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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.

Astrocytes respond and contribute to neuroinflammation by adopting inflammatory reactive states. Although recent efforts have characterized the gene expression signatures associated with these reactive states, the cell biology underlying inflammatory reactive astrocyte phenotypes remains under-explored. Here, we used CRISPR-based screening in human iPSC-derived astrocytes to identify mTOR activation a driver of cytokine-induced endolysosomal system remodeling, manifesting as alkalinization of endolysosomal compartments, decreased autophagic flux, and increased exocytosis of certain endolysosomal cargos. Through endolysosomal proteomics, we identified and focused on one such cargo-IL-32, a disease-associated pro-inflammatory cytokine not present in rodents, whose secretion mechanism is not well understood. We found that IL-32 was partially secreted in extracellular vesicles likely to be exosomes. Furthermore, we found that IL-32 was involved in the polarization of inflammatory reactive astrocyte states and was upregulated in astrocytes in multiple sclerosis lesions. We believe that our results advance our understanding of cell biological pathways underlying inflammatory reactive astrocyte phenotypes and identify potential therapeutic targets.

Understanding the normal function of the Huntingtin (HTT) protein is of significance in the design and implementation of therapeutic strategies for Huntington's disease (HD). Expansion of the CAG repeat in the HTT gene, encoding an expanded polyglutamine (polyQ) repeat within the HTT protein, causes HD and may compromise HTT's normal activity contributing to HD pathology. Here, we investigated the previously defined role of HTT in autophagy specifically through studying HTT's association with ubiquitin. We find that HTT interacts directly with ubiquitin in vitro. Tandem affinity purification was used to identify ubiquitinated and ubiquitin-associated proteins that copurify with a HTT N-terminal fragment under basal conditions. Copurification is enhanced by HTT polyQ expansion and reduced by mimicking HTT serine 421 phosphorylation. The identified HTT-interacting proteins include RNA-binding proteins (RBPs) involved in mRNA translation, proteins enriched in stress granules, the nuclear proteome, the defective ribosomal products (DRiPs) proteome and the brain-derived autophagosomal proteome. To determine whether the proteins interacting with HTT are autophagic targets, HTT knockout (KO) cells and immunoprecipitation of lysosomes were used to investigate autophagy in the absence of HTT. HTT KO was associated with reduced abundance of mitochondrial proteins in the lysosome, indicating a potential compromise in basal mitophagy, and increased lysosomal abundance of RBPs which may result from compensatory up-regulation of starvation-induced macroautophagy. We suggest HTT is critical for appropriate basal clearance of mitochondrial proteins and RBPs, hence reduced HTT proteostatic function with mutation may contribute to the neuropathology of HD.

Heparan sulfate (HS) is degraded in lysosome by a series of glycosidases. Before the glycosidases can act, the terminal glucosamine of HS must be acetylated by the integral lysosomal membrane enzyme heparan-α-glucosaminide N-acetyltransferase (HGSNAT). Mutations of HGSNAT cause HS accumulation and consequently mucopolysaccharidosis IIIC, a devastating lysosomal storage disease characterized by progressive neurological deterioration and early death where no treatment is available. HGSNAT catalyzes a unique transmembrane acetylation reaction where the acetyl group of cytosolic acetyl-CoA is transported across the lysosomal membrane and attached to HS in one reaction. However, the reaction mechanism remains elusive. Here we report six cryo-EM structures of HGSNAT along the reaction pathway. These structures reveal a dimer arrangement and a unique structural fold, which enables the elucidation of the reaction mechanism. We find that a central pore within each monomer traverses the membrane and controls access of cytosolic acetyl-CoA to the active site at its luminal mouth where glucosamine binds. A histidine-aspartic acid catalytic dyad catalyzes the transfer reaction via a ternary complex mechanism. Furthermore, the structures allow the mapping of disease-causing variants and reveal their potential impact on the function, thus creating a framework to guide structure-based drug discovery efforts.

Recessive Stargardt disease (STGD1) is an inherited juvenile maculopathy caused by mutations in the ABCA4 gene, for which there is no suitable treatment. Loss of functional ABCA4 in the retinal pigment epithelium (RPE) alone, without contribution from photoreceptor cells, was shown to induce STGD1 pathology. Here, we identified cathepsin D (CatD), the primary RPE lysosomal protease, as a key molecular player contributing to endo-lysosomal dysfunction in STGD1 using a newly developed "disease-in-a-dish" RPE model from confirmed STGD1 patients. Induced pluripotent stem cell (iPSC)-derived RPE originating from three STGD1 patients exhibited elevated lysosomal pH, as previously reported in Abca4-/- mice. CatD protein maturation and activity were impaired in RPE from STGD1 patients and Abca4-/- mice. Consequently, STGD1 RPE cells have reduced photoreceptor outer segment degradation and abnormal accumulation of α-synuclein, the natural substrate of CatD. Furthermore, dysfunctional ABCA4 in STGD1 RPE cells results in intracellular accumulation of autofluorescent material and phosphatidylethanolamine (PE). The altered distribution of PE associated with the internal membranes of STGD1 RPE cells presumably compromises LC3-associated phagocytosis, contributing to delayed endo-lysosomal degradation activity. Drug-mediated re-acidification of lysosomes in the RPE of STGD1 restores CatD functional activity and reduces the accumulation of immature CatD protein loads. This preclinical study validates the contribution of CatD deficiencies to STGD1 pathology and provides evidence for an efficacious therapeutic approach targeting RPE cells. Our findings support a cell-autonomous RPE-driven pathology, informing future research aimed at targeting RPE cells to treat ABCA4-mediated retinopathies.

Heat shock can be a lethal stressor. Previously, we described a CUL-6/cullin-ring ubiquitin ligase complex in the nematode Caenorhabditis elegans that is induced by intracellular intestinal infection and proteotoxic stress and that promotes improved survival upon heat shock (thermotolerance). Here, we show that CUL-6 promotes thermotolerance by targeting the heat shock protein HSP-90 for degradation. We show that CUL-6-mediated lowering of HSP-90 protein levels, specifically in the intestine, improves thermotolerance. Furthermore, we show that lysosomal function is required for CUL-6-mediated promotion of thermotolerance and that CUL-6 directs HSP-90 to lysosome-related organelles upon heat shock. Altogether, these results indicate that a CUL-6 ubiquitin ligase promotes organismal survival upon heat shock by promoting HSP-90 degradation in intestinal lysosomes. Thus, HSP-90, a protein commonly associated with protection against heat shock and promoting degradation of other proteins, is itself degraded to protect against heat shock.

