Lysosomal Biogenesis Research Articles

Lysosomal/mitochondrial interaction promotes tumor growth in squamous cell carcinoma of the head and neck.

Communication between intracellular organelles including lysosomes and mitochondria has recently been shown to regulate cellular proliferation and fitness. The way lysosomes and mitochondria communicate with each other (lysosomal/mitochondrial interaction, LMI) is, emerging as a major determinant of tumor proliferation and growth. About 30% of squamous carcinomas (including squamous cell carcinoma of the head and neck, SCCHN) overexpress TMEM16A, a calcium-activated chloride channel, which promotes cellular growth and negatively correlates with patient survival. We have recently shown that TMEM16A drives lysosomal biogenesis, but its impact on mitochondrial function has not been explored. Here, we show that in the context of high TMEM16A SCCHN, (1) patients display increased mitochondrial content, specifically complex I; (2) In vitro and in vivo models uniquely depend on mitochondrial complex I activity for growth and survival; (3) NRF2 signaling is a critical linchpin that drives mitochondrial function, and (4) mitochondrial complex I and lysosomal function are codependent for proliferation. Taken together, our data demonstrate that coordinated lysosomal and mitochondrial activity and biogenesis via LMI drive tumor proliferation and facilitates a functional interaction between lysosomal and mitochondrial networks. Therefore, inhibition of LMI instauration may serve as a therapeutic strategy for patients with SCCHN. Implications: Intervention of lysosome-mitochondria interaction may serve as a therapeutic approach for patients with high TMEM16A expressing SCCHN.

Second-Generation Antipsychotics Induce Metabolic Disruption in Adipose Tissue-Derived Mesenchymal Stem Cells Through an aPKC-Dependent Pathway

Metabolic syndrome (MetS) is a cluster of metabolic abnormalities, including visceral obesity, dyslipidemia, and insulin resistance. In this regard, visceral white adipose tissue (vWAT) plays a critical role, influencing energy metabolism, immunomodulation, and oxidative stress. Adipose-derived stem cells (ADSCs) are key players in these processes within vWAT. While second-generation antipsychotics (SGAs) have significantly improved treatments for mental health disorders, their chronic use is associated with an increased risk of MetS. In this study, we explored the impact of SGAs on ADSCs to better understand their role in MetS and identify potential therapeutic targets. Our findings reveal that olanzapine disrupts lipid droplet formation during adipogenic differentiation, impairing insulin receptor endocytosis, turnover, and signaling. SGAs also alter the endolysosomal compartment, leading to acidic vesicle accumulation and increased lysosomal biogenesis through TFEB activation. PKCζ is crucial for the SGA-induced nuclear translocation of TFEB and acidic vesicle formation. Notably, inhibiting PKCζ restored insulin receptor tyrosine phosphorylation, normalized receptor turnover, and improved downstream signaling following olanzapine treatment. This activation of PKCζ by olanzapine is driven by increased phosphatidic acid synthesis via phospholipase D (PLD), following G protein-coupled receptor (GPCR) signaling activation. Overall, olanzapine and clozapine disrupt endolysosomal homeostasis and insulin signaling in a PKCζ-dependent manner. These findings highlight SGAs as valuable tools for uncovering cellular dysfunction in vWAT during MetS and may guide the development of new therapeutic strategies to mitigate the metabolic side effects of these drugs.

The cGAS-STING pathway activates transcription factor TFEB to stimulate lysosome biogenesis and pathogen clearance.

