines tested. Overall, our results underscore the differences between the fly and human GCase enzymes, as evidenced by the distinct phenotypic outcomes of mutations in flies compared to those observed in human GD patients.

V-ATPase Dysfunction in the Brain: Genetic Insights and Therapeutic Opportunities.

Vacuolar-type ATPase (v-ATPase) is a multimeric protein complex that regulates H+ transport across membranes and intra-cellular organelle acidification. Catabolic processes, such as endocytic degradation and autophagy, strictly rely on v-ATPase-dependent luminal acidification in lysosomes. The v-ATPase complex is expressed at high levels in the brain and its impairment triggers neuronal dysfunction and neurodegeneration. Due to their post-mitotic nature and highly specialized function and morphology, neurons display a unique vulnerability to lysosomal dyshomeostasis. Alterations in genes encoding subunits composing v-ATPase or v-ATPase-related proteins impair brain development and synaptic function in animal models and underlie genetic diseases in humans, such as encephalopathies, epilepsy, as well as neurodevelopmental, and degenerative disorders. This review presents the genetic and functional evidence linking v-ATPase subunits and accessory proteins to various brain disorders, from early-onset developmental epileptic encephalopathy to neurodegenerative diseases. We highlight the latest emerging therapeutic strategies aimed at mitigating lysosomal defects associated with v-ATPase dysfunction.

TRPML1 ameliorates seizures-related neuronal injury by regulating autophagy and lysosomal biogenesis via Ca2+/TFEB signaling pathway

Alterations in autophagy have been observed in epilepsy, although their exact etiopathogenesis remains elusive. Transient Receptor Potential Mucolipin Protein 1 (TRPML1) is an ion channel protein that regulates autophagy and lysosome biogenesis. To explore the role of TRPML1 in seizures-induced neuronal injury and the potential mechanisms involved, an hyperexcitable neuronal model induced by Mg2+-free solution was used for the study. Our results revealed that TRPML1 expression was upregulated after seizures, which was accompanied by intracellular ROS accumulation, mitochondrial damage, and neuronal apoptosis. Activation of TRPML1 by ML-SA1 diminished intracellular ROS, restored mitochondrial function, and subsequently alleviated neuronal apoptosis. Conversely, inhibition of TRPML1 had the opposite effect. Further examination revealed that the accumulation of ROS and damaged mitochondria was associated with interrupted mitophagy flux and enlarged defective lysosomes, which were attenuated by TRPML1 activation. Mechanistically, TRPML1 activation allows more Ca2+ to permeate from the lysosome into the cytoplasm, resulting in the dephosphorylation of TFEB and its nuclear translocation. This process further enhances autophagy initiation and lysosomal biogenesis. Additionally, the expression of TRPML1 is positively regulated by WTAP-mediated m6A modification. Our findings highlighted crucial roles of TRPML1 and autophagy in seizures-induced neuronal injury, which provides a new target for epilepsy treatment.

Pseudoginsenoside-F11 reduces cognitive impairment and white matter injury in vascular dementia by alleviating autophagy-lysosomal pathway deficiency

BackgroundVascular dementia (VaD) resulting from chronic cerebral hypoperfusion (CCH) induces cognitive impairment and white matter injury (WMI). We previously found that CCH induces dysfunction of the autophagy-lysosomal pathway (ALP) in white matter (WM) of rats. Enhancing oligodendrocyte autophagy to counteract ALP deficiency is beneficial for cognitive recovery. Pseudogenoside-F11 (PF11), a saponin extracted from Panax quinquefolium l., provides neuroprotective benefits in many animal models of cerebral ischemia and dementia. PurposeTo investigate how PF11 affects cognitive deterioration in rats with VaD induced by two vessel occlusion (2VO), and to determine if PF11 regulates ALP dysfunction in WM. MethodsCCH-related VaD was induced in rats using the 2VO method. PF11 (6, 12, 24 mg/kg, intragastric administration) was given continuously for 4 weeks postoperatively. Behavioral tests related to cognitive function were performed on the 28th day following 2VO. Transmission electron microscopy, immunofluorescence, western blotting and Luxol fast blue staining were used to assess the WMI and the mechanism of action of PF11 in 2VO-induced VaD. ResultsPF11 (12 mg/kg) ameliorated 2VO-induced cognitive impairment. PF11 also alleviated WMI on the 28th day following 2VO, as characterized by reduction of neuronal axonal demyelination and axonal loss. Furthermore, PF11 prevented mature oligodendrocytes death by attenuating ALP deficiency in WM on the 14th day following 2VO, as manifested by enhancement of mechanistic target of rapamycin-mediated autophagy and lysosomal function, thereby reducing the aberrant accumulation of autophagy substrates and increasing the level of autophagosomes in WM. In addition, PF11 also prevented microglia and astrocytes from activating in WM on the 28th day following 2VO. ConclusionPF11 significantly ameliorates cognitive impairment and WMI, and the mechanism is at least partly related to lessening ALP dysfunction in WM by enhancing autophagy and reducing lysosomal defects in oligodendrocytes.