The prion-like spread of protein aggregates is a leading hypothesis for the propagation of neurofibrillary lesions in the brain, including the spread of tau inclusions associated with Alzheimers disease. The mechanisms of cellular uptake of tau seeds and subsequent nucleated polymerization of cytosolic tau are major questions in the field, and the potential for coupling between the entry and nucleation mechanisms has been little explored. We found that in primary astrocytes and neurons, endocytosis of tau seeds leads to their accumulation in lysosomes. This in turn leads to lysosomal swelling, deacidification, and recruitment of ESCRT proteins, but not Galectin-3, to the lysosomal membrane. These observations are consistent with nanoscale damage of the lysosomal membrane. Live cell imaging and STORM superresolution microscopy further show that the nucleation of cytosolic tau occurs primarily at the lysosome membrane under these conditions. These data suggest that tau seeds escape from lysosomes via nanoscale damage rather than wholesale rupture and that nucleation of cytosolic tau commences as soon as tau fibril ends emerge from the lysosomal membrane.

Mycobacterium tuberculosis (Mtb) infects lung myeloid cells, but the specific Mtb-permissive cells and host mechanisms supporting Mtb persistence during chronic infection are incompletely characterized. We report that after the development of T cell responses, CD11clo monocyte-derived cells harbor more live Mtb than alveolar macrophages (AM), neutrophils, and CD11chi monocyte-derived cells. Transcriptomic and functional studies revealed that the lysosome pathway is underexpressed in this highly permissive subset, characterized by less lysosome content, acidification, and proteolytic activity than AM, along with less nuclear TFEB, a regulator of lysosome biogenesis. Mtb infection does not drive lysosome deficiency in CD11clo monocyte-derived cells but promotes recruitment of monocytes that develop into permissive lung cells, mediated by the Mtb ESX-1 secretion system. The c-Abl tyrosine kinase inhibitor nilotinib activates TFEB and enhances lysosome functions of macrophages in vitro and in vivo, improving control of Mtb infection. Our results suggest that Mtb exploits lysosome-poor lung cells for persistence and targeting lysosome biogenesis is a potential host-directed therapy for tuberculosis.

The etiology of preeclampsia (PE), a severe complication of pregnancy with several clinical manifestations and a high incidence of maternal and fetal morbidity and mortality, remains unclear. This issue is a major hurdle for effective treatment strategies. We recently demonstrated that PE exhibits an Alzheimer-like etiology of impaired autophagy and proteinopathy in the placenta. Targeting of these pathological pathways may be a novel therapeutic strategy for PE. Stimulation of autophagy with the natural disaccharide trehalose and its lacto analog lactotrehalose in hypoxia-exposed primary human trophoblasts restored autophagy, inhibited the accumulation of toxic protein aggregates, and restored the ultrastructural features of autophagosomes and autolysosomes. Importantly, trehalose and lactotrehalose inhibited the onset of PE-like features in a humanized mouse model by normalizing autophagy and inhibiting protein aggregation in the placenta. These disaccharides restored the autophagy-lysosomal biogenesis machinery by increasing nuclear translocation of the master transcriptional regulator TFEB. RNA-seq analysis of the placentas of mice with PE indicated the normalization of the PE-associated transcriptome profile in response to trehalose and lactotrehalose. In summary, our results provide a novel molecular rationale for impaired autophagy and proteinopathy in patients with PE and identify treatment with trehalose and its lacto analog as promising therapeutic options for this severe pregnancy complication.

BACKGROUND: Cathepsin D (CatD) is a lysosomal protease that degrades both the amyloid-β protein (Aβ) and the microtubule-associated protein, tau, which accumulate pathognomonically in Alzheimer disease (AD), but few studies have examined the role of CatD in the development of Aβ pathology and tauopathy in vivo. METHODS: CatD knockout (KO) mice were crossed to human amyloid precursor protein (hAPP) transgenic mice, and amyloid burden was quantified by ELISA and immunohistochemistry (IHC). Tauopathy in CatD-KO mice, as initially suggested by Gallyas silver staining, was further characterized by extensive IHC and biochemical analyses. Controls included human tau transgenic mice (JNPL3) and another mouse model of a disease (Krabbe A) characterized by pronounced lysosomal dysfunction. Additional experiments examined the effects of CatD inhibition on tau catabolism in vitro and in cultured neuroblastoma cells with inducible expression of human tau. RESULTS: Deletion of CatD in hAPP transgenic mice triggers large increases in cerebral Aβ, manifesting as intense, exclusively intracellular aggregates; extracellular Aβ deposition, by contrast, is neither triggered by CatD deletion, nor affected in older, haploinsufficient mice. Unexpectedly, CatD-KO mice were found to develop prominent tauopathy by just ∼ 3 weeks of age, accumulating sarkosyl-insoluble, hyperphosphorylated tau exceeding the pathology present in aged JNPL3 mice. CatD-KO mice exhibit pronounced perinuclear Gallyas silver staining reminiscent of mature neurofibrillary tangles in human AD, together with widespread phospho-tau immunoreactivity. Striking increases in sarkosyl-insoluble phospho-tau (∼ 1250%) are present in CatD-KO mice but notably absent from Krabbe A mice collected at an identical antemortem interval. In vitro and in cultured cells, we show that tau catabolism is slowed by blockade of CatD proteolytic activity, including via competitive inhibition by Aβ42. CONCLUSIONS: Our findings support a major role for CatD in the proteostasis of both Aβ and tau in vivo. To our knowledge, the CatD-KO mouse line is the only model to develop detectable Aβ accumulation and profound tauopathy in the absence of overexpression of hAPP or human tau with disease-associated mutations. Given that tauopathy emerges from disruption of CatD, which can itself be potently inhibited by Aβ42, our findings suggest that impaired CatD activity may represent a key mechanism linking amyloid accumulation and tauopathy in AD.

Glycosphingolipids (GSLs) are key constituents of membrane bilayers playing a role in structural integrity, cell signalling in microdomains, endosomes and lysosomes, and cell death pathways. Conversion of ceramide into GSLs is controlled by GCS (glucosylceramide synthase) and inhibitors of this enzyme for the treatment of lipid storage disorders and specific cancers. With a diverse range of functions attributed to GSLs, the ability of the GSC inhibitor, eliglustat, to reduce myeloma bone disease was investigated. In pre-clinical models of multiple myeloma, osteoclast-driven bone loss was reduced by eliglustat in a mechanistically separate manner to zoledronic acid, a bisphosphonate that prevents osteoclast-mediated bone destruction. Autophagic degradation of TNF receptor-associated factor 3 (TRAF3), a key step for osteoclast differentiation, was inhibited by eliglustat as evidenced by TRAF3 lysosomal and cytoplasmic accumulation. By altering GSL composition, eliglustat prevented lysosomal degradation whilst exogenous addition of missing GSLs rescued TRAF3 degradation to restore osteoclast formation in bone marrow cells from myeloma patients. This work highlights the clinical potential of eliglustat as a therapy for myeloma bone disease. Furthermore, using eliglustat as a lysosomal inhibitor in osteoclasts may widen its therapeutic uses to other bone disorders such as bone metastasis, osteoporosis and inflammatory bone loss.

