Salmonella actively modulates TFEB in murine macrophages in a growth-phase and time-dependent manner.

IMPORTANCE: Activation of the host transcription factor TFEB helps mammalian cells adapt to stresses such as starvation and infection by upregulating lysosome, autophagy, and immuno-protective gene expression. Thus, TFEB is generally thought to protect host cells. However, it may also be that pathogenic bacteria like Salmonella orchestrate TFEB in a spatio-temporal manner to harness its functions to grow intracellularly. Indeed, the relationship between Salmonella and TFEB is controversial since some studies showed that Salmonella actively promotes TFEB, while others have observed that Salmonella degrades TFEB and that compounds that promote TFEB restrict bacterial growth. Our work provides a path to resolve these apparent discordant observations since we showed that stationary-grown Salmonella actively delays TFEB after infection, while late-log Salmonella is permissive of TFEB activation. Nevertheless, the exact function of this manipulation remains unclear, but conditions that erase the conditional control of TFEB by Salmonella may be detrimental to the microbe.

Cholesteryl Hemiazelate Present in Cardiovascular Disease Patients Causes Lysosome Dysfunction in Murine Fibroblasts.

There is growing evidence supporting the role of fibroblasts in all stages of atherosclerosis, from the initial phase to fibrous cap and plaque formation. In the arterial wall, as with macrophages and vascular smooth muscle cells, fibroblasts are exposed to a myriad of LDL lipids, including the lipid species formed during the oxidation of their polyunsaturated fatty acids of cholesteryl esters (PUFA-CEs). Recently, our group identified the final oxidation products of the PUFA-CEs, cholesteryl hemiesters (ChE), in tissues from cardiovascular disease patients. Cholesteryl hemiazelate (ChA), the most prevalent lipid of this family, is sufficient to impact lysosome function in macrophages and vascular smooth muscle cells, with consequences for their homeostasis. Here, we show that the lysosomal compartment of ChA-treated fibroblasts also becomes dysfunctional. Indeed, fibroblasts exposed to ChA exhibited a perinuclear accumulation of enlarged lysosomes full of neutral lipids. However, this outcome did not trigger de novo lysosome biogenesis, and only the lysosomal transcription factor E3 (TFE3) was slightly transcriptionally upregulated. As a consequence, autophagy was inhibited, probably via mTORC1 activation, culminating in fibroblasts' apoptosis. Our findings suggest that the impairment of lysosome function and autophagy and the induction of apoptosis in fibroblasts may represent an additional mechanism by which ChA can contribute to the progression of atherosclerosis.

A water-soluble fluorescent pH probe and its application for monitoring lysosomal pH changes in living cells.

Intracellular pH plays a crucial role in many cellular processes, and abnormal intracellular pH has been linked to common diseases such as cancer and Alzheimer's. To address this issue, a water-soluble fluorescent pH probe was designed based on the protonation/deprotonation of the 4-methylpiperazin-1-yl group, using dicyanoisophorone as the fluorophore. In the neutral form of the probe, fluorescence is quenched due to charge transfer from the 4-methylpiperazin-1-yl group to the fluorophore upon excitation. Under acidic conditions, protonation of the 4-methylpiperazin-1-yl group inhibits the photoinduced electron transfer process, leading to an increase in fluorescence intensity. Density-functional theory calculations also verified the fluorescence OFF-ON mechanism. The probe exhibits high selectivity, photostability, fast response to pH changes, and low cytotoxicity to cells. Additionally, the probe selectively accumulates in lysosomes, with a high Pearson coefficient (0.95) using LysoTracker Green DND-26 as a reference. Notably, the probe can monitor lysosomal pH changes in living cells and track pH changes stimulated by chloroquine. We anticipate that the probe has potential for diagnosing pH-related diseases.

LYST deficiency impairs autophagic lysosome reformation in neurons and alters lysosome number and size.