Multisystem disorder associated with a pathogenic variant in CLCN7 in the absence of osteopetrosis.

We clinically and genetically evaluated a Taiwanese boy presenting with developmental delay, organomegaly, hypogammaglobulinemia and hypopigmentation without osteopetrosis. Whole-exome sequencing revealed a de novo gain-of-function variant, p.Tyr715Cys, in the C-terminal domain of ClC-7 encoded by CLCN7. Nicoli etal. (2019) assessed the functional impact of p.Tyr715Cys by heterologous expression in Xenopus oocytes and evaluating resulting currents. The variant led to increased outward currents, indicating it underlies the patient's phenotype of lysosomal hyperacidity, storage defects and vacuolization. This demonstrates the crucial physiological role of ClC-7 antiporter activity in maintaining appropriate lysosomal pH. Elucidating mechanisms by which CLCN7 variants lead to lysosomal dysfunction will advance understanding of genotype-phenotype correlations. Identifying modifier genes and compensatory pathways may reveal therapeutic targets. Ongoing functional characterization of variants along with longitudinal clinical evaluations will continue advancing knowledge of ClC-7's critical roles and disease mechanisms resulting from its dysfunction. Expanded cohort studies are warranted to delineate the full spectrum of associated phenotypes.

Rab27b promotes lysosomal function and alpha-synuclein clearance in neurons.

Alpha-synuclein (αsyn) is the key pathogenic protein implicated in synucleinopathies including Parkinson's Disease (PD) and Dementia with Lewy Bodies (DLB). In these diseases, αsyn is thought to spread between cells where it accumulates and induces pathology; however, mechanisms that drive its propagation or aggregation are poorly understood. We have previously reported that the small GTPase Rab27b is elevated in human PD and DLB and that it can mediate the autophagic clearance and toxicity of αsyn in a paracrine αsyn cell culture neuronal model. Here, we expanded our previous work and further characterized a role for Rab27b in neuronal lysosomal processing and αsyn clearance. We found that Rab27b KD in this αsyn inducible neuronal model resulted in lysosomal dysfunction and increased αsyn levels in lysosomes. Similar lysosomal proteolytic defects and enzymatic dysfunction were observed in both primary neuronal cultures and brain lysates from Rab27b knockout (KO) mice. αSyn aggregation was exacerbated in Rab27b KO neurons upon treatment with αsyn preformed fibrils. We found no changes in lysosomal counts or lysosomal pH in either model, but we did identify defects in acidic vesicle trafficking in Rab27b KO primary neurons which may drive lysosomal dysfunction and promote αsyn aggregation. Rab27b OE enhanced lysosomal activity and reduced insoluble αsyn accumulation. Finally we found elevated Rab27b levels in human postmortem incidental Lewy Body Disease (iLBD) subjects relative to healthy controls. These data suggest a role for Rab27b in neuronal lysosomal activity and identify it as a potential therapeutic target in synucleinopathies.

Urolithin A promotes p62-dependent lysophagy to prevent acute retinal neurodegeneration

