Initiation of acute pancreatitis in mice is independent of fusion between lysosomes and zymogen granules.

The co-localization of the lysosomal protease cathepsin B (CTSB) and the digestive zymogen trypsinogen is a prerequisite for the initiation of acute pancreatitis. However, the exact molecular mechanisms of co-localization are not fully understood. In this study, we investigated the role of lysosomes in the onset of acute pancreatitis by using two different experimental approaches. Using an acinar cell-specific genetic deletion of the ras-related protein Rab7, important for intracellular vesicle trafficking and fusion, we analyzed the subcellular distribution of lysosomal enzymes and the severity of pancreatitis in vivo and ex vivo. Lysosomal permeabilization was performed by the lysosomotropic agent Glycyl-L-phenylalanine 2-naphthylamide (GPN). Acinar cell-specific deletion of Rab7 increased endogenous CTSB activity and despite the lack of re-distribution of CTSB from lysosomes to the secretory vesicles, the activation of CTSB localized in the zymogen compartment still took place leading to trypsinogen activation and pancreatic injury. Disease severity was comparable to controls during the early phase but more severe at later time points. Similarly, GPN did not prevent CTSB activation inside the secretory compartment upon caerulein stimulation, while lysosomal CTSB shifted to the cytosol. Intracellular trypsinogen activation was maintained leading to acute pancreatitis similar to controls. Our results indicate that initiation of acute pancreatitis seems to be independent of the presence of lysosomes and that fusion of lysosomes and zymogen granules is dispensable for the disease onset. Intact lysosomes rather appear to have protective effects at later disease stages.

Physiological and pathological functions of TMEM106B in neurodegenerative diseases.

As an integral lysosomal transmembrane protein, transmembrane protein 106B (TMEM106B) regulates several aspects of lysosomal function and is associated with neurodegenerative diseases. The TMEM106B gene mutations lead to lysosomal dysfunction and accelerate the pathological progression of Neurodegenerative diseases. Yet, the precise mechanism of TMEM106B in Neurodegenerative diseases remains unclear. Recently, different research teams discovered that TMEM106B is an amyloid protein and the C-terminal domain of TMEM106B forms amyloid fibrils in various Neurodegenerative diseases and normally elderly individuals. In this review, we discussed the physiological functions of TMEM106B. We also included TMEM106B gene mutations that cause neurodegenerative diseases. Finally, we summarized the identification and cryo-electronic microscopic structure of TMEM106B fibrils, and discussed the promising therapeutic strategies aimed at TMEM106B fibrils and the future directions for TMEM106B research in neurodegenerative diseases.

Enhancing antitumor efficacy of NIR-I region zinc phthalocyanine@upconversion nanoparticle through lysosomal escape and mitochondria targeting.

Accurately visualizing the intracellular trafficking of upconversion nanoparticles (UCNPs) loaded with phthalocyanines and achieving precise photodynamic therapy (PDT) using near-infrared (NIR) laser irradiation still present challenges. In this study, a novel NIR laser-triggered upconversion luminescence (UCL) imaging-guided nanoparticle called FA@TPA-NH-ZnPc@UCNPs (FTU) was developed for PDT. FTU consisted of UCNPs, folic acid (FA), and triphenylamino-phenylaniline zinc phthalocyanine (TPA-NH-ZnPc). Notably, TPA-NH-ZnPc showcases aggregation-induced emission (AIE) characteristic and NIR absorption properties at 741 nm, synthesized initially via molybdenum-catalyzed condensation reaction. The UCL emitted by FTU enable real-time visualization of their subcellular localization and intracellular trafficking within ovarian cancer HO-8910 cells. Fluorescence images revealed that FTU managed to escape from lysosomes due to the "proton sponge" effect of TPA-NH-ZnPc. The FA ligands on the surface of FTU further directed their transport and accumulation within mitochondria. When excited by a 980 nm laser, FTU exhibited UCL and activated TPA-NH-ZnPc, consequently generating cytotoxic singlet oxygen (1O2), disrupted mitochondrial function and induced apoptosis in cancer cells, which demonstrated great potential for tumor ablation.