Chediak-Higashi syndrome (CHS) is a rare, autosomal recessive disorder caused by biallelic mutations in the lysosomal trafficking regulator (LYST) gene. Even though enlarged lysosomes and/or lysosome-related organelles (LROs) are the typical cellular hallmarks of CHS, they have not been investigated in human neuronal models. Moreover, how and why the loss of LYST function causes a lysosome phenotype in cells has not been elucidated. We report that the LYST-deficient human neuronal model exhibits lysosome depletion accompanied by hyperelongated tubules extruding from enlarged autolysosomes. These results have also been recapitulated in neurons differentiated from CHS patients' induced pluripotent stem cells (iPSCs), validating our model system. We propose that LYST ensures the correct fission/scission of the autolysosome tubules during autophagic lysosome reformation (ALR), a crucial process to restore the number of free lysosomes after autophagy. We further demonstrate that LYST is recruited to the lysosome membrane, likely to facilitate the fission of autolysosome tubules. Together, our results highlight the key role of LYST in maintaining lysosomal homeostasis following autophagy and suggest that ALR dysregulation is likely associated with the neurodegenerative CHS phenotype.

PTEN Deficiency Facilitates Exosome Secretion and Metastasis in Cholangiocarcinoma by Impairing TFEB-mediated Lysosome Biogenesis.

BACKGROUND & AIMS: In eukaryotes, the ubiquitin-proteasome system and the autophagy-lysosome pathway are essential for maintaining cellular proteostasis and associated with cancer progression. Our previous studies have demonstrated that phosphatase and tensin homolog (PTEN), one of the most frequently mutated genes in human cancers, limits proteasome abundance and determines chemosensitivity to proteasome inhibitors in cholangiocarcinoma (CCA). However, whether PTEN regulates the lysosome pathway remains unclear. METHODS: We tested the effects of PTEN on lysosome biogenesis and exosome secretion using loss- and gain-of-function strategies in CCA cell lines. Using in vitro dephosphorylation assays, we explored the regulatory mechanism between PTEN and the key regulator of lysosome biogenesis, transcription factor EB (TFEB). Using the migration assays, invasion assays, and trans-splenic liver metastasis mouse models, we evaluated the function of PTEN deficiency, TFEB-mediated lysosome biogenesis, and exosome secretion on tumor metastasis. Moreover, we investigated the clinical significance of PTEN expression and exosome secretion by retrospective analysis. RESULTS: PTEN facilitated lysosome biogenesis and acidification through its protein phosphatase activity to dephosphorylate TFEB at Ser211. Notably, PTEN deficiency increased exosome secretion by reducing lysosome-mediated degradation of multi-vesicular bodies, which further facilitated the proliferation and invasion of CCA. TFEB agonist curcumin analog C1 restrained the metastatic phenotype caused by PTEN deficiency in mouse models, and we highlighted the correlation between PTEN deficiency and exosome secretion in clinical cohorts. CONCLUSIONS: In CCA, PTEN deficiency impairs lysosome biogenesis to facilitate exosome secretion and cancer metastasis in a TFEB phosphorylation-dependent manner.

ATP13A2 modifies mitochondrial localization of overexpressed TOM20 to autolysosomal pathway.

Mutations in ATP13A2 cause Kufor-Rakeb Syndrome (KRS), a juvenile form of Parkinson's Disease (PD). The gene product belongs to a diverse family of ion pumps and mediates polyamine influx from lysosomal lumen. While the biochemical and structural studies highlight its unique mechanics, how PD pathology is linked to ATP13A2 function remains unclear. Here we report that localization of overexpressed TOM20, a mitochondrial outer-membrane protein, is significantly altered upon ATP13A2 expression to partially merge with lysosome. Using Halo-fused version of ATP13A2, ATP13A2 was identified in lysosome and autophagosome. Upon ATP13A2 co-expression, overexpressed TOM20 was found not only in mitochondria but also within ATP13A2-containing autolysosome. This modification of TOM20 localization was inhibited by adding 1-methyl-4-phenylpyridinium (MPP+) and not accompanied with mitophagy induction. We suggest that ATP13A2 may participate in the control of overexpressed proteins targeted to mitochondrial outer-membrane.