Induction of autophagy is an ancient function of the cyclic GMP-AMP (cGAMP) synthase (cGAS)-stimulator of interferon genes (STING) pathway through which autophagic cargoes are delivered to lysosomes for degradation. However, whether lysosome function is also modulated by the cGAS-STING pathway remains unknown. Here, we discovered that the cGAS-STING pathway upregulated lysosomal activity by stimulating lysosome biogenesis independently of the downstream protein kinase TANK-binding kinase 1 (TBK1). STING activation enhanced lysosome biogenesis through inducing the nuclear translocation of transcription factor EB (TFEB) as well as its paralogs transcription factor E3 (TFE3) and microphthalmia-associated transcription factor (MITF). STING-induced lipidation of GABA type A receptor-associated protein (GABARAP), an autophagy-related protein, on STING vesicles was responsible for TFEB activation. Membrane-bound GABARAP sequestered the GTPase-activating protein folliculin (FLCN) and FLCN-interacting protein (FNIP) complex to block its function toward the Rag GTPases Ras-related GTP-binding C and D (RagC and RagD), abolishing mechanistic target of rapamycin (mTOR) complex 1 (mTORC1)-dependent phosphorylation and inactivation of TFEB. Functionally, STING-induced lysosome biogenesis within cells facilitated the clearance of cytoplasmic DNA and invading pathogens. Thus, our findings reveal that induction of lysosome biogenesis is another important function of the cGAS-STING pathway.

Polygoni Cuspidati Rhizoma et Radix extract activates TFEB and alleviates hepatic steatosis by promoting autophagy

Hepatic steatosis is a metabolic disorder characterized by excessive accumulation of lipid in the liver. The activation of autophagy shows a promising effect in the elimination of intracellular lipids, potentially improving steatosis. Polygoni Cuspidati Rhizoma et Radix (PCRR) has been used for thousands of years in treating atherosclerosis, hepatitis, and gallstone disease. We recently found that PCRR exerts a potent anti-hepatic steatosis effect, but the active compounds responsible for its effect and underlying mechanism remain unknown. This study aims to investigate whether PCRR water extract intervention improves steatosis by regulating hepatic autophagic flux. We demonstrated that PCRR water extract promoted autophagic flux, enhanced lysosomal biogenesis, and alleviated steatosis in hepatocytes and in the livers of rats with steatosis through autophagy induction. Mechanistic investigation revealed that PCRR water extract inhibited the activity of MTORC1, thereby dephosphorylating and hence inducing nuclear translocation of the lipophagy inducer TFEB. Notably, knockdown of TFEB attenuated the promoting effects of PCRR on lipophagy in hepatocytes. Furthermore, blockage of autophagy by chloroquine inhibited the therapeutic effect of PCRR against hepatic steatosis in HFD-fed rats. Our findings demonstrate the potential of PCRR water extract as a novel autophagy enhancer for treating hepatic steatosis.

Late-Life Alcohol Exposure Does Not Exacerbate Age-Dependent Reductions in Mouse Spatial Memory and Brain TFEB Activity

Alcohol consumption is believed to affect Alzheimer’s disease (AD) risk, but the contributing mechanisms are not well understood. A potential mediator of the proposed alcohol-AD connection is autophagy, a degradation pathway that maintains organelle and protein homeostasis. Autophagy is regulated through the activity of Transcription factor EB (TFEB), which promotes lysosome and autophagy-related gene expression. The purpose of this study is to explore whether chronic alcohol consumption worsens the age-related decline in TFEB-mediated lysosomal biogenesis in the brain and exacerbates cognitive decline associated with aging. To explore the effect of alcohol on brain TFEB and autophagy, we exposed young (3-month-old) and aged (23-month-old) mice to two alcohol-feeding paradigms and assessed biochemical, transcriptome, histology, and behavioral endpoints. In young mice, alcohol decreased hippocampal nuclear TFEB staining but increased SQSTM1/p62, LC3-II, ubiquitinated proteins, and phosphorylated Tau. Hippocampal TFEB activity was lower in aged mice than it was in young mice, and Gao-binge alcohol feeding did not worsen the age-related reduction in TFEB activity. Morris Water and Barnes Maze spatial memory tasks were used to characterize the effects of aging and chronic alcohol exposure (mice fed alcohol for 4 weeks). The aged mice showed worse spatial memory acquisition in both tests. Alcohol feeding slightly impaired spatial memory in the young mice, but had little effect or even slightly improved spatial memory acquisition in the aged mice. In conclusion, aging produces greater reductions in brain autophagy flux and impairment of spatial memory than alcohol consumption.

TMBIM6/BI-1 is an intracellular environmental regulator that induces paraptosis in cancer via ROS and Calcium-activated ERAD II pathways.

