MCT4 and CD147 colocalize with MMP14 in invadopodia and support matrix degradation and invasion by breast cancer cells.

Expression levels of the lactate-H+ cotransporter MCT4 (also known as SLC16A3) and its chaperone CD147 (also known as basigin) are upregulated in breast cancers, correlating with decreased patient survival. Here, we test the hypothesis that MCT4 and CD147 favor breast cancer invasion through interdependent effects on extracellular matrix (ECM) degradation. MCT4 and CD147 expression and membrane localization were found to be strongly reciprocally interdependent in MDA-MB-231 breast cancer cells. Overexpression of MCT4 and/or CD147 increased, and their knockdown decreased, migration, invasion and the degradation of fluorescently labeled gelatin. Overexpression of both proteins led to increases in gelatin degradation and appearance of the matrix metalloproteinase (MMP)-generated collagen-I cleavage product reC1M, and these increases were greater than those observed upon overexpression of each protein alone, suggesting a concerted role in ECM degradation. MCT4 and CD147 colocalized with invadopodia markers at the plasma membrane. They also colocalized with MMP14 and the lysosomal marker LAMP1, as well as partially with the autophagosome marker LC3, in F-actin-decorated intracellular vesicles. We conclude that MCT4 and CD147 reciprocally regulate each other and interdependently support migration and invasiveness of MDA-MB-231 breast cancer cells. Mechanistically, this involves MCT4-CD147-dependent stimulation of ECM degradation and specifically of MMP-mediated collagen-I degradation. We suggest that the MCT4-CD147 complex is co-delivered to invadopodia with MMP14.

LAMP1 controls CXCL10-CXCR3 axis mediated inflammatory regulation of macrophage polarization during inflammatory stimulation.

Increased expression of CXCL10 and its receptor CXCR3 represents an inflammatory response in cells and tissues. Macrophage polarization and autophagy are major functions in inflammatory macrophages; however, the cellular functions of the CXCL10-CXCR3 axis in macrophages are not well understood. Here, we examined the role of CXCL10-CXCR3-axis-regulated autophagy in macrophage polarization. First, in non-inflammatory macrophages, whereas CXCL10 promotes M2 polarization and inhibits M1 polarization, CXCR3 antagonist AMG487 induces the opposite macrophage polarization. Next, CXCL10 promotes the expression of autophagy proteins (Atg5-Atg12 complex, p62, LC3-II, and LAMP1) and AMG487 inhibits their expression. Knockdown of LAMP1 by short interfering RNA switches the CXCL10-induced polarization from M2 to M1 in non-inflammatory macrophages. Furthermore, in inflammatory macrophages stimulated by poly(I:C), CXCL10 induces M1 polarization and AMG487 induces M2 polarization in association with a decrease in LAMP1. Finally, AMG487 alleviates lung injury after poly(I:C) treatment in mice. In conclusion, CXCL10-CXCR3 axis differentially directs macrophage polarization in inflammatory and non-inflammatory states, and autophagy protein LAMP1 acts as the switch controlling the direction of macrophage polarization by CXCL10-CXCR3.

Enterovirus A71 does not meet the uncoating receptor SCARB2 at the cell surface.

Enterovirus A71 (EV-A71) infection involves a variety of receptors. Among them, two transmembrane protein receptors have been investigated in detail and shown to be critical for infection: P-selectin glycoprotein ligand-1 (PSGL-1) in lymphocytes (Jurkat cells), and scavenger receptor class B member 2 (SCARB2) in rhabdomyosarcoma (RD) cells. PSGL-1 and SCARB2 have been reported to be expressed on the surface of Jurkat and RD cells, respectively. In the work reported here, we investigated the roles of PSGL-1 and SCARB2 in the process of EV-A71 entry. We first examined the expression of SCARB2 in Jurkat cells, and detected it within the cytoplasm, but not on the cell surface. Further, using PSGL-1 and SCARB2 knockout cells, we found that although both PSGL-1 and SCARB2 are essential for virus infection of Jurkat cells, virus attachment to these cells requires only PSGL-1. These results led us to evaluate the cell surface expression and the roles of SCARB2 in other EV-A71-susceptible cell lines. Surprisingly, in contrast to the results of previous studies, we found that SCARB2 is absent from the surface of RD cells and other susceptible cell lines we examined, and that although SCARB2 is essential for infection of these cells, it is dispensable for virus attachment. These results indicate that a receptor other than SCARB2 is responsible for virus attachment to the cell and probably for internalization of virions, not only in Jurkat cells but also in RD cells and other EV-A71-susceptible cells. SCARB2 is highly concentrated in lysosomes and late endosomes, where it is likely to trigger acid-dependent uncoating of virions, the critical final step of the entry process. Our results suggest that the essential interactions between EV-A71 and SCARB2 occur, not at the cell surface, but within the cell.