Lysosomes at the Crossroads of Cell Metabolism, Cell Cycle, and Stemness.

Initially described as lytic bodies due to their degradative and recycling functions, lysosomes play a critical role in metabolic adaptation to nutrient availability. More recently, the contribution of lysosomal proteins to cell signaling has been established, and lysosomes have emerged as signaling hubs that regulate diverse cellular processes, including cell proliferation and cell fate. Deciphering these signaling pathways has revealed an extensive crosstalk between the lysosomal and cell cycle machineries that is only beginning to be understood. Recent studies also indicate that a number of lysosomal proteins are involved in the regulation of embryonic and adult stem cell fate and identity. In this review, we will focus on the role of the lysosome as a signaling platform with an emphasis on its function in integrating nutrient sensing with proliferation and cell cycle progression, as well as in stemness-related features, such as self-renewal and quiescence.

The concept of intrinsic versus extrinsic apoptosis.

Regulated cell death is a vital and dynamic process in multicellular organisms that maintains tissue homeostasis and eliminates potentially dangerous cells. Apoptosis, one of the better-known forms of regulated cell death, is activated when cell-surface death receptors like Fas are engaged by their ligands (the extrinsic pathway) or when BCL-2-family pro-apoptotic proteins cause the permeabilization of the mitochondrial outer membrane (the intrinsic pathway). Both the intrinsic and extrinsic pathways of apoptosis lead to the activation of a family of proteases, the caspases, which are responsible for the final cell demise in the so-called execution phase of apoptosis. In this review, I will first discuss the most common types of regulated cell death on a morphological basis. I will then consider in detail the molecular pathways of intrinsic and extrinsic apoptosis, discussing how they are activated in response to specific stimuli and are sometimes overlapping. In-depth knowledge of the cellular mechanisms of apoptosis is becoming more and more important not only in the field of cellular and molecular biology but also for its translational potential in several pathologies, including neurodegeneration and cancer.

JIP3 links lysosome transport to regulation of multiple components of the axonal cytoskeleton.

Lysosome axonal transport is important for the clearance of cargoes sequestered by the endocytic and autophagic pathways. Building on observations that mutations in the JIP3 (MAPK8IP3) gene result in lysosome-filled axonal swellings, we analyzed the impact of JIP3 depletion on the cytoskeleton of human neurons. Dynamic focal lysosome accumulations were accompanied by disruption of the axonal periodic scaffold (spectrin, F-actin and myosin II) throughout each affected axon. Additionally, axonal microtubule organization was locally disrupted at each lysosome-filled swelling. This local axonal microtubule disorganization was accompanied by accumulations of both F-actin and myosin II. These results indicate that transport of axonal lysosomes is functionally interconnected with mechanisms that control the organization and maintenance of the axonal cytoskeleton. They have potential relevance to human neurological disease arising from JIP3 mutations as well as for neurodegenerative diseases associated with the focal accumulations of lysosomes within axonal swellings such as Alzheimer's disease.

De Novo Design of A Membrane-Anchored Probe for Multidimensional Quantification of Endocytic Dynamics.

As a process of cellular uptake, endocytosis, with gradient acidity in different endocytic vesicles, is vital for the homeostasis of intracellular nutrients and other functions. To study the dynamics of endocytic pathway, a membrane-anchored pH probe, ECGreen, is synthesized to visualize endocytic vesicles under structured illumination microscopy (SIM), a super-resolution technology. Being sensitive to acidity with increasing fluorescence at low pH, ECGreen can differentiate early and late endosomes as well as endolysosomes. Meanwhile, membrane anchoring not only improves the durability of ECGreen, but also provides an excellent anti-photobleaching property for long-time imaging with SIM. Moreover, by taking these advantages of ECGreen, a multidimensional analysis model containing spatial, temporal, and pH information is successfully developed for elucidating the dynamics of endocytic vesicles and their interactions with mitochondria during autophagy, and reveals a fast conversion of endosomes near the plasma membrane.

Intravitreal gene therapy restores the autophagy-lysosomal pathway and attenuates retinal degeneration in cathepsin D-deficient mice.