Transmembrane B cell lymphoma 2-associated X protein inhibitor motif-containing (TMBIM) 6, also known as Bax Inhibitor-1 (BI-1), has been heavily researched for its cytoprotective functions. TMBIM6 functional diversity includes modulating cell survival, stress, metabolism, cytoskeletal dynamics, organelle function, regulating cytosolic acidification, calcium, and reactive oxygen species (ROS). Clinical research shows TMBIM6 plays a key role in many of the world's top diseases/injuries (i.e., Alzheimer's, Parkinson's, diabetes, obesity, brain injury, liver disease, heart disease, aging, etc.), including cancer, where TMBIM6 expression impacts patient survival, chemoresistance, cancer progression, and metastasis. We show TMBIM6 is activated by, and undergoes, different conformational changes that dictate its function following a significant change in the cell's IntraCellular Environment (ICE). TMBIM6 agonism, following ICE change, can help the cell overcome multiple stresses including toxin exposure, viral infection, wound healing, and excitotoxicity. However, in cancer cells TMBIM6 agonism results in rapid paraptotic induction irrespective of the cancer type, sub-type, genotype or phenotype. Furthermore, the level of TMBIM6 expression in cancer did not dictate the level of paraptotic induction; however, it did dictate the rate at which paraptosis occurred. TMBIM6 agonism did not induce paraptosis in cancer via canonical routes involving p38 MAPK, JNK, ERK, UPR, autophagy, proteasomes, or Caspase-9. Instead, TMBIM6 agonism in cancer upregulates cytosolic Ca2+ and ROS, activates lysosome biogenesis, and induces paraptosis via ERAD II mechanisms. In xenograft models, we show TMBIM6 agonism induces rapid cancer cell death with no toxicity, even at high doses of TMBIM6 agonist (>450 mg/kg). In summary, this study shows TMBIM6's functional diversity is only activated by severe ICE change in diseased/injured cells, highlighting its transformative potential as a therapeutic target across various diseases and injuries, including cancer.

The Parkinson’s disease risk gene cathepsin B promotes fibrillar alpha-synuclein clearance, lysosomal function and glucocerebrosidase activity in dopaminergic neurons

BackgroundVariants in the CTSB gene encoding the lysosomal hydrolase cathepsin B (catB) are associated with increased risk of Parkinson’s disease (PD). However, neither the specific CTSB variants driving these associations nor the functional pathways that link catB to PD pathogenesis have been characterized. CatB activity contributes to lysosomal protein degradation and regulates signaling processes involved in autophagy and lysosome biogenesis. Previous in vitro studies have found that catB can cleave monomeric and fibrillar alpha-synuclein, a key protein involved in the pathogenesis of PD that accumulates in the brains of PD patients. However, truncated synuclein isoforms generated by catB cleavage have an increased propensity to aggregate. Thus, catB activity could potentially contribute to lysosomal degradation and clearance of pathogenic alpha synuclein from the cell, but also has the potential of enhancing synuclein pathology by generating aggregation-prone truncations. Therefore, the mechanisms linking catB to PD pathophysiology remain to be clarified.MethodsHere, we conducted genetic analyses of the association between common and rare CTSB variants and risk of PD. We then used genetic and pharmacological approaches to manipulate catB expression and function in cell lines, induced pluripotent stem cell-derived dopaminergic neurons and midbrain organoids and assessed lysosomal activity and the handling of aggregated synuclein fibrils.ResultsWe find that catB inhibition impairs autophagy, reduces glucocerebrosidase (encoded by GBA1) activity, and leads to an accumulation of lysosomal content. In cell lines, reduction of CTSB gene expression impairs the degradation of pre-formed alpha-synuclein fibrils, whereas CTSB gene activation enhances fibril clearance. In midbrain organoids and dopaminergic neurons treated with alpha-synuclein fibrils, catB inhibition potentiates the formation of inclusions which stain positively for phosphorylated alpha-synuclein.ConclusionsThese results indicate that the reduction of catB function negatively impacts lysosomal pathways associated with PD pathogenesis, while conversely catB activation could promote the clearance of pathogenic alpha-synuclein.