Orphan solute carrier (SLC) represents a group of membrane transporters whose exact functions and substrate specificities are not known. Elucidating the function and regulation of orphan SLC transporters is not only crucial for advancing our knowledge of cellular and molecular biology but can potentially lead to the development of new therapeutic strategies. Here, we provide evidence for the biological function of a ubiquitous orphan lysosomal SLC, the Major Facilitator Superfamily Domain-containing Protein 1 (MFSD1), which has remained phylogenetically unassigned. Targeted metabolomics revealed that dipeptides containing either lysine or arginine residues accumulate in lysosomes of cells lacking MFSD1. Whole-cell patch-clamp electrophysiological recordings of HEK293-cells expressing MFSD1 on the cell surface displayed transport affinities for positively charged dipeptides in the lower mM range, while dipeptides that carry a negative net charge were not transported. This was also true for single amino acids and tripeptides, which MFSD1 failed to transport. Our results identify MFSD1 as a highly selective lysosomal lysine/arginine/histidine-containing dipeptide exporter, which functions as a uniporter.

ATG5 plays a pivotal role in membrane Atg8ylation, influencing downstream processes encompassing canonical autophagy and noncanonical processes. Remarkably, genetic ablation of ATG5 in myeloid cells leads to an exacerbated pathological state in murine models of tuberculosis, characterized by an early surge in mortality much more severe when compared to the depletion of other components involved in Atg8ylation or canonical autophagy. This study shows that in the absence of ATG5, but not other core canonical autophagy factors, endolysosomal organelles display a lysosomal hypersensitivity phenotype when subjected to damage. This is in part due to a compromised recruitment of ESCRT proteins to lysosomes in need of repair. Mechanistically, in the absence of ATG5, the ESCRT protein PDCD6IP/ALIX is sequestered by the alternative conjugate ATG12-ATG3, contributing to excessive exocytic processes while not being available for lysosomal repair. Specifically, this condition increases secretion of extracellular vesicles and particles, and leads to excessive degranulation in neutrophils. Our findings uncover unique functions of ATG5 outside of the autophagy and Atg8ylation paradigm. This finding is of in vivo relevance for tuberculosis pathogenesis as modeled in mice.Abbreviations: Atg5: autophagy related 5; ESCRT: endosomal sorting complex required for transport; EVPs: extracellular vesicles and particles; FPR1: formyl peptide receptor 1; LyHYP: lysosomal hypersensitivity phenotype; LysoIP: lysosome immunopurification; Mtb: Mycobacterium tuberculosis; ORF3a: open reading frame 3a protein; PDCD6IP/ALIX: programmed cell death 6 interacting protein; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2, TFEB: transcription factor EB.

Non-neovascular or dry age-related macular degeneration (AMD) is a multi-factorial disease with degeneration of the aging retinal-pigmented epithelium (RPE). Lysosomes play a crucial role in RPE health via phagocytosis and autophagy, which are regulated by transcription factor EB/E3 (TFEB/E3). Here, we find that increased AKT2 inhibits PGC-1α to downregulate SIRT5, which we identify as an AKT2 binding partner. Crosstalk between SIRT5 and AKT2 facilitates TFEB-dependent lysosomal function in the RPE. AKT2/SIRT5/TFEB pathway inhibition in the RPE induced lysosome/autophagy signaling abnormalities, disrupted mitochondrial function and induced release of debris contributing to drusen. Accordingly, AKT2 overexpression in the RPE caused a dry AMD-like phenotype in aging Akt2 KI mice, as evident from decline in retinal function. Importantly, we show that induced pluripotent stem cell-derived RPE encoding the major risk variant associated with AMD (complement factor H; CFH Y402H) express increased AKT2, impairing TFEB/TFE3-dependent lysosomal function. Collectively, these findings suggest that targeting the AKT2/SIRT5/TFEB pathway may be an effective therapy to delay the progression of dry AMD.

To stimulate cell growth, the protein kinase complex mTORC1 requires intracellular amino acids for activation. Amino-acid sufficiency is relayed to mTORC1 by Rag GTPases on lysosomes, where growth factor signaling enhances mTORC1 activity via the GTPase Rheb. In the absence of amino acids, GATOR1 inactivates the Rags, resulting in lysosomal detachment and inactivation of mTORC1. We demonstrate that in human cells, the release of mTORC1 from lysosomes depends on its kinase activity. In accordance with a negative feedback mechanism, activated mTOR mutants display low lysosome occupancy, causing hypo-phosphorylation and nuclear localization of the lysosomal substrate TFE3. Surprisingly, mTORC1 activated by Rheb does not increase the cytoplasmic/lysosomal ratio of mTORC1, indicating the existence of mTORC1 pools with distinct substrate specificity. Dysregulation of either pool results in aberrant TFE3 activity and may explain nuclear accumulation of TFE3 in epileptogenic malformations in focal cortical dysplasia type II (FCD II) and tuberous sclerosis (TSC). [Display omitted] • Pharmacological inhibition prevents lysosomal mTORC1 release during amino acid depletion • A specific mTORC1 pool controls lysosomal mTORC1 levels in a Rag-dependent manner • Activating mTOR mutations result in hypo-phosphorylated and nuclear TFE3 • Nuclear TFE3 is found in dysmorphic cells in brain malformations with active mTOR Zwakenberg et al. show that mTORC1 auto-regulates its presence on lysosomes. Inhibition of mTOR results in lysosomal mTORC1 accumulation. Active mTOR mutants, on the other hand, have a high cytosolic/lysosomal ratio, leading to nuclear TFE3 accumulation. Auto-regulation is mediated by an mTORC1 pool distinct from the one activated through loss of TSC and may involve GATOR1. [ABSTRACT FROM AUTHOR]