Loss of vision due to progressive retinal degeneration is a hallmark of neuronal ceroid lipofuscinoses (NCL), a group of fatal neurodegenerative lysosomal storage diseases. Enzyme substitution therapies represent promising treatment options for NCLs caused by dysfunctions of soluble lysosomal enzymes. Here, we compared the efficacy of a cell-based enzyme substitution strategy and a gene therapy approach to attenuate the retinal pathology in cathepsin D- (CTSD) deficient mice, an animal model of CLN10 disease. Levels of enzymatically active CTSD in mutant retinas were significantly higher after an adeno-associated virus vector-mediated CTSD transfer to retinal glial cells and retinal pigment epithelial cells than after intravitreal transplantations of a CTSD overexpressing clonal neural stem cell line. In line with this finding, the gene therapy treatment restored the disrupted autophagy-lysosomal pathway more effectively than the cell-based approach, as indicated by a complete clearance of storage, significant attenuation of lysosomal hypertrophy, and normalized levels of the autophagy marker sequestosome 1/p62 and microtubule-associated protein 1 light chain 3-II. While the cell-based treatment did not prevent the rapidly progressing loss of various retinal cell types, the gene therapy approach markedly attenuated retinal degeneration as demonstrated by a pronounced rescue of photoreceptor cells and rod bipolar cells.

Protocol for labeling and fixation of intact lysosomes with esterified amino acid analogs to assess lysosomal expansion in living eukaryotic cells.

The lysosomal compartment is a key hub for cell metabolism, and morphological alterations have been described in several pathological conditions. Here, we describe the use of amino acid analogs modified by the presence of a methyl ester group that accumulates within lysosomes. This generates an intraluminal osmotic effect able to trigger a rapid and selective expansion of the lysosomal compartment within 2 h of treatment. We also present protocols to preserve lysosomal morphology, which yields a more accurate size measurement. For complete details on the use and execution of this protocol, please refer to Scerra et al. (2021).

ORF3a of SARS-CoV-2 promotes lysosomal exocytosis-mediated viral egress.

Viral entry and egress are important determinants of virus infectivity and pathogenicity. ß-coronaviruses, including the COVID-19 virus SARS-CoV-2 and mouse hepatitis virus (MHV), exploit the lysosomal exocytosis pathway for egress. Here, we show that SARS-CoV-2 ORF3a, but not SARS-CoV ORF3a, promotes lysosomal exocytosis. SARS-CoV-2 ORF3a facilitates lysosomal targeting of the BORC-ARL8b complex, which mediates trafficking of lysosomes to the vicinity of the plasma membrane, and exocytosis-related SNARE proteins. The Ca2+ channel TRPML3 is required for SARS-CoV-2 ORF3a-mediated lysosomal exocytosis. Expression of SARS-CoV-2 ORF3a greatly elevates extracellular viral release in cells infected with the coronavirus MHV-A59, which itself lacks ORF3a. In SARS-CoV-2 ORF3a, Ser171 and Trp193 are critical for promoting lysosomal exocytosis and blocking autophagy. When these residues are introduced into SARS-CoV ORF3a, it acquires the ability to promote lysosomal exocytosis and inhibit autophagy. Our results reveal a mechanism by which SARS-CoV-2 interacts with host factors to promote its extracellular egress.

Enhanced enzymatic production of cholesteryl 6'-acylglucoside impairs lysosomal degradation for the intracellular survival of Helicobacter pylori.