Ultraphotostable Phosphorescent Nanosensors for Sensing the Lysosomal pH at the Single-Cell Level over Long Durations.

Ultraphotostable phosphorescent nanosensors have been designed for continuously sensing the lysosome pH over a long duration. The nanosensors exhibited excellent photostability, high accuracy, and capability to measure pH values during cell proliferation for up to 7 days. By arranging a metal-ligand complex of long phosphorescence lifetime and pH indicator in silica nanoparticles, we discover efficient Förster resonance energy transfer, which converts the pH-responsive UV-vis absorption signal of the pH indicator into a phosphorescent signal. Both the phosphorescent intensity and lifetime change at different pH values, and intracellular pH values can be accurately measured by our custom-built rapid phosphorescent lifetime imaging microscopy. The excellent photostability, high accuracy, and good biocompatibility prove that these nanosensors are a useful tool for tracing the fluctuation of pH values during endocytosis. The methodology can be easily adapted to design new nanosensors with different pKa or for different kinds of intracellular ions, as there are hundreds of pH and ion indicators readily available.

Giant organelle vesicles to uncover intracellular membrane mechanics and plasticity.

Tools for accessing and studying organelles remain underdeveloped. Here, we present a method by which giant organelle vesicles (GOVs) are generated by submitting cells to a hypotonic medium followed by plasma membrane breakage. By this means, GOVs ranging from 3 to over 10 µm become available for micromanipulation. GOVs are made from organelles such as the endoplasmic reticulum, endosomes, lysosomes and mitochondria, or in contact with one another such as giant mitochondria-associated ER membrane vesicles. We measure the mechanical properties of each organelle-derived GOV and find that they have distinct properties. In GOVs procured from Cos7 cells, for example, bending rigidities tend to increase from the endoplasmic reticulum to the plasma membrane. We also found that the mechanical properties of giant endoplasmic reticulum vesicles (GERVs) vary depending on their interactions with other organelles or the metabolic state of the cell. Lastly, we demonstrate GERVs' biochemical activity through their capacity to synthesize triglycerides and assemble lipid droplets. These findings underscore the potential of GOVs as valuable tools for studying the biophysics and biology of organelles.

RUFY4 deletion prevents pathological bone loss by blocking endo-lysosomal trafficking of osteoclasts.

Mature osteoclasts degrade bone matrix by exocytosis of active proteases from secretory lysosomes through a ruffled border. However, the molecular mechanisms underlying lysosomal trafficking and secretion in osteoclasts remain largely unknown. Here, we show with GeneChip analysis that RUN and FYVE domain-containing protein 4 (RUFY4) is strongly upregulated during osteoclastogenesis. Mice lacking Rufy4 exhibited a high trabecular bone mass phenotype with abnormalities in osteoclast function in vivo. Furthermore, deleting Rufy4 did not affect osteoclast differentiation, but inhibited bone-resorbing activity due to disruption in the acidic maturation of secondary lysosomes, their trafficking to the membrane, and their secretion of cathepsin K into the extracellular space. Mechanistically, RUFY4 promotes late endosome-lysosome fusion by acting as an adaptor protein between Rab7 on late endosomes and LAMP2 on primary lysosomes. Consequently, Rufy4-deficient mice were highly protected from lipopolysaccharide- and ovariectomy-induced bone loss. Thus, RUFY4 plays as a new regulator in osteoclast activity by mediating endo-lysosomal trafficking and have a potential to be specific target for therapies against bone-loss diseases such as osteoporosis.

TASL mediates keratinocyte differentiation by regulating intracellular calcium levels and lysosomal function.

Maintaining epidermal homeostasis relies on a tightly organized process of proliferation and differentiation of keratinocytes. While past studies have primarily focused on calcium regulation in keratinocyte differentiation, recent research has shed light on the crucial role of lysosome dysfunction in this process. TLR adaptor interacting with SLC15A4 on the lysosome (TASL) plays a role in regulating pH within the endo-lysosome. However, the specific role of TASL in keratinocyte differentiation and its potential impact on proliferation remains elusive. In our study, we discovered that TASL deficiency hinders the proliferation and migration of keratinocytes by inducing G1/S cell cycle arrest. Also, TASL deficiency disrupts proper differentiation process in TASL knockout human keratinocyte cell line (HaCaT) by affecting lysosomal function. Additionally, our research into calcium-induced differentiation showed that TASL deficiency affects calcium modulation, which is essential for keratinocyte regulation. These findings unveil a novel role of TASL in the proliferation and differentiation of keratinocytes, providing new insights into the intricate regulatory mechanisms of keratinocyte biology.