Nutrient signaling converges on mTORC1, which, in turn, orchestrates a physiological cellular response. A key determinant of mTORC1 activity is its shuttling between the lysosomal surface and the cytoplasm, with nutrients promoting its recruitment to lysosomes by the Rag GTPases. Active mTORC1 regulates various cellular functions by phosphorylating distinct substrates at different subcellular locations. Importantly, how mTORC1 that is activated on lysosomes is released to meet its non-lysosomal targets and whether mTORC1 activity itself impacts its localization remain unclear. Here, we show that, in human cells, mTORC1 inhibition prevents its release from lysosomes, even under starvation conditions, which is accompanied by elevated and sustained phosphorylation of its lysosomal substrate TFEB. Mechanistically, "inactive" mTORC1 causes persistent Rag activation, underlining its release as another process actively mediated via the Rags. In sum, we describe a mechanism by which mTORC1 controls its own localization, likely to prevent futile cycling on and off lysosomes. [Display omitted] • Inhibition of mTORC1 augments its lysosomal presence due to impaired release • Inactive mTORC1 is strongly lysosomal even upon starvation or lysosome dysfunction • Enhanced lysosomal mTORC1 concomitantly impacts TFEB phosphorylation and activation • mTORC1 autoregulates its cycling on/off lysosomes via changes in RagA activation The activation of mTORC1 is intricately linked to its subcellular localization, with the Rag GTPases controlling its lysosomal recruitment in response to nutrient cues. Acharya and Demetriades uncover another layer of regulation of mTORC1 localization, wherein its intrinsic kinase activity governs its own lysosomal tethering by influencing RagA nucleotide loading. [ABSTRACT FROM AUTHOR]

Autophagy is a highly conserved 'self-digesting' mechanism used in eukaryotes to degrade and recycle cellular components by enclosing them in a double membrane compartment and delivering them to lytic organelles (lysosomes or vacuoles). Extensive studies in plants have revealed how autophagy is intricately linked to essential aspects of metabolism and growth, in both normal and stress conditions, including cellular and organelle homeostasis, nutrient recycling, development, responses to biotic and abiotic stresses, senescence and cell death. However, knowledge regarding autophagic processes in other photosynthetic organisms remains limited. In this review, we attempt to summarize the current understanding of autophagy in algae from a metabolic, molecular and evolutionary perspective. We focus on the composition and conservation of the autophagy molecular machinery in eukaryotes and discuss the role of autophagy in metabolic regulation, cellular homeostasis and stress adaptation in algae. This article is part of the theme issue 'The evolution of plant metabolism'. [ABSTRACT FROM AUTHOR]

The analysis of gangliosides and glycosphingolipids is crucial for understanding cellular membrane structure and function as well as to accurately diagnose certain inborn errors of metabolism. GM2-gangliosidosis represents a rare and fatal group of lysosomal storage disorders characterized by accumulation of GM2 gangliosides in various tissues and organs. These disorders arise due to deficiency or functional impairment of the β-hexosaminidase A or B enzymes, which are responsible for degradation of GM2 ganglioside. Deficient enzyme activity primarily leads to the accumulation of GM2 gangliosides within the lysosomes of cells. Accurate and rapid diagnostic methods that detect increased levels of GM2 gangliosides in patients with GM2-gangliosidosis can play a significant role in early diagnosis and appropriate treatment of this condition. To address this need, we developed a multiplexed liquid chromatography-tandem mass spectrometry method targeting 84 species of gangliosides and other glycosphingolipids involved in ganglioside metabolism. Reproducibility, linearity, extraction efficiency, and sample stability were evaluated and proof-of-concept data obtained from analysis of serum samples from confirmed cases of GM2-gangliosidosis. This method has the potential to simultaneously monitor the biosynthesis of gangliosides and the lysosomal catabolic pathway serving as a valuable tool for screening and diagnosing an important group of lysosomal storage disorders. [ABSTRACT FROM AUTHOR]

Phospholipase D3 (PLD3) has emerged as an important 5′‐exonuclease in charge of removing single‐stranded DNA in lysosomes. Rare genetic variants of the gene encoding PLD3 have been implicated in late‐onset Alzheimer's disease (AD). Ishii et al. have produced the soluble domain of human PLD3 with the aim of determining its three‐dimensional structure using X‐ray crystallography. The high‐resolution structure (2.3 Å) provides new insights into the biochemical properties of the enzyme and paves the way to a deeper understanding of amino acid replacements affecting the stability and activity of the enzyme. [ABSTRACT FROM AUTHOR]

Biondi bodies are filamentous amyloid inclusions of unknown composition in ependymal cells of the choroid plexuses, ependymal cells lining cerebral ventricles and ependymal cells of the central canal of the spinal cord. Their formation is age-dependent and they are commonly associated with a variety of neurodegenerative conditions, including Alzheimer's disease and Lewy body disorders. Here, we show that Biondi bodies are strongly immunoreactive with TMEM239, an antibody specific for inclusions of transmembrane protein 106B (TMEM106B). Biondi bodies were labelled by both this antibody and the amyloid dye pFTAA. Many Biondi bodies were also labelled for TMEM106B and the lysosomal markers Hexosaminidase A and Cathepsin D. By transmission immuno-electron microscopy, Biondi bodies of choroid plexuses were decorated by TMEM239 and were associated with structures that resembled residual bodies or secondary lysosomes. By electron cryo-microscopy, TMEM106B filaments from Biondi bodies of choroid plexuses were similar (Biondi variant), but not identical, to the fold I that was previously identified in filaments from brain parenchyma. [ABSTRACT FROM AUTHOR]

Lysosomes are important cellular structures for human health as centers for recycling, signaling, metabolism and stress adaptation. However, the potential role of lysosomes in stress‐related emotions has long been overlooked. Here, it is found that lysosomal morphology in astrocytes is altered in the medial prefrontal cortex (mPFC) of susceptible mice after chronic social defeat stress. A screen of lysosome‐related genes revealed that the expression of the mucolipin 1 gene (Mcoln1; protein: mucolipin TRP channel 1) is decreased in susceptible mice and depressed patients. Astrocyte‐specific knockout of mucolipin TRP channel 1 (TRPML1) induced depressive‐like behaviors by inhibiting lysosomal exocytosis‐mediated adenosine 5′‐triphosphate (ATP) release. Furthermore, this stress response of astrocytic lysosomes is mediated by the transcription factor EB (TFEB), and overexpression of TRPML1 rescued depressive‐like behaviors induced by astrocyte‐specific knockout of TFEB. Collectively, these findings reveal a lysosomal stress‐sensing signaling pathway contributing to the development of depression and identify the lysosome as a potential target organelle for antidepressants. [ABSTRACT FROM AUTHOR]