Plasmodium berghei liver stage parasites exploit host GABARAP proteins for TFEB activation

Plasmodium, the causative agent of malaria, infects hepatocytes prior to establishing a symptomatic blood stage infection. During this liver stage development, parasites reside in a parasitophorous vacuole (PV), whose membrane acts as the critical interface between the parasite and the host cell. It is well-established that host cell autophagy-related processes significantly impact the development of Plasmodium liver stages. Expression of genes related to autophagy and lysosomal biogenesis is orchestrated by transcription factor EB (TFEB). In this study, we explored the activation of host cell TFEB in Plasmodium berghei-infected cells during the liver stage of the parasite. Our results unveiled a critical role of proteins belonging to the Gamma-aminobutyric acid receptor-associated protein subfamily (GABARAP) of ATG8 proteins (GABARAP/L1/L2 and LC3A/B/C) in recruiting the TFEB-blocking FLCN-FNIP (Folliculin-Folliculin-interacting protein) complex to the PVM. Remarkably, the sequestration of FLCN-FNIP resulted in a robust activation of TFEB, reliant on conjugation of ATG8 proteins to single membranes (CASM) and GABARAP proteins. Our findings provide novel mechanistic insights into host cell signaling occurring at the PVM, shedding light on the complex interplay between Plasmodium parasites and the host cell during the liver stage of infection.

An increase of lysosomes through EGF-triggered endocytosis attenuated zinc-mediated lysosomal membrane permeabilization and neuronal cell death

In the context of acute brain injuries, where zinc neurotoxicity and oxidative stress are acknowledged contributors to neuronal damage, we investigated the pivotal role of lysosomes as a potential protective mechanism. Our research commenced with an exploration of epidermal growth factor (EGF) and its impact on lysosomal dynamics, particularly its neuroprotective potential against zinc-induced cytotoxicity. Using primary mouse cerebrocortical cultures, we observed the rapid induction of EGFR endocytosis triggered by EGF, resulting in a transient increase in lysosomal vesicles. Furthermore, EGF stimulated lysosomal biogenesis, evident through elevated expression of lysosomal-associated membrane protein 1 (LAMP-1) and the induction and activation of prominent lysosomal proteases, particularly cathepsin B (CTSB). This process of EGFR endocytosis was found to promote lysosomal augmentation, thus conferring protection against zinc-induced lysosomal membrane permeabilization (LMP) and subsequent neuronal death. Notably, the neuroprotective effects and lysosomal enhancement induced by EGF were almost completely reversed by the inhibition of clathrin-mediated and caveolin-mediated endocytosis pathways, along with the disruption of retrograde trafficking. Furthermore, tyrosine kinase inhibition of EGFR nullified EGFR endocytosis, resulting in the abrogation of EGF-induced lysosomal upregulation and neuroprotection. An intriguing aspect of our study is the successful replication of EGF’s neuroprotective effects through the overexpression of LAMP-1, which significantly reduced zinc-induced LMP and cell death, demonstrated in both primary mouse cerebrocortical neuronal cultures and human embryonic kidney (HEK) cells. Our research extended beyond zinc-induced neurotoxicity, as we observed EGF’s protective effects against other oxidative stressors linked to intracellular zinc release, including hydrogen peroxide (H2O2) and 1-methyl-4-phenylpyridinium ion (MPP+). Collectively, our findings unveil the intricate interplay between EGF-triggered EGFR endocytosis, lysosomal upregulation, an increase in the regulatory capacity for zinc homeostasis, and the subsequent alleviation of zinc-induced neurotoxicity. These results present promising avenues for therapeutic interventions to enhance neuroprotection by targeting lysosomal augmentation.

Lysosomal enzyme binding to the cation-independent mannose 6-phosphate receptor is regulated allosterically by insulin-like growth factor 2.