Impairing hydrolase transport machinery prevents human melanoma metastasis.

Metastases are the major cause of cancer-related death, yet, molecular weaknesses that could be exploited to prevent tumor cells spreading are poorly known. Here, we found that perturbing hydrolase transport to lysosomes by blocking either the expression of IGF2R, the main receptor responsible for their trafficking, or GNPT, a transferase involved in the addition of the specific tag recognized by IGF2R, reduces melanoma invasiveness potential. Mechanistically, we demonstrate that the perturbation of this traffic, leads to a compensatory lysosome neo-biogenesis devoided of degradative enzymes. This regulatory loop relies on the stimulation of TFEB transcription factor expression. Interestingly, the inhibition of this transcription factor playing a key role of lysosome production, restores melanomas' invasive potential in the absence of hydrolase transport. These data implicate that targeting hydrolase transport in melanoma could serve to develop new therapies aiming to prevent metastasis by triggering a physiological response stimulating TFEB expression in melanoma.

The lysosomal trafficking regulator "LYST": an 80-year traffic jam.

Lysosomes and lysosome related organelles (LROs) are dynamic organelles at the intersection of various pathways involved in maintaining cellular hemostasis and regulating cellular functions. Vesicle trafficking of lysosomes and LROs are critical to maintain their functions. The lysosomal trafficking regulator (LYST) is an elusive protein important for the regulation of membrane dynamics and intracellular trafficking of lysosomes and LROs. Mutations to the LYST gene result in Chédiak-Higashi syndrome, an autosomal recessive immunodeficiency characterized by defective granule exocytosis, cytotoxicity, etc. Despite eight decades passing since its initial discovery, a comprehensive understanding of LYST's function in cellular biology remains unresolved. Accumulating evidence suggests that dysregulation of LYST function also manifests in other disease states. Here, we review the available literature to consolidate available scientific endeavors in relation to LYST and discuss its relevance for immunomodulatory therapies, regenerative medicine and cancer applications.

Response to Comments on "Increasing Enzyme Mannose-6-Phosphate Levels but Not Miglustat Coadministration Enhances the Efficacy of Enzyme Replacement Therapy in Pompe Mice".

We thank Dr. Weimer and her colleagues for their comments related to our recent work (Anding et al., 2023) and are grateful for the opportunity to further discuss the importance of efficient lysosomal targeting of enzyme-replacement therapies (ERT) for the treatment of Pompe disease. Patients with Pompe disease have mutations in the gene that encodes for acid α glucosidase (GAA), a lysosomal enzyme necessary for the breakdown of glycogen. The first-generation ERT, alglucosidase alfa, provides a lifesaving therapy for the severe form of the disease (infantile onset Pompe disease) and improves or stabilizes respiratory and motor function in patients with less severe disease (late onset Pompe disease). Despite these gains, significant unmet need remains, particularly in patients who display respiratory and motor decline following years of treatment. Poor tissue uptake and lysosomal targeting via inefficient binding of the cation-independent mannose-6-phosphate (M6P) receptor (CIMPR) in skeletal muscle contributed to this suboptimal treatment response, prompting the development of new ERTs with increased levels of M6P.

Failure of Autophagy in Pompe Disease.

Autophagy is an evolutionarily conserved lysosome-dependent degradation of cytoplasmic constituents. The system operates as a critical cellular pro-survival mechanism in response to nutrient deprivation and a variety of stress conditions. On top of that, autophagy is involved in maintaining cellular homeostasis through selective elimination of worn-out or damaged proteins and organelles. The autophagic pathway is largely responsible for the delivery of cytosolic glycogen to the lysosome where it is degraded to glucose via acid α-glucosidase. Although the physiological role of lysosomal glycogenolysis is not fully understood, its significance is highlighted by the manifestations of Pompe disease, which is caused by a deficiency of this lysosomal enzyme. Pompe disease is a severe lysosomal glycogen storage disorder that affects skeletal and cardiac muscles most. In this review, we discuss the basics of autophagy and describe its involvement in the pathogenesis of muscle damage in Pompe disease. Finally, we outline how autophagic pathology in the diseased muscles can be used as a tool to fast track the efficacy of therapeutic interventions.