Sortilin‐related receptor 1 (SorL1) deficiency is a genetic predisposition to familial Alzheimer's disease (AD), but its pathology is poorly understood. In SorL1‐null rats, a disorder of the global endosome‐lysosome network (ELN) is found in hippocampal neurons. Deletion of amyloid precursor protein (APP) in SorL1‐null rats could not completely rescue the neuronal abnormalities in the ELN of the hippocampus and the impairment of spatial memory in SorL1‐null young rats. These in vivo observations indicated that APP is one of the cargoes of SorL1 in the regulation of the ELN, which affects hippocampal‐dependent memory. When SorL1 is depleted, the endolysosome takes up more of the lysosome flux and damages lysosomal digestion, leading to pathological lysosomal storage and disturbance of cholesterol and iron homeostasis in the hippocampus. These disturbances disrupt the original homeostasis of the material‐energy‐subcellular structure and reprogram energy metabolism based on fatty acids in the SorL1‐null hippocampus, instead of glucose. Although fatty acid oxidation increases ATP supply, it cannot reduce the levels of the harmful byproduct ROS during oxidative phosphorylation, as it does in glucose catabolism. Therefore, the SorL1‐null rats exhibit hippocampal degeneration, and their spatial memory is impaired. Our research sheds light on the pathology of SorL1 deficiency in AD. [ABSTRACT FROM AUTHOR]

Deleterious mutations in the lipopolysaccharide responsive beige-like anchor protein (LRBA) gene cause severe childhood immune dysregulation. The complexity of the symptoms involving multiple organs and the broad range of unpredictable clinical manifestations of LRBA deficiency complicate the choice of therapeutic interventions. Although LRBA has been linked to Rab11-dependent trafficking of the immune checkpoint protein CTLA-4, its precise cellular role remains elusive. We show that LRBA, however, only slightly colocalizes with Rab11. Instead, LRBA is recruited by members of the small GTPase Arf protein family to the TGN and to Rab4+ endosomes, where it controls intracellular traffic. In patient-derived fibroblasts, loss of LRBA led to defects in the endosomal pathway promoting the accumulation of enlarged endolysosomes and lysosome secretion. Thus, LRBA appears to regulate flow through the endosomal system on Rab4+ endosomes. Our data strongly suggest functions of LRBA beyond CTLA-4 trafficking and provide a conceptual framework to develop new therapies for LRBA deficiency. [ABSTRACT FROM AUTHOR]

Aggressive solid malignancies, including pancreatic ductal adenocarcinoma (PDAC), can exploit lysosomal exocytosis to modify the tumor microenvironment, enhance motility, and promote invasiveness. However, the molecular pathways through which lysosomal functions are co-opted in malignant cells remain poorly understood. In this study, we demonstrate that inositol polyphosphate 4-phosphatase, Type II (INPP4B) overexpression in PDAC is associated with PDAC progression. We show that INPP4B overexpression promotes peripheral dispersion and exocytosis of lysosomes resulting in increased migratory and invasive potential of PDAC cells. Mechanistically, INPP4B overexpression drives the generation of PtdIns(3,5)P2 on lysosomes in a PIKfyve-dependent manner, which directs TRPML-1 to trigger the release of calcium ions (Ca2+). Our findings offer a molecular understanding of the prognostic significance of INPP4B overexpression in PDAC through the discovery of a novel oncogenic signaling axis that orchestrates migratory and invasive properties of PDAC via the regulation of lysosomal phosphoinositide homeostasis. [ABSTRACT FROM AUTHOR]

A series of donor‐acceptor (D–π–A) substituted diphenylbutadienes exhibiting solvatochromic emission and a large Stokes shift (100–200 nm) were designed and synthesized for distinctive organelle labelling, enabling real‐time monitoring of organelle behaviour such as lysosomal dynamics, mitophagy monitoring, and stress responses. The morpholine‐substituted D–A–D diphenylbutadiene (M2) was employed to investigate selective imaging of lysosomes, the uptake of damaged mitochondria through mitophagy, and monitoring lysosomal viscosity or pH changes. Other diphenylbutadiene derivatives (M1, M3, M4) selectively accumulated in lipid droplets. All the synthesized derivatives demonstrated significant uptake in 5‐day post‐fertilization zebrafish larvae, with M2 showing maximum uptake in the enterocyte‐containing heart and intestinal regions, which include the lysosomes. [ABSTRACT FROM AUTHOR]

Multiple system atrophy is characterized pathologically by the accumulation of alpha-synuclein (aSyn) into glial cytoplasmic inclusions (GCIs). The mechanism underlying the formation of GCIs is not well understood. In this study, correlative light and electron microscopy was employed to investigate aSyn pathology in the substantia nigra and putamen of post-mortem multiple system atrophy brain donors. Three distinct types of aSyn immuno-positive inclusions were identified in oligodendrocytes, neurons and dark cells presumed to be dark microglia. Oligodendrocytes contained fibrillar GCIs that were consistently enriched with lysosomes and peroxisomes, supporting the involvement of the autophagy pathway in aSyn aggregation in multiple system atrophy. Neuronal cytoplasmic inclusions exhibited ultrastructural heterogeneity resembling both fibrillar and membranous inclusions, linking multiple systems atrophy and Parkinson's disease. The novel aSyn pathology identified in the dark cells, displayed GCI-like fibrils or non-GCI-like ultrastructures suggesting various stages of aSyn accumulation in these cells. The observation of GCI-like fibrils within dark cells suggests these cells may be an important contributor to the origin or spread of pathological aSyn in multiple system atrophy. Our results suggest a complex interplay between multiple cell types that may underlie the formation of aSyn pathology in multiple system atrophy brain and highlight the need for further investigation into cell-specific disease pathologies in multiple system atrophy. [ABSTRACT FROM AUTHOR]

The editorial "Special Issue: Molecular Advance on Reproduction and Fertility of Aquatic Animals" explores the complexities of reproduction and fertility in commercial aquatic species, emphasizing the importance of sexual dimorphism and monosexual culture for aquaculture efficiency. The document delves into the molecular mechanisms of sex determination and gonadal development in aquatic animals, highlighting the role of genes, markers, and epigenetic modifications in sex differentiation. Additionally, it discusses the impact of environmental factors, such as food availability, on gonad maturation mechanisms, providing insights into adaptation strategies under starvation conditions. The research presented in this special issue contributes to a deeper understanding of reproduction and fertility in aquatic animals through advanced sequencing technologies and genetic analyses. [Extracted from the article]