Mechanisms regulating the intracellular trafficking and release of CLN5 and CTSD.

Ceroid lipofuscinosis neuronal 5 (CLN5) and cathepsin D (CTSD) are soluble lysosomal enzymes that also localize extracellularly. In humans, homozygous mutations in CLN5 and CTSD cause CLN5 disease and CLN10 disease, respectively, which are two subtypes of neuronal ceroid lipofuscinosis (commonly known as Batten disease). The mechanisms regulating the intracellular trafficking of CLN5 and CTSD and their release from cells are not well understood. Here, we used the social amoeba Dictyostelium discoideum as a model system to examine the pathways and cellular components that regulate the intracellular trafficking and release of the D. discoideum homologs of human CLN5 (Cln5) and CTSD (CtsD). We show that both Cln5 and CtsD contain signal peptides for secretion that facilitate their release from cells. Like Cln5, extracellular CtsD is glycosylated. In addition, Cln5 release is regulated by the amount of extracellular CtsD. Autophagy induction promotes the release of Cln5, and to a lesser extent CtsD. Release of Cln5 requires the autophagy proteins Atg1, Atg5, and Atg9, as well as autophagosomal-lysosomal fusion. Atg1 and Atg5 are required for the release of CtsD. Together, these data support a model where Cln5 and CtsD are actively released from cells via their signal peptides for secretion and pathways linked to autophagy. The release of Cln5 and CtsD from cells also requires microfilaments and the D. discoideum homologs of human AP-3 complex mu subunit, the lysosomal-trafficking regulator LYST, mucopilin-1, and the Wiskott-Aldrich syndrome-associated protein WASH, which all regulate lysosomal exocytosis in this model organism. These findings suggest that lysosomal exocytosis also facilitates the release of Cln5 and CtsD from cells. In addition, we report the roles of ABC transporters, microtubules, osmotic stress, and the putative D. discoideum homologs of human sortilin and cation-independent mannose-6-phosphate receptor in regulating the intracellular/extracellular distribution of Cln5 and CtsD. In total, this study identifies the cellular mechanisms regulating the release of Cln5 and CtsD from D. discoideum cells and provides insight into how altered trafficking of CLN5 and CTSD causes disease in humans.

Characterization of two human induced pluripotent stem cell lines derived from Batten disease patient fibroblasts harbouring CLN5 mutations.

The neuronal ceroid lipofuscinoses (NCLs) are a group of common inherited neurodegenerative disorders of childhood. All forms of NCLs are life-limiting with no curative treatments. Most of the 13 NCL genes encode proteins residing in endolysosomal pathways, such as CLN5, a potential lysosomal enzyme. Two induced pluripotent stem cell lines (hiPSCs) were generated from skin fibroblasts of CLN5 disease patients via non-integrating Sendai virus reprogramming. They demonstrate typical stem cell morphology, express pluripotency markers, exhibit trilineage differentiation potential and also successfully differentiate into neurons. These hiPSCs represent a potential resource to model CLN5 disease in a human context and investigate potential therapies.

MiR-526b-3p enhances sensitivity of head and neck squamous cell carcinoma cells to radiotherapy via suppressing exosomal LAMP3-mediated autophagy.