BackgroundAge-related macular degeneration (AMD) is the leading cause of blindness in elderly people in the developed world, and the number of people affected is expected to almost double by 2040. The retina presents one of the highest metabolic demands in our bodies that is partially or fully fulfilled by mitochondria in the neuroretina and retinal pigment epithelium (RPE), respectively. Together with its post-mitotic status and constant photooxidative damage from incoming light, the retina requires a tightly-regulated housekeeping system that involves autophagy. The natural polyphenol Urolithin A (UA) has shown neuroprotective benefits in several models of aging and age-associated disorders, mostly attributed to its ability to induce mitophagy and mitochondrial biogenesis. Sodium iodate (SI) administration recapitulates the late stages of AMD, including geographic atrophy and photoreceptor cell death.MethodsA combination of in vitro, ex vivo and in vivo models were used to test the neuroprotective potential of UA in the SI model. Functional assays (OCT, ERGs), cellular analysis (flow cytometry, qPCR) and fine confocal microscopy (immunohistochemistry, tandem selective autophagy reporters) helped address this question.ResultsUA alleviated neurodegeneration and preserved visual function in SI-treated mice. Simultaneously, we observed severe proteostasis defects upon SI damage induction, including autophagosome accumulation, that were resolved in animals that received UA. Treatment with UA restored autophagic flux and triggered PINK1/Parkin-dependent mitophagy, as previously reported in the literature. Autophagy blockage caused by SI was caused by severe lysosomal membrane permeabilization. While UA did not induce lysosomal biogenesis, it did restore upcycling of permeabilized lysosomes through lysophagy. Knockdown of the lysophagy adaptor SQSTM1/p62 abrogated viability rescue by UA in SI-treated cells, exacerbated lysosomal defects and inhibited lysophagy.ConclusionsCollectively, these data highlight a novel putative application of UA in the treatment of AMD whereby it bypasses lysosomal defects by promoting p62-dependent lysophagy to sustain proteostasis.Graphical

Atlastin-1 regulates endosomal tubulation and lysosomal proteolysis in human cortical neurons

Mutation of the ATL1 gene is one of the most common causes of hereditary spastic paraplegia (HSP), a group of genetic neurodegenerative conditions characterised by distal axonal degeneration of the corticospinal tract axons. Atlastin-1, the protein encoded by ATL1, is one of three mammalian atlastins, which are homologous dynamin-like GTPases that control endoplasmic reticulum (ER) morphology by fusing tubules to form the three-way junctions that characterise ER networks. However, it is not clear whether atlastin-1 is required for correct ER morphology in human neurons and if so what the functional consequences of lack of atlastin-1 are. Using CRISPR-inhibition we generated human cortical neurons lacking atlastin-1. We demonstrate that ER morphology was altered in these neurons, with a reduced number of three-way junctions. Neurons lacking atlastin-1 had longer endosomal tubules, suggestive of defective tubule fission. This was accompanied by reduced lysosomal proteolytic capacity. As well as demonstrating that atlastin-1 is required for correct ER morphology in human neurons, our results indicate that lack of a classical ER-shaping protein such as atlastin-1 may cause altered endosomal tubulation and lysosomal proteolytic dysfunction. Furthermore, they strengthen the idea that defective lysosome function contributes to the pathogenesis of a broad group of HSPs, including those where the primary localisation of the protein involved is not at the endolysosomal system.

Human tNeurons reveal aging-linked proteostasis deficits driving Alzheimer's phenotypes.

Aging is a prominent risk factor for Alzheimer's disease (AD), but the cellular mechanisms underlying neuronal phenotypes remain elusive. Both accumulation of amyloid plaques and neurofibrillary tangles in the brain1 and age-linked organelle deficits2-7 are proposed as causes of AD phenotypes but the relationship between these events is unclear. Here, we address this question using a transdifferentiated neuron (tNeuron) model directly from human dermal fibroblasts. Patient-derived tNeurons retain aging hallmarks and exhibit AD-linked deficits. Quantitative tNeuron proteomic analyses identify aging and AD-linked deficits in proteostasis and organelle homeostasis, particularly affecting endosome-lysosomal components. The proteostasis and lysosomal homeostasis deficits in aged tNeurons are exacerbated in sporadic and familial AD tNeurons, promoting constitutive lysosomal damage and defects in ESCRT-mediated repair. We find deficits in neuronal lysosomal homeostasis lead to inflammatory cytokine secretion, cell death and spontaneous development of Aß and phospho-Tau deposits. These proteotoxic inclusions co-localize with lysosomes and damage markers and resemble inclusions in brain tissue from AD patients and APP-transgenic mice. Supporting the centrality of lysosomal deficits driving AD phenotypes, lysosome-function enhancing compounds reduce AD-associated cytokine secretion and Aβ deposits. We conclude that proteostasis and organelle deficits are upstream initiating factors leading to neuronal aging and AD phenotypes.