Neurons become more vulnerable to stress factors with age, which leads to increased oxidative DNA damage, decreased activity of mitochondria and lysosomes, increased levels of p16, decreased LaminB1 proteins, and the depletion of the dendritic tree. These changes are exacerbated in vulnerable neuronal populations during the development of neurodegenerative diseases. Glu-Asp-Arg (EDR) and Lys-Glu-Asp (KED), and Ala-Glu-Asp-Gly (AEDG) peptides have previously demonstrated neuroprotective effects in various models of Alzheimer's disease. In this study, we investigated the influence of EDR, KED, and AEDG peptides on the aging of fibroblast-derived induced neurons. We used a new in vitro cellular model of human neuronal aging based on the transdifferentiation of aged dermal fibroblasts from elderly donors into induced cortical neurons. All peptides promote the arborization of the dendritic tree, increasing both the number of primary processes and the total length of dendrites. Tripeptides have no effect on the activity of mitochondria and lysosomes and the level of p16 protein in induced neurons. EDR peptide reduces oxidative DNA damage in induced neurons derived from elderly donor fibroblasts. Short peptides partially protect induced neurons from age-related changes and stimulate dendritogenesis in neurons. They can be recommended for use as neuroprotective agents. [ABSTRACT FROM AUTHOR]

Mitochondria are dynamic, intracellular organelles with a separate genome originating from prokaryotes. They perform numerous functions essential for cellular metabolism and energy production. Mitochondrial‐derived vesicles (MDVs) are single or double membrane‐enclosed vesicles, formed and released from the mitochondrial sub‐compartments into the cytosol, in response to various triggers. MDVs interact with other organelles such as lysosomes and peroxisomes or may be incorporated and excreted via extracellular vesicles (EVs). MDVs selectively incorporate diverse protein and lipid cargoes and are involved in various functions such as mitochondrial quality control, immunomodulation, energy complementation, and compartmentalization and transport. This review aims to provide a summary of the current knowledge of MDVs biogenesis, release, cargoes, and roles. [ABSTRACT FROM AUTHOR]

Lysosomes are involved in a myriad of cellular functions, such as degradation of macromolecules, endocytosis and exocytosis, modulation of several signaling pathways, and regulation of cell metabolism. To fulfill these diverse functions, lysosomes can undergo several dynamic changes in their content, size, pH, and location within cells. Here, we studied some of these parameters during embryonic chick skeletal muscle cells. We used an anti‐lysosome‐associated membrane protein 2 (LAMP2) antibody to specifically determine the intracellular localization of lysosomes in these cells. Our data shows that lysosomes are highly enriched in the perinuclear region of chick embryonic muscle cells. We also showed that the wingless signaling pathway (Wnt)/β‐catenin signaling pathway can modulate the location of LAMP2 in chick myogenic cells. Our results highlight the role of lysosomes during muscle differentiation and particularly the presence of a subcellular population of lysosomes that are concentrated in the perinuclear region of muscle cells. [ABSTRACT FROM AUTHOR]

Dynein cytoplasmic 1 light intermediate chain 1 (LIC1, DYNC1LI1) is a core subunit of the dynein motor complex. The LIC1 subunit also interacts with various cargo adaptors to regulate Rab-mediated endosomal recycling and lysosomal degradation. Defects in this gene are predicted to alter dynein motor function, Rab binding capabilities, and cytoplasmic cargo trafficking. Here, we have identified a dync1li1 zebrafish mutant, harboring a premature stop codon at the exon 12/13 splice acceptor site, that displays increased angiogenesis. In vitro, LIC1-deficient human endothelial cells display increases in cell surface levels of the pro-angiogenic receptor VEGFR2, SRC phosphorylation, and Rab11-mediated endosomal recycling. In vivo, endothelial-specific expression of constitutively active Rab11a leads to excessive angiogenesis, similar to the dync1li1 mutants. Increased angiogenesis is also evident in zebrafish harboring mutations in rilpl1/2, the adaptor proteins that promote Rab docking to Lic1 to mediate lysosomal targeting. These findings suggest that LIC1 and the Rab-adaptor proteins RILPL1 and 2 restrict angiogenesis by promoting degradation of VEGFR2-containing recycling endosomes. Disruption of LIC1- and RILPL1/2-mediated lysosomal targeting increases Rab11-mediated recycling endosome activity, promoting excessive SRC signaling and angiogenesis. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is a progressive neurogenerative movement disorder characterized by dopaminergic cell death within the substantia nigra pars compacta (SNpc) due to the aggregation-prone protein α-synuclein. Accumulation of α-synuclein is implicated in mitochondrial dysfunction and disruption of the autophagic turnover of mitochondria, or mitophagy, which is an essential quality control mechanism proposed to preserve mitochondrial fidelity in response to aging and stress. Yet, the precise relationship between α-synuclein accumulation, mitochondrial autophagy, and dopaminergic cell loss remains unresolved. Here, we determine the kinetics of α-synuclein overexpression and mitophagy using the pH-sensitive fluorescent mito-QC reporter. We find that overexpression of mutant A53T α-synuclein in either human SH-SY5Y cells or rat primary cortical neurons induces mitophagy. Moreover, the accumulation of mutant A53T α-synuclein in the SNpc of rats results in mitophagy dysregulation that precedes the onset of dopaminergic neurodegeneration. This study reveals a role for mutant A53T α-synuclein in inducing mitochondrial dysfunction, which may be an early event contributing to neurodegeneration. [ABSTRACT FROM AUTHOR]

Mercury is highly toxic, and appropriate amounts of the mercury‐selenium complex can protect plasma. However, when excessive mercury (II) ions (Hg2+) are exposed to human skin or ingested directly, it can lead to irreversible accumulation in the body. Therefore, detecting the presence of Hg2+ in cells is important. A novel fluorescent probe BTD‐Hg‐Lyso was designed and constructed based on the intramolecular charge transfer mechanism. Thiotaldehyde could specifically recognize Hg2+ so that the probe could produce a fluorescence enhancement effect. In addition, the fluorescent probe BTD‐Hg‐Lyso exhibited the advantages of large Stokes shift (210 nm) and good selectivity. More importantly, the probe BTD‐Hg‐Lyso could be used for the determination of Hg2+ in cellular lysosomes. BTD‐Hg‐Lyso was able to image Hg2+ in HeLa cells, zebrafish, and tobacco seedlings (rhizome and stem) successfully. [ABSTRACT FROM AUTHOR]