Cholesterol Modulation Attenuates the AD-like Phenotype Induced by Herpes Simplex Virus Type 1 Infection.

Cholesterol, a crucial component of cell membranes, influences various biological processes, including membrane trafficking, signal transduction, and host-pathogen interactions. Disruptions in cholesterol homeostasis have been linked to congenital and acquired conditions, including neurodegenerative disorders such as Alzheimer's disease (AD). Previous research from our group has demonstrated that herpes simplex virus type I (HSV-1) induces an AD-like phenotype in several cell models of infection. This study explores the interplay between cholesterol and HSV-1-induced neurodegeneration. The impact of cholesterol was determined by modulating its levels with methyl-beta-cyclodextrin (MßCD) using the neuroblastoma cell lines SK-N-MC and N2a. We have found that HSV-1 infection triggers the intracellular accumulation of cholesterol in structures resembling endolysosomal/autophagic compartments, a process reversible upon MßCD treatment. Moreover, MßCD exhibits inhibitory effects at various stages of HSV-1 infection, underscoring the importance of cellular cholesterol levels, not only in the viral entry process but also in subsequent post-entry stages. MßCD also alleviated several features of AD-like neurodegeneration induced by viral infection, including lysosomal impairment and intracellular accumulation of amyloid-beta peptide (Aß) and phosphorylated tau. In conclusion, these findings highlight the connection between cholesterol, neurodegeneration, and HSV-1 infection, providing valuable insights into the underlying mechanisms of AD.

Exogenous Metabolic Modulators Improve Response to Carboplatin in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) lacks targeted therapies, leaving cytotoxic chemotherapy as the current standard treatment. However, chemotherapy resistance remains a major clinical challenge. Increased insulin-like growth factor 1 signaling can potently blunt chemotherapy response, and lysosomal processes including the nutrient scavenging pathway autophagy can enable cancer cells to evade chemotherapy-mediated cell death. Thus, we tested whether inhibition of insulin receptor/insulin-like growth factor 1 receptor with the drug BMS-754807 and/or lysosomal disruption with hydroxychloroquine (HCQ) could sensitize TNBC cells to the chemotherapy drug carboplatin. Using in vitro studies in multiple TNBC cell lines, in concert with in vivo studies employing a murine syngeneic orthotopic transplant model of TNBC, we show that BMS-754807 and HCQ each sensitized TNBC cells and tumors to carboplatin and reveal that exogenous metabolic modulators may work synergistically with carboplatin as indicated by Bliss analysis. Additionally, we demonstrate the lack of overt in vivo toxicity with our combination regimens and, therefore, propose that metabolic targeting of TNBC may be a safe and effective strategy to increase sensitivity to chemotherapy. Thus, we conclude that the use of exogenous metabolic modulators, such as BMS-754807 or HCQ, in combination with chemotherapy warrants additional study as a strategy to improve therapeutic responses in women with TNBC.

Insights into PCSK9-LDLR Regulation and Trafficking via the Differential Functions of MHC-I Proteins HFE and HLA-C.

PCSK9 is implicated in familial hypercholesterolemia via targeting the cell surface PCSK9-LDLR complex toward lysosomal degradation. The M2 repeat in the PCSK9's C-terminal domain is essential for its extracellular function, potentially through its interaction with an unidentified "protein X". The M2 repeat was recently shown to bind an R-x-E motif in MHC-class-I proteins (implicated in the immune system), like HLA-C, and causing their lysosomal degradation. These findings suggested a new role of PCSK9 in the immune system and that HLA-like proteins could be "protein X" candidates. However, the participation of each member of the MHC-I protein family in this process and their regulation of PCSK9's function have yet to be determined. Herein, we compared the implication of MHC-I-like proteins such as HFE (involved in iron homeostasis) and HLA-C on the extracellular function of PCSK9. Our data revealed that the M2 domain regulates the intracellular sorting of the PCSK9-LDLR complex to lysosomes, and that HFE is a new target of PCSK9 that inhibits its activity on the LDLR, whereas HLA-C enhances its function. This work suggests the potential modulation of PCSK9's functions through interactions of HFE and HLA-C.