The cation-independent mannose 6-phosphate receptor (CI-MPR) is clinically significant in the treatment of patients with lysosomal storage diseases because it functions in the biogenesis of lysosomes by transporting mannose 6-phosphate (M6P)-containing lysosomal enzymes to endosomal compartments. CI-MPR is multifunctional and modulates embryonic growth and fetal size by downregulating circulating levels of the peptide hormone insulin-like growth factor 2 (IGF2). The extracellular region of CI-MPR comprises 15 homologous domains with binding sites for M6P-containing ligands located in domains 3, 5, 9, and 15, whereas IGF2 interacts with residues in domain 11. How a particular ligand affects the receptor's conformation or its ability to bind other ligands remains poorly understood. To address these questions, we purified a soluble form of the receptor from newborn calf serum, carried out glycoproteomics to define the N-glycans at its 19 potential glycosylation sites, probed its ability to bind lysosomal enzymes in the presence and absence of IGF2 using surface plasmon resonance, and assessed its conformation in the presence and absence of IGF2 by negative-staining electron microscopy and hydroxyl radical protein footprinting studies. Together, our findings support the hypothesis that IGF2 acts as an allosteric inhibitor of lysosomal enzyme binding by inducing global conformational changes of CI-MPR.

Activation of lysosomal Ca2+ channels mitigates mitochondrial damage and oxidative stress.

Elevated levels of plasma-free fatty acids and oxidative stress have been identified as putative primary pathogenic factors in endothelial dysfunction etiology, though their roles are unclear. In human endothelial cells, we found that saturated fatty acids (SFAs)-including the plasma-predominant palmitic acid (PA)-cause mitochondrial fragmentation and elevation of intracellular reactive oxygen species (ROS) levels. TRPML1 is a lysosomal ROS-sensitive Ca2+ channel that regulates lysosomal trafficking and biogenesis. Small-molecule agonists of TRPML1 prevented PA-induced mitochondrial damage and ROS elevation through activation of transcriptional factor EB (TFEB), which boosts lysosome biogenesis and mitophagy. Whereas genetically silencing TRPML1 abolished the protective effects of TRPML1 agonism, TRPML1 overexpression conferred a full resistance to PA-induced oxidative damage. Pharmacologically activating the TRPML1-TFEB pathway was sufficient to restore mitochondrial and redox homeostasis in SFA-damaged endothelial cells. The present results suggest that lysosome activation represents a viable strategy for alleviating oxidative damage, a common pathogenic mechanism of metabolic and age-related diseases.

Critical role of ROCK1 in AD pathogenesis via controlling lysosomal biogenesis and acidification

BackgroundLysosomal homeostasis and functions are essential for the survival of neural cells. Impaired lysosomal biogenesis and acidification in Alzheimer’s disease (AD) pathogenesis leads to proteolytic dysfunction and neurodegeneration. However, the key regulatory factors and mechanisms of lysosomal homeostasis in AD remain poorly understood.MethodsROCK1 expression and its co-localization with LAMP1 and SQSTM1/p62 were detected in post-mortem brains of healthy controls and AD patients. Lysosome-related fluorescence probe staining, transmission electron microscopy and immunoblotting were performed to evaluate the role of ROCK1 in lysosomal biogenesis and acidification in various neural cell types. The interaction between ROCK1 and TFEB was confirmed by surface plasmon resonance and in situ proximity ligation assay (PLA). Moreover, we performed AAV-mediated ROCK1 downregulation followed by immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and behavioral tests to unveil the effects of the ROCK1–TFEB axis on lysosomes in APP/PS1 transgenic mice.ResultsROCK1 level was significantly increased in the brains of AD individuals, and was positively correlated with lysosomal markers and Aβ. Lysosomal proteolysis was largely impaired by the high abundance of ROCK1, while ROCK1 knockdown mitigated the lysosomal dysfunction in neurons and microglia. Moreover, we verified ROCK1 as a previously unknown upstream kinase of TFEB independent of m-TOR or GSK-3β. ROCK1 elevation resulted in abundant extracellular Aβ deposition which in turn bound to Aβ receptors and activated RhoA/ROCK1, thus forming a vicious circle of AD pathogenesis. Genetically downregulating ROCK1 lowered its interference with TFEB, promoted TFEB nuclear distribution, lysosomal biogenesis and lysosome-mediated Aβ clearance, and eventually prevented pathological traits and cognitive deficits in APP/PS1 mice.ConclusionIn summary, our results provide a mechanistic insight into the critical role of ROCK1 in lysosomal regulation and Aβ clearance in AD by acting as a novel upstream serine kinase of TFEB.