BACKGROUND: During autophagy defense against invading microbes, certain lipid types are indispensable for generating specialized membrane-bound organelles. The lipid composition of autophagosomes remains obscure, as does the issue of how specific lipids and lipid-associated enzymes participate in autophagosome formation and maturation. Helicobacter pylori is auxotrophic for cholesterol and converts cholesterol to cholesteryl glucoside derivatives, including cholesteryl 6'-O-acyl-α-D-glucoside (CAG). We investigated how CAG and its biosynthetic acyltransferase assist H. pylori to escape host-cell autophagy. METHODS: We applied a metabolite-tagging method to obtain fluorophore-containing cholesteryl glucosides that were utilized to understand their intracellular locations. H. pylori 26695 and a cholesteryl glucosyltransferase (CGT)-deletion mutant (ΔCGT) were used as the standard strain and the negative control that contains no cholesterol-derived metabolites, respectively. Bacterial internalization and several autophagy-related assays were conducted to unravel the possible mechanism that H. pylori develops to hijack the host-cell autophagy response. Subcellular fractions of H. pylori-infected AGS cells were obtained and measured for the acyltransferase activity. RESULTS: The imaging studies of fluorophore-labeled cholesteryl glucosides pinpointed their intracellular localization in AGS cells. The result indicated that CAG enhances the internalization of H. pylori in AGS cells. Particularly, CAG, instead of CG and CPG, is able to augment the autophagy response induced by H. pylori. How CAG participates in the autophagy process is multifaceted. CAG was found to intervene in the degradation of autophagosomes and reduce lysosomal biogenesis, supporting the idea that intracellular H. pylori is harbored by autophago-lysosomes in favor of the bacterial survival. Furthermore, we performed the enzyme activity assay of subcellular fractions of H. pylori-infected AGS cells. The analysis showed that the acyltransferase is mainly distributed in autophago-lysosomal compartments. CONCLUSIONS: Our results support the idea that the acyltransferase is mainly distributed in the subcellular compartment consisting of autophagosomes, late endosomes, and lysosomes, in which the acidic environment is beneficial for the maximal acyltransferase activity. The resulting elevated level of CAG can facilitate bacterial internalization, interfere with the autophagy flux, and causes reduced lysosomal biogenesis.

Exercise and Sestrin Mediate Speed and Lysosomal Activity in Drosophila by Partially Overlapping Mechanisms.

Chronic exercise is widely recognized as an important contributor to healthspan in humans and in diverse animal models. Recently, we have demonstrated that Sestrins, a family of evolutionarily conserved exercise-inducible proteins, are critical mediators of exercise benefits in flies and mice. Knockout of Sestrins prevents exercise adaptations to endurance and flight in Drosophila, and similarly prevents benefits to endurance and metabolism in exercising mice. In contrast, overexpression of dSestrin in muscle mimics several of the molecular and physiological adaptations characteristic of endurance exercise. Here, we extend those observations to examine the impact of dSestrin on preserving speed and increasing lysosomal activity. We find that dSestrin is a critical factor driving exercise adaptations to climbing speed, but is not absolutely required for exercise to increase lysosomal activity in Drosophila. The role of Sestrin in increasing speed during chronic exercise requires both the TORC2/AKT axis and the PGC1α homolog spargel, while dSestrin requires interactions with TORC1 to cell-autonomously increase lysosomal activity. These results highlight the conserved role of Sestrins as key factors that drive diverse physiological adaptations conferred by chronic exercise.

RORα Enhances Lysosomal Acidification and Autophagic Flux in the Hepatocytes.

Lysosomes are intracellular acidic organelles with catabolic functions that contribute to the activation of autophagy. Although autophagy abnormality is associated with defects in lysosomal acidification during the progression of nonalcoholic fatty liver disease (NAFLD), the mechanisms of control of lysosomal acidification are not well understood at the molecular level. Thus, we aimed to elucidate the role of the orphan nuclear receptor retinoic acid-related orphan receptor α (RORα) in lysosomal acidification and autophagic flux, particularly in nutrition-enriched hepatocytes. First, lysosomal acidity was much lower in the hepatocytes obtained from hepatocyte-specific RORα-deleted (RORα-LKO) mice, whereas the infusion of an adenovirus encoding RORα in wild-type hepatocytes increased lysosomal acidity, as determined by LysoSensor. Second, the lysosomal translocation of the mechanistic target of rapamycin was increased and immature cathepsin D was accumulated in the liver of RORα-LKO mice. Third, the accumulation of LC3-II, p62/sequestosome 1 (SQSTM1), and neighbor of BRCA1 gene 1 (NBR1) was increased in the livers of RORα-LKO mice, indicating an impaired autophagic flux in the livers. Consistently, the number of autolysosomes containing mitochondria and lipid droplets was dramatically reduced in the RORα-deleted hepatocytes. Finally, we found that RORα induced the transcription of genes involved in lysosomal function, such as Atp6v1g1, a vacuolar H+ -ATPase (v-ATPase) subunit, which were largely down-regulated in the livers of mice with high-fat diet-induced NAFLD and patients with hepatitis. Conclusion: Targeting RORα may be a potential therapeutic strategy to restore lysosomal acidification, which inhibits the progression of NAFLD.