Lysosomal associated membrane protein 3 (LAMP3) has been reported to be a tumour promoter in multiple cancer types by modulating tumour cell autophagy. However, the potential mechanism of LAMP3 in radio-resistance of head and neck squamous cell carcinoma (HNSCC) remains unknown. Therefore, our current study aims to detect the impacts of LAMP3 on the resistance of HNSCC cells to radiotherapy and meanwhile explore its functional mechanism. Through RT-Qpcr examination, LAMP3 expression was identified to be expressed at a significantly high level in irradiation-resistant HNSCC cell lines compared with irradiation-sensitive HNSCC cell lines. Functional assays including CCK-8, colony formation and Transwell assays demonstrated that LAMP3 enhanced the radio-resistance through inducing autophagy to promote HNSCC cell growth. Furthermore, irradiation-resistant HNSCC cells could transfer exosomal LAMP3 to elevate LAMP3 expression in irradiation-sensitive HNSCC cells. Mechanistically, microRNA (miRNA) miR-526b-3p could inhibit LAMP3 expression so as to strengthen sensitivity of HNSCC cells to radiotherapy. In a word, exosomal LAMP3 expression promoted radioresistance of HNSCC cells via inducing autophagy, while this effect could be suppressed by miR-526b-3p in a targeted manner.

The Batten disease gene product CLN5 is the lysosomal bis(monoacylglycero)phosphate synthase.

Lysosomes critically rely on bis(monoacylglycero)phosphate (BMP) to stimulate lipid catabolism, cholesterol homeostasis, and lysosomal function. Alterations in BMP levels in monogenic and complex neurodegeneration suggest an essential function in human health. However, the site and mechanism responsible for BMP synthesis have been subject to debate for decades. Here, we report that the Batten disease gene product CLN5 is the elusive BMP synthase (BMPS). BMPS-deficient cells exhibited a massive accumulation of the BMP synthesis precursor lysophosphatidylglycerol (LPG), depletion of BMP species, and dysfunctional lipid metabolism. Mechanistically, we found that BMPS mediated synthesis through an energy-independent base exchange reaction between two LPG molecules with increased activity on BMP-laden vesicles. Our study elucidates BMP biosynthesis and reveals an anabolic function of late endosomes/lysosomes.

LAMP1/2 as potential diagnostic and prognostic marker for brain lower grade glioma: A review.

Brain lower grade glioma (LGG) is a common type of glioma. The current treatment methods still have some limitations, and some LGG patients will inevitably continue to deteriorate after treatment. We found the value of lysosomal associated membrane proteins (LAMPs) in the diagnosis and prognosis of LGG, which helps to enhance the clinical understanding of LGG treatment and improved prognosis. We assess the role of LAMPs in LGG, via the publicly available TCGA database. We explored expression levels of LAMPs in LGG using GEPIA2, cBioPortal, and UALCAN databases. The correction of LAMPs expression levels with immune cell infiltration in LGG patient was assessed by TIMER database. The Lysosomal associated membrane protein 1 (LAMP1)/2/4 mRNA levels were significantly higher in LGG patients than in healthy controls. Morover, high mRNA expressions of LAMP1/2/Lysosomal associated membrane protein 3 were associated with poor overall survival. We found that the immune invasion of LGG was almost significantly correlated with the expression of LAMPs. The results suggested that mRNA expressions of LAMP1 and LAMP4 were significantly associated with histological subtypes in LGG patients. lysosomal associated membrane protein 2 and LAMP5 were significantly down-regulated expression in samples of TP53 mutant in LGG compared to TP53 wild type. In addition, Lysosomal associated membrane protein 3 and LAMP4 were significantly overexpressed in samples of TP53 mutant in LGG Enrichment analysis applied to each component indicated that biological function was primarily associated with series of pathways in synapse and immunity.

Lysosomal LAMP proteins regulate lysosomal pH by direct inhibition of the TMEM175 channel.

Maintaining a highly acidic lysosomal pH is central to cellular physiology. Here, we use functional proteomics, single-particle cryo-EM, electrophysiology, and in vivo imaging to unravel a key biological function of human lysosome-associated membrane proteins (LAMP-1 and LAMP-2) in regulating lysosomal pH homeostasis. Despite being widely used as a lysosomal marker, the physiological functions of the LAMP proteins have long been overlooked. We show that LAMP-1 and LAMP-2 directly interact with and inhibit the activity of the lysosomal cation channel TMEM175, a key player in lysosomal pH homeostasis implicated in Parkinson's disease. This LAMP inhibition mitigates the proton conduction of TMEM175 and facilitates lysosomal acidification to a lower pH environment crucial for optimal hydrolase activity. Disrupting the LAMP-TMEM175 interaction alkalinizes the lysosomal pH and compromises the lysosomal hydrolytic function. In light of the ever-increasing importance of lysosomes to cellular physiology and diseases, our data have widespread implications for lysosomal biology.