Itaconate uptake via SLC13A3 improves hepatic antibacterial innate immunity.

Itaconate is an immunoregulatory metabolite produced by the mitochondrial enzyme immune-responsive gene 1 (IRG1) in inflammatory macrophages. We recently identified an important mechanism by which itaconate is released from inflammatory macrophages. However, it remains unknown whether extracellular itaconate is taken up by non-myeloid cells to exert immunoregulatory functions. Here, we used a custom-designed CRISPR screen to identify the dicarboxylate transporter solute carrier family 13 member 3 (SLC13A3) as an itaconate importer and to characterize the role of SLC13A3 in itaconate-improved hepatic antibacterial innate immunity. Functionally, liver-specific deletion of Slc13a3 impairs hepatic antibacterial innate immunity invivo and invitro. Mechanistically, itaconate uptake via SLC13A3 induces transcription factor EB (TFEB)-dependent lysosomal biogenesis and subsequently improves antibacterial innate immunity in mouse hepatocytes. These findings identify SLC13A3 as a key itaconate importer in mouse hepatocytes and will aid in the development of potent itaconate-based antibacterial therapeutics.

Stromal softness confines pancreatic cancer growth through lysosomal-cathepsin mediated YAP1 degradation

The progression and malignancy of many tumors are associated with increased tissue stiffness. Conversely, the oncogenically transformed cells can be confined in soft stroma. Yet, the underlying mechanisms by which soft matrix confines tumorigenesis and metastasis remain elusive. Here, we show that pancreatic cancer cells are suppressed in the soft extracellular matrix, which is associated with YAP1 degradation through autophagic-lysosomal pathway rather than Hippo signal mediated proteasome pathway. In the soft stroma, PTEN is upregulated and activated, which consequently promotes lysosomal biogenesis, leading to the activation of cysteine-cathepsins for YAP1 degradation. In vitro, purified cathepsin L can directly digest YAP1 under acidic conditions. Lysosomal stress, either caused by chloroquine or overexpression of cystatin A/B, results in YAP1 accumulation and malignant transformation. Likewise, liver fibrosis induced stiffness can promote malignant potential in mice. Clinical data show that down-regulation of lysosomal biogenesis is associated with pancreatic fibrosis and stiffness, YAP1 accumulation, and poor prognosis in PDAC patients. Together, our findings suggest that soft stroma triggers lysosomal flux-mediated YAP1 degradation and induces cancer cell dormancy.Graphical

CDK4/6 inhibition initiates cell cycle arrest by nuclear translocation of RB and induces a multistep molecular response

CDK4/6 inhibitors are standard of care in the treatment of metastatic breast cancer. Treatment regimen consists of a combination with endocrine therapy, since their therapeutic efficacy as monotherapy in most clinical trials was rather limited. Thus, understanding the molecular mechanisms that underlie response to therapy might allow for the development of an improved therapy design. We analyzed the response to the CDK4/6 inhibitor palbociclib in bladder cancer cells over a 48-hour time course using RNA sequencing and identified a multi-step mechanism of response. We next translated these results to the molecular mechanism in bladder cancer cells upon PD treatment. The initial step is characterized by translocation of the RB protein into the nucleus by activation of importin α/β, a mechanism that requires the NLS sequence. In parallel, RB is proteolyzed in the cytoplasm, a process regulated by gankyrin and the SCF complex. Only hypophosphorylated RB accumulates in the nucleus, which is an essential step for an efficient therapy response by initiating G1 arrest. This might explain the poor response in RB negative or mutated patients. At later stages during therapy, increased expression of the MiT/TFE protein family leads to lysosomal biogenesis which is essential to maintain this response. Lastly, cancer cells either undergo senescence and apoptosis or develop mechanisms of resistance following CDK4/6 inhibition.