Upregulation of cholesterol synthesis by lysosomal defects requires a functional mitochondrial respiratory chain

Mitochondria and lysosomes are two organelles that carry out both signaling and metabolic roles in cells. Recent evidence has shown that mitochondria and lysosomes are dependent on one another, as primary defects in one cause secondary defects in the other. Although there are functional impairments in both cases, the signaling consequences of primary mitochondrial dysfunction and lysosomal defects are dissimilar. Here, we used RNA sequencing to obtain transcriptomes from cells with primary mitochondrial or lysosomal defects to identify the global cellular consequences associated with mitochondrial or lysosomal dysfunction. We used these data to determine the pathways affected by defects in both organelles, which revealed a prominent role for the cholesterol synthesis pathway. We observed a transcriptional upregulation of this pathway in cellular and murine models of lysosomal defects, while it is transcriptionally downregulated in cellular and murine models of mitochondrial defects. We identified a role for the posttranscriptional regulation of transcription factor SREBF1, a master regulator of cholesterol and lipid biosynthesis, in models of mitochondrial respiratory chain deficiency. Furthermore, we found that retention of Ca2+ in lysosomes of cells with mitochondrial respiratory chain defects contributes to the differential regulation of the cholesterol synthesis pathway in the mitochondrial and lysosomal defects tested. Finally, we verified invivo, using a model of mitochondria-associated disease in Caenorhabditis elegans that normalization of lysosomal Ca2+ levels results in partial rescue of the developmental delay induced by the respiratory chain deficiency.

Microplastics and Oxidative Stress-Current Problems and Prospects.

Microplastics (MPs) are plastic particles between 0.1 and 5000 µm in size that have attracted considerable attention from the scientific community and the general public, as they threaten the environment. Microplastics contribute to various harmful effects, including lipid peroxidation, DNA damage, activation of mitogen-activated protein kinase pathways, cell membrane breakages, mitochondrial dysfunction, lysosomal defects, inflammation, and apoptosis. They affect cells, tissues, organs, and overall health, potentially contributing to conditions like cancer and cardiovascular disease. They pose a significant danger due to their widespread occurrence in food. In recent years, information has emerged indicating that MPs can cause oxidative stress (OS), a known factor in accelerating the aging of organisms. This comprehensive evaluation exposed notable variability in the reported connection between MPs and OS. This work aims to provide a critical review of whether the harmfulness of plastic particles that constitute environmental contaminants may result from OS through a comprehensive analysis of recent research and existing scientific literature, as well as an assessment of the characteristics of MPs causing OS. Additionally, the article covers the analytical methodology used in this field. The conclusions of this review point to the necessity for further research into the effects of MPs on OS.

Intersection of chronic contractile activity and lysosomal defects in mediating lysosomal adaptations in skeletal muscle cells

Mitophagy is the cellular process that serves to degrade mitochondria when these organelles lose their potential for ATP production, generate excessive reactive oxygen species, and exhibit a reduced membrane potential. The terminal step of mitophagy is mediated by the lysosome, the organelle that degrades defective cellular cargos. Electron microscopy evidence shows that lysosomes become defective in aging muscle, as well as in lysosomal storage diseases, ultimately leading to cellular pathology. Recent research has shown that chronic contractile activity can induce rapid increases in lysosomal gene and protein expression in muscle. Our objective is to investigate whether this adaptation leads to greater lysosomal content and function, thus enhancing the capacity to degrade defective mitochondria. This result would suggest that reduced lysosomal function in muscle could be rescued by exercise. However, the underlying mechanisms mediating lysosomal turnover and function are not clear. To study this, we simulated lysosomal dysfunction in C2C12 myotubes using short-interfering RNA targeting the lysosomal calcium channel mucolipin-1 (MCOLN1) or the autophagosome-lysosome fusion protein, lysosomal-associated membrane protein 2 (LAMP2). We achieved a 60-70% knockdown, as well as a 40-50% knockdown, after a 24-hour transfection, in each lysosomal marker, respectively. The cells were then subjected to four days of electrical stimulation-induced chronic contractile activity. Changes in gene expression were measured via qPCR, while protein content was measured via western blotting techniques, in addition to lysosomal content and functional assessments via confocal microscopy and flow cytometry. Silencing of MCOLN1 or LAMP2 both resulted in a compensatory increase in the transcript and protein levels of lysosomal genes, including the lysosomal transcription factors TFEB, TFE3, as well as lysosomal cathepsins B and D. Interestingly, contractile activity superimposed on the MCOLN1 or LAMP2 knockdown did not further enhance these compensatory increases. This suggests that contractile activity and the cellular signaling that arises from lysosomal defects, operate similarly to initiate the biogenesis of lysosomes as a negative feedback mechanism to enhance lysosomal degradation capacity. The nature of these signaling pathways, potentially involving changes in lysosome-derived intracellular calcium driving organelle synthesis, recycling, or degradation represent novel avenues for upcoming research investigating skeletal muscle phenotypic adaptations. This work is supported by NSERC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Defective Lamtor5 Leads to Autoimmunity by Deregulating v-ATPase and Lysosomal Acidification.