Characterization of neuropathology in ovine CLN5 and CLN6 neuronal ceroid lipofuscinoses (Batten disease).

Sheep with naturally occurring CLN5 and CLN6 forms of neuronal ceroid lipofuscinoses (Batten disease) share the key clinical features of the human disease and represent an ideal model system in which the clinical efficacy of gene therapies is developed and test. However, it was first important to characterize the neuropathological changes that occur with disease progression in affected sheep. This study compared neurodegeneration, neuroinflammation, and lysosomal storage accumulation in CLN5 affected Borderdale, CLN6 affected South Hampshire, and Merino sheep brains from birth to end-stage disease at ≤24 months of age. Despite very different gene products, mutations, and subcellular localizations, the pathogenic cascade was remarkably similar for all three disease models. Glial activation was present at birth in affected sheep and preceded neuronal loss, with both spreading from the visual and parieto-occipital cortices most prominently associated with clinical symptoms to the entire cortical mantle by end-stage disease. In contrast, the subcortical regions were less involved, yet lysosomal storage followed a near-linear increase across the diseased sheep brain with age. Correlation of these neuropathological changes with published clinical data identified three potential therapeutic windows in affected sheep-presymptomatic (3 months), early symptomatic (6 months), and a later symptomatic disease stage (9 months of age)-beyond which the extensive depletion of neurons was likely to diminish any chance of therapeutic benefit. This comprehensive natural history of the neuropathological changes in ovine CLN5 and CLN6 disease will be integral in determining what impact treatment has at each of these disease stages.

LAMP5 may promote MM progression by activating p38.

Multiple myeloma (MM) is the second most common tumor of the hematologic system. MM remains incurable at this time. In this study, we used bioinformatics analysis to find key genes in the pathogenesis of MM. We first found that Lysosome associated membrane protein 5 (LAMP5) expression was sequentially increased in healthy donors (HD), monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM) and newly diagnosed MM (NDMM), relapsed MM (RMM). We collected bone marrow from patients with NDMM, HD and post-treatment MM (PTMM) and performed qPCR analysis of LAMP5, and found that the expression of LAMP5 is stronger in NDMM than in HD, and decreases after treatment. Western blotting assay also found more expression of LAMP5 in NDMM than in HD. Patients with high LAMP5 expression have a higher DS (Durie-Salmon) stage and worse prognosis. We next verified the expression of LAMP5 in four MM cell lines and silenced LAMP5 expression in RPMI-8226 and AMO-1, and explored the effects of LAMP5 silencing on MM cell apoptosis and cell cycle by flow cytometry and western blotting. Knockdown of LAMP5 promoted apoptosis in MM cells, but had no effect on the cell cycle. Mechanistically, LAMP5 may exert its pro-tumor effects in MM in part through activation of p38 protein. We screened LAMP5 for the first time as a key gene for MM progression and recurrence, and found that LAMP5 may exert its pro-tumor effects in MM through activation of p38 protein.

LAMP3 expression in the liver is involved in T cell activation and adaptive immune regulation in hepatitis B virus infection.