Rethinking lysosomes and lysosomal disease.

Lysosomal storage diseases were recognized and defined over a century ago as a class of disorders affecting mostly children and causing systemic disease often accompanied by major neurological consequences. Since their discovery, research focused on understanding their causes has been an important driver of our ever-expanding knowledge of cell biology and the central role that lysosomes play in cell function. Today we recognize over 50 so-called storage diseases, with most understood at the level of gene, protein and pathway involvement, but few fully clarified in terms of how the defective lysosomal function causes brain disease; even fewer have therapies that can effectively rescue brain function. Importantly, we also recognize that storage diseases are not simply a class of lysosomal disorders all by themselves, as increasingly a critical role for the greater lysosomal system with its endosomal, autophagosomal and salvage streams has also emerged in a host of neurodevelopmental and neurodegenerative diseases. Despite persistent challenges across all aspects of these complex disorders, and as reflected in this and other articles focused on lysosomal storage diseases in this special issue of Neuroscience Letters, the progress and promise to both understand and effectively treat these conditions has never been greater.

LOXL3 Silencing Affected Cell Adhesion and Invasion in U87MG Glioma Cells.

Lysyl oxidase-like 3 (LOXL3), belonging to the lysyl oxidase family, is responsible for the crosslinking in collagen or elastin. The cellular localization of LOXL3 is in the extracellular space by reason of its canonical function. In tumors, the presence of LOXL3 has been associated with genomic stability, cell proliferation, and metastasis. In silico analysis has shown that glioblastoma was among tumors with the highest LOXL3 expression levels. LOXL3 silencing of U87MG cells by siRNA led to the spreading of the tumor cell surface, and the transcriptome analysis of these cells revealed an upregulation of genes coding for extracellular matrix, cell adhesion, and cytoskeleton components, convergent to an increase in cell adhesion and a decrease in cell invasion observed in functional assays. Significant correlations of LOXL3 expression with genes coding for tubulins were observed in the mesenchymal subtype in the TCGA RNA-seq dataset of glioblastoma (GBM). Conversely, genes involved in endocytosis and lysosome formation, along with MAPK-binding proteins related to focal adhesion turnover, were downregulated, which may corroborate the observed decrease in cell viability and increase in the rate of cell death. Invasiveness is a major determinant of the recurrence and poor outcome of GBM patients, and downregulation of LOXL3 may contribute to halting the tumor cell invasion.

ß-Cell autophagy in the pathogenesis of type 1 diabetes.

Type 1 diabetes is an insulin-dependent, autoimmune disease where the pancreatic ß cells are destroyed resulting in hyperglycemia. This multifactorial disease involves multiple environmental and genetic factors, and has no clear etiology. Accumulating evidence suggests that early signaling defects within the ß cells may promote a change in the local immune milieu leading to autoimmunity. Therefore, many studies have been focused on intrinsic ß-cell mechanisms that aid in the restoration of cellular homeostasis under environmental conditions that cause dysfunction. One of these intrinsic mechanisms to promote homeostasis is autophagy, defects which are clearly linked with ß-cell dysfunction in the context of type 2 diabetes. Recent studies have now also pointed towards ß-cell autophagy defects in the context of type 1 diabetes. In this perspectives review, we will discuss the evidence supporting a role for ß-cell autophagy in the pathogenesis of type 1 diabetes, including a potential role for unconventional secretion of autophagosomes/lysosomes in the changing dialogue between the ß cell and immune cells.