TFEB agonist clomiphene citrate activates the autophagy-lysosomal pathway and ameliorates Alzheimer's disease symptoms in mice

Autophagy is a conserved eukaryotic cellular clearance and recycling process through the lysosome-mediated degradation of damaged organelles and protein aggregates to maintain homeostasis. Impairment of the autophagy-lysosomal pathway is implicated in the pathogenesis of Alzheimer’s disease (AD). Transcription factor EB (TFEB) is a master regulator of autophagy and lysosomal biogenesis. Therefore, activating TFEB and autophagy provides a novel strategy for AD treatment. We previously described that clomiphene citrate (CC) promotes nuclear translocation of TFEB and increases autophagy and lysosomal biogenesis. In this study, 7 and 3-month-old APP/PS1 mice were treated with TFEB agonist CC and assessed. The behavioral tests were performed using Morris water maze and open field test. Additional changes in Aβ pathology, autophagy and inflammatory response were determined. We found that CC activated TFEB and the autophagy-lysosomal pathway in neuronal cells. Moreover, using mouse model of Alzheimer's disease, CC treatment promoted clearance of Aβ plaques and ameliorated cognitive function in both 7 and 3-month-old APP/PS1 mice. The CC-induced activation of TFEB occurs by promoting acetylation of TFEB for nuclear translocation. These findings provide a molecular mechanism for the TFEB-mediated activation of the autophagy-lysosome pathway by CC, which has the potential to be repurposed and applied in the treatment or prevention of AD.

Ubiquitin regulatory X (UBX) domain-containing protein 6 is essential for autophagy induction and inflammation control in macrophages

Ubiquitin regulatory X (UBX) domain-containing protein 6 (UBXN6) is an essential cofactor for the activity of the valosin-containing protein p97, an adenosine triphosphatase associated with diverse cellular activities. Nonetheless, its role in cells of the innate immune system remains largely unexplored. In this study, we report that UBXN6 is upregulated in humans with sepsis and may serve as a pivotal regulator of inflammatory responses via the activation of autophagy. Notably, the upregulation of UBXN6 in sepsis patients was negatively correlated with inflammatory gene profiles but positively correlated with the expression of Forkhead box O3, an autophagy-driving transcription factor. Compared with those of control mice, the macrophages of mice subjected to myeloid cell-specific UBXN6 depletion exhibited exacerbated inflammation, increased mitochondrial oxidative stress, and greater impairment of autophagy and endoplasmic reticulum-associated degradation pathways. UBXN6-deficient macrophages also exhibited immunometabolic remodeling, characterized by a shift to aerobic glycolysis and elevated levels of branched-chain amino acids. These metabolic shifts amplify mammalian target of rapamycin pathway signaling, in turn reducing the nuclear translocation of the transcription factor EB and impairing lysosomal biogenesis. Together, these data reveal that UBXN6 serves as an activator of autophagy and regulates inflammation to maintain immune system suppression during human sepsis.

Transcriptome and proteome profiling reveals TREM2-dependent and -independent glial response and metabolic perturbation in an Alzheimer’s mouse model

Elucidating the intricate molecular mechanisms of Alzheimer's disease (AD) requires a multidimensional analysis incorporating various omics data. In this study, we employed transcriptome and proteome profiling of AppNL-G-F, a human APP knock-in model of amyloidosis, at the early and mid-stages of amyloid-beta (Aβ) pathology to delineate the impacts of Aβ deposition on brain cells. By contrasting AppNL-G-F mice with TREM2 (Triggering receptor expressed on myeloid cells 2) knockout models, our study further investigates the role of TREM2, a well-known AD risk gene, in influencing microglial responses to Aβ pathology. Our results highlight altered microglial states as a central feature of Aβ pathology, characterized by the significant upregulation of microglia-specific genes related to immune responses such as complement system and antigen presentation, and catabolic pathways such as phagosome formation and lysosome biogenesis. The absence of TREM2 markedly diminishes the induction of these genes, impairs Aβ clearance, and exacerbates dystrophic neurite formation. Importantly, TREM2 is required for the microglial engagement with Aβ plaques and the formation of compact Aβ plaque cores. Furthermore, this study reveals substantial disruptions in energy metabolism and protein synthesis, signaling a shift from anabolism to catabolism in response to Aβ deposition. This metabolic alteration, coupled with a decrease in synaptic protein abundance, occurs independently of TREM2, suggesting the direct effects of Aβ deposition on synaptic integrity and plasticity. In summary, our findings demonstrate altered microglial states and metabolic disruption following Aβ deposition, offering mechanistic insights into Aβ pathology and highlighting the potential of targeting these pathways in AD therapy.