Despite accumulating evidence linking defective lysosome function with autoimmune diseases, how the catabolic machinery is regulated to maintain immune homeostasis remains unknown. Late endosomal/lysosomal adaptor, MAPK and mTOR activator 5 (Lamtor5) is a subunit of the Ragulator mediating mechanistic target of rapamycin complex 1 (mTORC1) activation in response to amino acids, but its action mode and physiological role are still unclear. Here it is demonstrated that Lamtor5 level is markedly decreased in peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE). In parallel, the mice with myeloid Lamtor5 ablation developed SLE-like manifestation. Impaired lysosomal function and aberrant activation of mTORC1 are evidenced in Lamtor5 deficient macrophages and PBMCs of SLE patients, accompanied by blunted autolysosomal pathway and undesirable inflammatory responses. Mechanistically, it is shown that Lamtor5 is physically associated with ATP6V1A, an essential subunit of vacuolar H+-ATPase(v-ATPase), and promoted the V0/V1 holoenzyme assembly to facilitate lysosome acidification. The binding of Lamtor5 to v-ATPase affected the lysosomal tethering of Rag GTPase and weakened its interaction with mTORC1 for activation. Overall, Lamtor5 is identified as a critical factor for immune homeostasis by intergrading v-ATPase activity, lysosome function, and mTOR pathway. The findings provide a potential therapeutic target for SLE and/or other autoimmune diseases.

EIF2α-mediated integrated stress response links multiple intracellular signaling pathways to reprogram vascular smooth muscle cell fate in carotid artery plaque

BackgroundCarotid arterial atherosclerotic stenosis is a well-recognized pathological basis of ischemic stroke; however, its underlying molecular mechanisms remain unknown. Vascular smooth muscle cells (VSMCs) play fundamental roles in the initiation and progression of atherosclerosis. Organelle dynamics have been reported to affect atherosclerosis development. However, the association between organelle dynamics and various cellular stresses in atherosclerotic progression remain ambiguous. MethodsIn this study, we conducted transcriptomics and bioinformatics analyses of stable and vulnerable carotid plaques. Primary VSMCs were isolated from carotid plaques and subjected to histopathological staining to determine their expression profiles. Endoplasmic reticulum (ER), mitochondria, and lysosome dynamics were observed in primary VSMCs and VSMC cell lines using live-cell imaging. Moreover, the mechanisms underlying disordered organelle dynamics were investigated using comprehensive biological approaches. ResultsER whorls, a representative structural change under ER stress, are prominent dynamic reconstructions of VSMCs between vulnerable and stable plaques, followed by fragmented mitochondria and enlarged lysosomes, suggesting mitochondrial stress and lysosomal defects, respectively. Induction of mitochondrial stress alleviated ER stress and autophagy in an eukaryotic translation initiation factor (eIF)-2α-dependent manner. Furthermore, the effects of eIF2α on ER stress, mitochondrial stress, and lysosomal defects were validated using clinical samples. ConclusionOur results indicate that morphological and functional changes in VSMC organelles, especially in ER whorls, can be used as reliable biomarkers for atherosclerotic progression. Moreover, eIF2α plays an important role in integrating multiple stress-signaling pathways to determine the behavior and fate of VSMCs.

Hydroxychloroquine Induced Cardiotoxicity: A Case Series.

Hydroxychloroquine (HCQ) induced cardiotoxicity is a rare diagnosis and is often associated with chronic use of the medication. It has been shown that chronic HCQ use is associated with a drug-induced cardiomyopathy mainly driven by acquired lysosomal storage defects leading to hypertrophy and conduction abnormalities. As the only proven treatment is the discontinuation of the offending agent, prompt recognition is required to avoid further exposure to the drug and potential progression of disease. History, physical examination and advanced imaging modalities are useful diagnostic tools, but more invasive testing with an endomyocardial biopsy is required for definitive diagnosis. We present a descriptive case series of ten patients that were diagnosed with biopsy proven HCQ cardiotoxicity.