Heterologous HSPC Transplantation Rescues Neuroinflammation and Ameliorates Peripheral Manifestations in the Mouse Model of Lysosomal Transmembrane Enzyme Deficiency, MPS IIIC.

Mucopolysaccharidosis III type C (MPS IIIC) is an untreatable neuropathic lysosomal storage disease caused by a genetic deficiency of the lysosomal N-acetyltransferase, HGSNAT, catalyzing a transmembrane acetylation of heparan sulfate. HGSNAT is a transmembrane enzyme incapable of free diffusion between the cells or their cross-correction, which limits development of therapies based on enzyme replacement and gene correction. Since our previous work identified neuroinflammation as a hallmark of the CNS pathology in MPS IIIC, we tested whether it can be corrected by replacement of activated brain microglia with neuroprotective macrophages/microglia derived from a heterologous HSPC transplant. Eight-week-old MPS IIIC (HgsnatP304L) mice were transplanted with HSPC from congenic wild type mice after myeloablation with Busulfan and studied using behavior test battery, starting from the age of 6 months. At the age of ~8 months, mice were sacrificed to study pathological changes in the brain, heparan sulfate storage, and other biomarkers of the disease. We found that the treatment corrected several behavior deficits including hyperactivity and reduction in socialization, but not memory decline. It also improved several features of CNS pathology such as microastroglyosis, expression of pro-inflammatory cytokine IL-1ß, and accumulation of misfolded amyloid aggregates in cortical neurons. At the periphery, the treatment delayed development of terminal urinary retention, potentially increasing longevity, and reduced blood levels of heparan sulfate. However, we did not observe correction of lysosomal storage phenotype in neurons and heparan sulfate brain levels. Together, our results demonstrate that neuroinflammation in a neurological lysosomal storage disease, caused by defects in a transmembrane enzyme, can be effectively ameliorated by replacement of microglia bearing the genetic defect with cells from a normal healthy donor. They also suggest that heterologous HSPC transplant, if used together with other methods, such as chaperone therapy or substrate reduction therapy, may constitute an effective combination therapy for MPS IIIC and other disorders with a similar etiology.

Small-molecule Molephantin induces apoptosis and mitophagy flux blockage through ROS production in glioblastoma.

Glioblastoma (GBM), one of the most malignant brain tumors in the world, has limited treatment options and a dismal survival rate. Effective and safe disease-modifying drugs for glioblastoma are urgently needed. Here, we identified a small molecule, Molephantin (EM-5), effectively penetrated the blood-brain barrier (BBB) and demonstrated notable antitumor effects against GBM with good safety profiles both in vitro and in vivo. Mechanistically, EM-5 not only inhibits the proliferation and invasion of GBM cells but also induces cell apoptosis through the reactive oxygen species (ROS)-mediated PI3K/Akt/mTOR pathway. Furthermore, EM-5 causes mitochondrial dysfunction and blocks mitophagy flux by impeding the fusion of mitophagosomes with lysosomes. It is noteworthy that EM-5 does not interfere with the initiation of autophagosome formation or lysosomal function. Additionally, the mitophagy flux blockage caused by EM-5 was driven by the accumulation of intracellular ROS. In vivo, EM-5 exhibited significant efficacy in suppressing tumor growth in a xenograft model. Collectively, our findings not only identified EM-5 as a promising, effective, and safe lead compound for treating GBM but also uncovered its underlying mechanisms from the perspective of apoptosis and mitophagy.

Bacteria-organelle communication in physiology and disease.