Clear cell renal cell carcinoma (ccRCC) is tightly associated with VHL (von Hippel-Lindau tumor suppressor) mutation and dysregulated angiogenesis. Accumulating evidence indicates that antiangiogenic treatment abolishing tumor angiogenesis can achieve longer disease-free survival in patients with ccRCC. Atractylenolide I (ATL-I) is one of the main active compounds in Atractylodes macrocephala root extract and exhibits various pharmacological effects, including anti-inflammatory and antitumor effects. In this study, we revealed the potent antitumor activity of ATL-I in ccRCC. ATL-I exhibited robust antiangiogenic capacity by inhibiting EPAS1/HIF2α-mediated VEGFA production in VHL-deficient ccRCC, and it promoted autophagic degradation of EPAS1 by upregulating the ATPase subunit ATP6V0D2 (ATPase H+ transporting V0 subunit d2) to increase lysosomal function and facilitated fusion between autophagosomes and lysosomes. Mechanistically, ATP6V0D2 directly bound to RAB7 and VPS41 and promoted the RAB7-HOPS interaction, facilitating SNARE complex assembly and autophagosome-lysosome fusion. Moreover, ATP6V0D2 promoted autolysosome degradation by increasing the acidification and activity of lysosomes during the later stages of macroautophagy/autophagy. Additionally, we found that ATL-I could decrease the level of EPAS1, which was upregulated in sunitinib-resistant cells, thus reversing sunitinib resistance. Collectively, our findings demonstrate that ATL-I is a robust antiangiogenic and antitumor lead compound with potential clinical application for ccRCC therapy.Abbreviations: ATL-I: atractylenolide I; ATP6V0D2: ATPase H+ transporting V0 subunit d2; CAM: chick chorioallantoic membrane; ccRCC: clear cell renal cell carcinoma; CTSB: cathepsin B; CTSD: cathepsin D; GO: Gene Ontology; HIF-1: HIF1A-ARNT heterodimer; HOPS: homotypic fusion and protein sorting; KDR/VEGFR: kinase insert domain receptor; KEGG: Kyoto Encyclopedia of Genes and Genomes; RCC: renal cell carcinoma; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TCGA: The Cancer Genome Atlas; TEM: transmission electron microscopy; TKI: tyrosine kinase inhibitor; V-ATPase: vacuolar-type H±translocating ATPase; VEGF: vascular endothelial growth factor; VHL: von Hippel-Lindau tumor suppressor. [ABSTRACT FROM AUTHOR]

Stimulator of interferon genes (STING) is activated in many pathophysiological conditions, leading to TBK1-dependent interferon production in higher organisms. However, primordial functions of STING independent of TBK1 are poorly understood. Here, through proteomics and bioinformatics approaches, we identify lysosomal biogenesis as an unexpected function of STING. Transcription factor EB (TFEB), an evolutionarily conserved regulator of lysosomal biogenesis and host defense, is activated by STING from multiple species, including humans, mice, and frogs. STING-mediated TFEB activation is independent of TBK1, but it requires STING trafficking and its conserved proton channel. GABARAP lipidation, stimulated by the channel of STING, is key for STING-dependent TFEB activation. STING stimulates global upregulation of TFEB-target genes, mediating lysosomal biogenesis and autophagy. TFEB supports cell survival during chronic sterile STING activation, a common condition in aging and age-related diseases. These results reveal a primordial function of STING in the biogenesis of lysosomes, essential organelles in immunity and cellular stress resistance. [Display omitted] • TFEB and TFE3 are conserved effectors of STING, predating type I interferon signaling • Noncanonical GABARAP lipidation, triggered by the channel of STING, activates TFEB/TFE3 • STING stimulates cytoprotective upregulation of lysosomal biogenesis and autophagy • The cGAS-STING-TFEB axis is a primordial form of the antiviral DNA-sensing pathway Lv et al. discover TFEB/TFE3 as evolutionarily ancient effectors of the cGAS-STING DNA-sensing innate immune pathway. TFEB/TFE3 activation via the channel of STING integrates cytosolic DNA sensing, a general cellular stress-sensing mechanism, with lysosomal biogenesis and autophagy upregulation, conserved and general strategies for host defense and stress clearance. [ABSTRACT FROM AUTHOR]

Autophagosome (AP)–lysosome/vacuole fusion is one of the hallmarks of macroautophagy. Membrane features and changes during the fusion process have mostly been described using two-dimensional (2D) models with one AP and one lysosome/vacuole. The outer membrane (OM) of a closed mature AP has been suggested to fuse with the lysosomal/vacuolar membrane. However, the descriptions in some studies for fusion-related issues are questionable or incomplete. The correct membrane features of APs and lysosomes/vacuoles are the prerequisite for describing the fusion process. We searched the literature for representative membrane features of AP-related structures based on electron microscopy (EM) graphs of both animal and yeast cells and re-evaluated the findings. We also summarized the main 2D models describing the membrane changes during AP–lysosome/vacuole fusion in the literature. We used three-dimensional (3D) models to characterize the known and unknown membrane changes during and after fusion of the most plausible 2D models. The actual situation is more complex, since multiple lysosomes may fuse with the same AP in mammalian cells, multiple APs may fuse with the same vacuole in yeast cells, and in some mutant cells, phagophores (unclosed APs) fuse with lysosomes/vacuoles. This review discusses the membrane features and highly dynamic changes during AP (phagophore)–lysosome/vacuole fusion. The resulting information will improve the understanding of AP–lysosome/vacuole fusion and direct the future research on AP–lysosome/vacuole fusion and regeneration. [ABSTRACT FROM AUTHOR]

Excessive sulfur dioxide (SO₂) disturbs physiology of lysosomes causing diseases and threatening human health. A fluorescent probe has been regarded as one of the most attractive approaches, which is compatible with living cells and possesses high sensitivity. However, most of fluorescent probes' reaction sites are activated before they reach the destination. In this work, an acid‐activatable fluorescent probe PT1 was synthesized, characterized, and used for SO2 detection. The introduction of oxazolines in PT1 enables the intelligent response of probe to release the activation stie for SO2 derivatives through Michael addition upon exposure to acid. In vitro studies showed a remarkable selectivity of PT1 to SO₂ derivatives than other biothiols with a limit of detection as low as 62 nM. By using this acidic pH‐controlled fluorescence responsiveness to SO₂, precise spatiotemporal identification of lysosomal SO2 fluctuations has been successfully performed. Furthermore, probe PT1 can be applied for monitoring SO₂ derivatives in traditional Chinese medicines. [ABSTRACT FROM AUTHOR]