Toxic Effect of Methyl-Thiophanate on Bombyx mori Based on Physiological and Transcriptomic Analysis

Background/Objectives: The utilization of methyl-thiophanate (MT) in vegetables and fruits is widespread due to its broad efficiency, yet its potential impact on silkworm growth remains uncertain. This study aims to examine the effects of MT on the growth of silkworms. Specifically, we assessed the weights of fifth-instar larvae that were fed mulberry leaves saturated with three concentrations (2.5, 5, and 10 mg/mL) of MT, as well as the weights of a control group. Methods: TEM was used to show the status of the silkworm midgut after MT supplementation. Oxidative stress was evaluated in the presence of MT. Furthermore, a transcriptomic sequencing experiment was conducted to investigate the mechanism through which the development of silkworms is induced by MT. Results: Our findings indicate that the supplementation of MT hindered larval growth compared to the control group, suggesting a toxic effect of MT on silkworms. The transmission electron microscopy (TEM) results show that MT supplementation induced autophagy in the silkworm midgut. MT was also found to induce oxidative stress in silkworms through the activation of reactive oxygen (ROS), superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities. Subsequent transcriptomic analysis revealed 1265 significantly differentially expressed genes (DEGs) in response to MT. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that these DEGs were associated with antioxidant defense, detoxification processes, lysosome biogenesis, and metabolic pathways. Conclusions: These findings suggest that MT toxicity in silkworm larvae is mediated through the induction of oxidative stress and alterations in metabolism. This study contributes to our understanding of the impacts of MT exposure on silkworms and provides insights into potential pesticides for use in mulberry gardens.

Phospholipase D3 regulates TFEB/TFE3 metabolism to maintain lysosomal homeostasis.

A coding variant in Phospholipase D3 ( PLD3 ) increases the risk of Alzheimer's disease (AD). PLD3 is a lysosomal protein, and endosomal and lysosomal abnormalities are linked to AD; however, the role of PLD3 in lysosomal homeostasis and its implications in AD remain poorly understood. To address this knowledge gap, we conducted comprehensive studies integrating transcriptomics, proteomics, and cell biology approaches. We observed significant enlargement of lysosomes in neurons lacking PLD3, accompanied by increased endocytosis and autophagy, but a decline in lysosomal proteolytic activity. Lysosomes of PLD3-deficient cells underwent proteome remodeling, manifested by an enrichment of proteins involved in lysosomal biogenesis, endocytosis and calcium signaling. Mechanistically, we discovered that PLD3 mediates TFEB/TFE3 degradation through the proteasome, and as a result, PLD3 deficiency leads to increased TFEB/TFE3 levels, nuclear translocation, and transcriptional activities. Notably, variants in PLD3, e.g., V232M or K486R, do not alter its impact on TFEB/TFE3 metabolism. Transcriptomic profiling further confirmed the enrichment of transcripts involved in lysosomal biogenesis, endocytosis, autophagy, mTOR signaling and AD in response to PLD3 loss. Additionally, PLD3 ablation has synergistic effects with β-amyloid in causing lysosomal abnormalities and modifying TFEB/TFE3 signaling. In conclusion, our findings demonstrate that PLD3 is involved in regulating lysosomal biogenesis via TFEB/TFE3 signaling, and lysosomal abnormalities resulting from PLD3 deficiency are potentially a risk factor for AD.