Bacteria, omnipresent in our environment and coexisting within our body, exert dual beneficial and pathogenic influences. These microorganisms engage in intricate interactions with the human body, impacting both human health and disease. Simultaneously, certain organelles within our cells share an evolutionary relationship with bacteria, particularly mitochondria, best known for their energy production role and their dynamic interaction with each other and other organelles. In recent years, communication between bacteria and mitochondria has emerged as a new mechanism for regulating the host's physiology and pathology. In this review, we delve into the dynamic communications between bacteria and host mitochondria, shedding light on their collaborative regulation of host immune response, metabolism, aging, and longevity. Additionally, we discuss bacterial interactions with other organelles, including chloroplasts, lysosomes, and the endoplasmic reticulum (ER).

Methods for investigating STAT3 regulation of lysosomal function in mammary epithelial cells.

The transcription factor STAT3 is activated by multiple cytokines and other extrinsic factors. It plays a key role in immune and inflammatory responses and, when dysregulated, in tumourigenesis. STAT3 is also an indispensable mediator of the cell death process that occurs during post-lactational regression of the mammary gland, one of the most dramatic examples of physiological cell death in adult mammals. During this involution of the gland, STAT3 powerfully enhances the lysosomal system to efficiently remove superfluous milk-producing mammary epithelial cells via a lysosomal-mediated programmed cell death pathway. The lysosome is a membrane-enclosed  cytoplasmic organelle that digests and recycles cellular waste, with an important role as a signalling centre that monitors cellular metabolism. Here, we describe key strategies for investigating the role of STAT3 in regulating lysosomal function using a mammary epithelial cell culture model system. These include protocols for lysosome enrichment and enzyme activity assays, in addition to microscopic analyses of the vesicular compartment in cell lines. Collectively, these approaches provide the tools to investigate multiple aspects of lysosome biogenesis and function, and to define both direct and indirect roles for STAT3.

Hsc70 promotes anti-tumor immunity by targeting PD-L1 for lysosomal degradation.

Immune checkpoint inhibition targeting the PD-1/PD-L1 pathway has become a powerful clinical strategy for treating cancer, but its efficacy is complicated by various resistance mechanisms. One of the reasons for the resistance is the internalization and recycling of PD-L1 itself upon antibody binding. The inhibition of lysosome-mediated degradation of PD-L1 is critical for preserving the amount of PD-L1 recycling back to the cell membrane. In this study, we find that Hsc70 promotes PD-L1 degradation through the endosome-lysosome pathway and reduces PD-L1 recycling to the cell membrane. This effect is dependent on Hsc70-PD-L1 binding which inhibits the CMTM6-PD-L1 interaction. We further identify an Hsp90α/ß inhibitor, AUY-922, which induces Hsc70 expression and PD-L1 lysosomal degradation. Either Hsc70 overexpression or AUY-922 treatment can reduce PD-L1 expression, inhibit tumor growth and promote anti-tumor immunity in female mice; AUY-922 can further enhance the anti-tumor efficacy of anti-PD-L1 and anti-CTLA4 treatment. Our study elucidates a molecular mechanism of Hsc70-mediated PD-L1 lysosomal degradation and provides a target and therapeutic strategies for tumor immunotherapy.

The complete assembly of human LAT1-4F2hc complex provides insights into its regulation, function and localisation.

The LAT1-4F2hc complex (SLC7A5-SLC3A2) facilitates uptake of essential amino acids, hormones and drugs. Its dysfunction is associated with many cancers and immune/neurological disorders. Here, we apply native mass spectrometry (MS)-based approaches to provide evidence of super-dimer formation (LAT1-4F2hc)2. When combined with lipidomics, and site-directed mutagenesis, we discover four endogenous phosphatidylethanolamine (PE) molecules at the interface and C-terminus of both LAT1 subunits. We find that interfacial PE binding is regulated by 4F2hc-R183 and is critical for regulation of palmitoylation on neighbouring LAT1-C187. Combining native MS with mass photometry (MP), we reveal that super-dimerization is sensitive to pH, and modulated by complex N-glycans on the 4F2hc subunit. We further validate the dynamic assemblies of LAT1-4F2hc on plasma membrane and in the lysosome. Together our results link PTM and lipid binding with regulation and localisation of the LAT1-4F2hc super-dimer.