Background: The disease burden caused by chronic hepatitis B virus (HBV) infection is still heavy, and the current treatment scheme has not achieved a complete cure. Changes in natural and adaptive immunity usually accompany chronic HBV infection. As a marker expressed on dendritic cells (DCs), whether lysosome-associated membrane glycoprotein 3 (LAMP3) participates in chronic HBV infection deserves further analysis. Methods: We retrieved chronic HBV infection transcriptional information from the Gene Expression Omnibus (GEO) database. The LAMP3 expression in the liver of patients with chronic hepatitis B (CHB) was analyzed in three GEO datasets and confirmed in our validation cohort (27 patients with CHB). Differentially expressed genes were obtained from one CHB cohort by comparing LAMP3high and LAMP3low expression subgroups. These genes underwent Gene Ontology, Kyoto Encyclopedia of Genes and Genomes analysis, and Gene Set Enrichment Analysis to decipher the influence of LAMP3 on the biological process and immunity changes in HBV infection. Furthermore, we investigated the potential relationship between LAMP3 levels, the abundance of infiltrating immune cells, and liver dysfunction. Results: Compared to healthy controls, LAMP3 expression was upregulated in the transcriptional profiles of the liver in patients with CHB. The high LAMP3 expression was related to T cell activation and the chemokine signaling pathway. The LAMP3 gene was positively linked to marker sets of infiltrating activated regulatory T cells (Treg), T cell exhaustion, monocytes, and DCs. Moreover, CHB patients with high LAMP3 expression had unfavorable liver dysfunction. Conclusions: LAMP3 is a gene related to HBV infection, which might be involved in HBV infection by regulating T cell activation and adaptive immune response.

The effects of SCARB2 and SELPLG gene polymorphisms on EV71 infection in hand, foot and mouth disease.

The same viral infection in different hosts may result in varying levels of clinical symptoms, which is related to the genetic background of the host itself. A total of 406 common cases and 452 severe cases of enterovirus 71 (EV71) infection in Yunnan Province were selected as the research subjects, and SNaPshot technology was used to detect genetic polymorphisms for 25 Tag single-nucleotide polymorphisms (TagSNPs) in the selectin P ligand (SELPLG) and scavenger receptor class B member 2 (SCARB2) genes. Our results demonstrate that SCARB2 polymorphisms (rs74719289, rs3733255 and rs17001551) are related to the severity of EV71 infection (A vs G: OR 0.330; 95% CI 0.115 - 0.947; T vs C: OR 0.336; 95% CI 0.118 - 0.958; and A vs G: OR 0.378; 95% CI 0.145 - 0.984). The SELPLG polymorphisms were not significantly different between common cases and severe cases. Therefore, we conclude that the SCARB2 gene has a protective effect on the course of hand, foot and mouth disease caused by EV71 infection and that SCARB2 gene mutations can reduce the severity of the disease.

Human SCARB2 Acts as a Cellular Associator for Helping Coxsackieviruses A10 Infection.

Coxsackievirus A10 (CVA10) causes hand, foot, and mouth disease (HFMD) and herpangina, which can result in severe neurological symptoms in children. CVA10 does not use the common enterovirus 71 (EV71) receptor, human SCARB2 (hSCARB2, scavenger receptor class B, member 2), for infection but instead uses another receptor, such as KREMEN1. Our research has shown that CVA10 can infect and replicate in mouse cells expressing human SCARB2 (3T3-SCARB2) but not in the parental NIH3T3 cells, which do not express hSCARB2 for CVA10 entry. Knocking down endogenous hSCARB2 and KREMEN1 with specific siRNAs inhibited CVA10 infection in human cells. Co-immunoprecipitation confirmed that VP1, a main capsid protein where virus receptors for attaching to the host cells, could physically interact with hSCARB2 and KREMEN1 during CVA10 infection. It is the efficient virus replication following virus attachment to its cellular receptor. It resulted in severe limb paralysis and a high mortality rate in 12-day-old transgenic mice challenged with CVA10 but not in wild-type mice of the same age. Massive amounts of CVA10 accumulated in the muscles, spinal cords, and brains of the transgenic mice. Formalin inactivated CVA10 vaccine-induced protective immunity against lethal CVA10 challenge and reduced the severity of disease and tissue viral loads. This is the first report to show that hSCARB2 serves as an associate to aid CVA10 infection. hSCARB2-transgenic mice could be useful in evaluating anti-CVA10 medications and studying the pathogenesis induced by CVA10.

Construction and evaluation of DNA vaccine encoding Crimean Congo hemorrhagic fever virus nucleocapsid protein, glycoprotein N-terminal and C-terminal fused with LAMP1.

Crimean-Congo hemorrhagic fever virus (CCHFV) can cause severe hemorrhagic fever in humans and is mainly transmitted by ticks. There is no effective vaccine for Crimean-Congo hemorrhagic fever (CCHF) at present. We developed three DNA vaccines encoding CCHFV nucleocapsid protein (NP), glycoprotein N-terminal (Gn) and C-terminal (Gc) fused with lysosome-associated membrane protein 1 (LAMP1) and assessed their immunogenicity and protective efficacy in a human MHC (HLA-A11/DR1) transgenic mouse model. The mice that were vaccinated three times with pVAX-LAMP1-CCHFV-NP induced balanced Th1 and Th2 responses and could most effectively protect mice from CCHFV transcription and entry-competent virus-like particles (tecVLPs) infection. The mice vaccinated with pVAX-LAMP1-CCHFV-Gc mainly elicited specific anti-Gc and neutralizing antibodies and provided a certain protection from CCHFV tecVLPs infection, but the protective efficacy was less than that of pVAX-LAMP1-CCHFV-NP. The mice vaccinated with pVAX-LAMP1-CCHFV-Gn only elicited specific anti-Gn antibodies and could not provide sufficient protection from CCHFV tecVLPs infection. These results suggest that pVAX-LAMP1-CCHFV-NP would be a potential and powerful candidate vaccine for CCHFV.

Thyroid hormone upregulates LAMP2 expression and lysosome activity.

Thyroid hormone (T3)-induced autophagy and its biological significance have been extensively investigated in recent years. However, limited studies to date have focused on the important role of lysosomes in autophagy. In this study, we explored the effects of T3 on lysosomal protein expression and trafficking in detail. Our findings showed that T3 activates rapid lysosomal turnover and expression of numerous lysosomal genes, including TFEB, LAMP2, ARSB, GBA, PSAP, ATP6V0B, ATP6V0D1, ATP6V1E1, CTSB, CTSH, CTSL, and CTSS, in a thyroid hormone receptor-dependent manner. In a murine model, LAMP2 protein was specifically induced in mice with hyperthyroidism. T3-promoted microtubule assembly was significantly disrupted by vinblastine, resulting in accumulation of the lipid droplet marker PLIN2. In the presence of the lysosomal autophagy inhibitors bafilomycin A1, chloroquine and ammonium chloride, we observed substantial accumulation of LAMP2 but not LAMP1 protein. T3 further enhanced the protein levels of ectopically expressed LAMP1 and LAMP2. Upon knockdown of LAMP2, cavities of lysosomes and lipid droplets accumulated in the presence of T3, although the changes in LAMP1 and PLIN2 expression were less pronounced. More specifically, the protective effect of T3 against ER stress-induced death was abolished by knockdown of LAMP2. Our collective results indicate that T3 not only promotes lysosomal gene expression but also LAMP protein stability and microtubule assembly, leading to enhancement of lysosomal activity in digesting any additional autophagosomal burden.

Salivary gland LAMP3 mRNA expression is a possible predictive marker in the response to hydroxychloroquine in Sjögren's disease.

Hydroxychloroquine (HCQ) is a lysosomotropic agent that is commonly used for treating Sjögren's disease (SjD). However, its efficacy is controversial because of the divergent response to the drug among patients. In a subgroup of SjD patients, lysosome-associated membrane protein 3 (LAMP3) is elevated in expression in the salivary glands and promotes lysosomal dysregulation and lysosome-dependent apoptotic cell death. In this study, chloroquine (CQ) and its derivative HCQ were tested for their ability to prevent LAMP3-induced apoptosis, in vitro and on a mouse model of SjD. In addition, efficacy of HCQ treatment was retrospectively compared between high LAMP3 mRNA expression in minor salivary glands and those with LAMP3 mRNA levels comparable with healthy controls. Study results show that CQ treatment stabilized the lysosomal membrane in LAMP3-overexpressing cells via deactivation of cathepsin B, resulting in decreased apoptotic cell death. In mice with established SjD-like phenotype, HCQ treatment also significantly decreased apoptotic cell death and ameliorated salivary gland hypofunction. Retrospective analysis of SjD patients found that HCQ tended to be more effective in improving disease activity index, symptom severity and hypergammaglobulinemia in patients with high LAMP3 expression compared those with normal LAMP3 expression. Taken together, these findings suggested that by determining salivary gland LAMP3 mRNA level, a patient's response to HCQ treatment could be predicted. This finding may provide a novel strategy for guiding the development of more personalized medicine for SjD.

Lysosomal dysfunction, autophagic defects, and CLN5 accumulation underlie the pathogenesis of KCTD7-mutated neuronal ceroid lipofuscinoses.

Lysosomes are essential catabolic organelles responsible for the degradation of biomacromolecules into low-molecular-weight materials for subsequent reuse. Neuronal ceroid lipofuscinoses (NCLs) are a group of fatal neurodegenerative lysosomal storage disorders characterized by the intracellular accumulation of lipoprotein aggregates (called ceroid lipofuscin) in neurons and other tissues. Mutations in KCTD7, which encodes a substrate-binding adaptor for the CUL3-RING E3 (CRL3) ubiquitin ligase complex, are categorized as a unique NCL subtype. However, the molecular mechanisms underlying the KCTD7-mutated NCLs remain unclear. In our recent study, we showed that KCTD7 deficiency leads to the accumulation of lysosomal storage deposits owing to lysosomal dysfunction and macroautophagic/autophagic defects. We identified CLN5 as an authentic substrate of CRL3-KCTD7 E3s. Wild-type KCTD7 targets CLN5 for ubiquitination and proteasomal degradation, whereas NCL patient-derived KCTD7 mutations disrupt the interaction between KCTD7-CUL3 or KCTD7-CLN5 and ultimately lead to excessive CLN5 accumulation in the endoplasmic reticulum. Accumulated CLN5 disrupts the interaction between CLN6-CLN8 and lysosomal enzymes, leading to impaired ER-to-Golgi trafficking of lysosomal enzymes. Thus, our findings indicate that KCTD7 is a key player in maintaining lysosomal and autophagic homeostasis and demonstrate that KCTD7 and CLN5, two NCL causative genes, are biochemically linked and function in a common neurodegenerative pathway.

LAMP3/CD63 Expression in Early and Late Endosomes in Human Vaginal Epithelial Cells Is Associated with Enhancement of HSV-2 Infection.

Herpes simplex virus 2 (HSV-2) is a lifelong sexually transmitted virus that disproportionately infects women through heterosexual transmission in the vaginal tract. The vaginal epithelium is known to be highly susceptible to HSV-2 infection; however, the cellular mechanism of HSV-2 uptake and replication in vaginal epithelium has not been extensively studied. Previously, we observed that lysosomal-associated membrane protein-3 (LAMP3/CD63) was among the highly upregulated genes during HSV-2 infection of human vaginal epithelial cell line VK2, leading us to posit that LAMP3/CD63 may play a role in HSV-2 infection. Consequently, we generated two gene-altered VK2-derived cell lines, a LAMP3-overexpressed (OE) line and a LAMP3 knockout (KO) line. The wild-type VK2 and the LAMP3 OE and KO cell lines were grown in air-liquid interface (ALI) cultures for 7 days and infected with HSV-2. Twenty-four hours postinfection, LAMP3 OE cells produced and released significantly higher numbers of HSV-2 virions than wild-type VK2 cells, while virus production was greatly attenuated in LAMP3 KO cells, indicating a functional association between LAMP3/CD63 expression and HSV-2 replication. Fluorescence microscopy of HSV-2-infected cells revealed that HSV-2 colocalized with LAMP3 in both early endosomes and lysosomal compartments. In addition, blocking endosomal maturation or late endosomal/lysosomal fusion using specific inhibitors resulted in reduced HSV-2 replication in VK2 cells. Similarly, LAMP3 KO cells exhibited very low viral entry and association with endosomes, while LAMP3 OE cells demonstrated large amounts of virus that colocalized with LAMP3/CD63 in endosomes and lysosomes. IMPORTANCE Collectively, these results showed that HSV-2 is taken up by human vaginal epithelial cells through an endosomal-lysosomal pathway in association with LAMP3, which plays a crucial role in the enhancement of HSV-2 replication. These findings provide the basis for the future design of antiviral agents for prophylactic measures against HSV-2 infection.