Autophagy is a preserved process in eukaryotes that allows large material degeneration and nutrient recovery via vacuoles or lysosomes in cytoplasm. Autophagy starts from the moment of induction during the formation of a phagophore. Degradation may occur in the autophagosomes even without fusion with lysosome or vacuole, particularly in microautophagosomes. This process is arbitrated by the conserved machinery of basic autophagy‐related genes (ATGs). In selective autophagy, specific materials are recruited by autophagosomes via receptors. Selective autophagy targets a vast variety of cellular components for degradation, i.e., old or damaged organelles, aggregates, and inactive or misfolded proteins. In optimal conditions, autophagy in plants ensures cellular homeostasis, proper plant growth, and fitness. Moreover, autophagy is essential during stress responses in plants and aids in survival of plants. Several biotic and abiotic stresses, i.e., pathogen infection, nutrient deficiency, plant senescence, heat stress, drought, osmotic stress, and hypoxia induce autophagy in plants. Cell death is not a stress, which induces autophagy but in contrast, sometimes it is a consequence of autophagy. In this way, autophagy plays a vital role in plant survival during harsh environmental conditions by maintaining nutrient concentration through elimination of useless cellular components. This review discussed the recent advances regarding regulatory functions of autophagy under normal and stressful conditions in plants and suggests future prospects in mitigating climate change. Autophagy in plants offers a viable way to increase plant resilience to climate change by increasing stress tolerance and nutrient usage efficiency. [ABSTRACT FROM AUTHOR]

Imidacloprid (IMI), a neonicotinoid pesticide, has been widely used due to its high efficiency against insect pests. However, its prolonged exposure may pose significant risks to non-target organisms, including mammals. Recent studies have raised concerns about its potential neurotoxicity, yet the underlying mechanisms remain poorly understood. This study aimed to assess the neurotoxic effects of chronic Imidacloprid exposure in Wistar rats, focusing on oxidative stress, mitochondrial dysfunction, and lysosomal disruption. Wistar rats were orally administered two doses of Imidacloprid (5 mg/kg and 50 mg/kg body weight) for three months. Neurotoxic effects were assessed by measuring key biochemical markers such as the enzymatic activities of catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), and glutathione S-transferase (GST). Non-enzymatic markers, including glutathione (GSH) levels and malondialdehyde (MDA) index, were also evaluated. Mitochondrial function was assessed by analyzing oxygen consumption, swelling, and membrane permeability and histopathological changes. Lysosomal stability was examined using the Neutral Red Retention Time (NRRT) assay. Neutral red is a dye that accumulates in the acidic environment of lysosomes. Healthy lysosomes retain the dye, while compromised lysosomes lose it, indicating destabilization. By measuring the amount of neutral red retained in lysosomes, the NRRT assay assesses lysosomal integrity. Lysosomal pH variations were also monitored to evaluate functional changes. Microscopic analysis provided insight into structural changes in lysosomes and other cell components. Lysosomal destabilization was further confirmed by morphological alterations observed through light microscopy, revealing a progressive, time-dependent degeneration of lysosomal structures, including lysosomal expansion, neutral red dye leakage, and cell rounding. These changes reflected a temporal evolution of lysosomal damage, progressing from minor structural disruptions to more severe alterations as exposure continued, observable at the microscopic level. During the study, clinical observations of intoxicated rats included symptoms such as lethargy, reduced activity levels, and impaired motor coordination. High-dose Imidacloprid exposure led to noticeable behavioral changes, including decreased exploratory behavior and altered grooming patterns. Additionally, signs of neurotoxic effects, such as tremors or ataxia, were observed in the rats exposed to the higher dose, reflecting the systemic impact of chronic pesticide exposure. The results revealed a significant decrease in the enzymatic activities of CAT, GPx, and SOD, accompanied by an increase in GST activity. A notable reduction in glutathione levels and a rise in MDA index were observed, indicating enhanced oxidative stress in the brain. Mitochondrial impairment was evidenced by disturbances in oxygen consumption, increased swelling, and altered membrane permeability. Lysosomal destabilization was confirmed by reduced retention of neutral red dye, structural changes in lysosomes, and a significant rise in lysosomal pH in the IMI-exposed groups. In addition, the histopathological features indicate that imidacloprid at the given dose and exposure duration may have caused notable neurotoxic effects in Wistar rat brain tissue. Chronic exposure to Imidacloprid induces oxidative stress, mitochondrial dysfunction, lysosomal disruption and histopathological alterations in the central nervous system of Wistar rats. These findings provide valuable insights into the neurotoxic mechanisms of neonicotinoid pesticides, highlighting the need for further research to understand the long-term effects of Imidacloprid exposure on mammalian health. [ABSTRACT FROM AUTHOR]

Lysosomes, crucial cellular organelles, undergo bidirectional transport along microtubules, mediated by motor proteins such as cytoplasmic dynein-1 (dynein) and various kinesins. While the kinesin-3 family member KIF1C is established in mediating anterograde vesicle transport, its role in lysosomal transport remains unclear. Our study reveals that KIF1C unexpectedly supports the retrograde transport of lysosomes, driven by dynein, and contributes to their perinuclear localization. Notably, while KIF1C facilitates this perinuclear positioning, its motor activity is not required and, instead, exerts an inhibitory effect on this process. Mechanistically, KIF1C facilitates this process by interacting with the dynein-activating adaptor Hook3, which associates with the lysosome-anchored protein RUFY3. This regulatory mechanism is critical for the efficient degradation of cargo in autophagic and endocytic pathways. Our findings identify an unconventional, non-motor role for KIF1C in activating dynein-driven lysosomal transport, expanding our understanding of its functional diversity in cellular trafficking. KIF1C mediates dynein-driven retrograde transport of lysosomes by interacting with Hook3 and RUFY3, independent of its motor activity. This ensures perinuclear lysosomal positioning, essential for efficient autophagic and endocytic degradation. [ABSTRACT FROM AUTHOR]

Lysosome-related organelles (LROs) are specialized lysosomes with cell type–specific roles in organismal homeostasis. Dysregulation of LROs leads to many human disorders, but the mechanisms underlying their biogenesis are not fully understood. Here, we identify a group of LYSMD proteins as evolutionarily conserved regulators of LROs. In Caenorhabditis elegans, mutations of LMD-2, a LysM domain–containing protein, reduce the levels of the Rab32 GTPase ortholog GLO-1 on intestine-specific LROs, the gut granules, leading to their abnormal enlargement and defective biogenesis. LMD-2 interacts with GLO-3, a subunit of GLO-1 guanine nucleotide exchange factor (GEF), thereby promoting GLO-1 activation. Mammalian homologs of LMD-2, LYSMD1, and LYSMD2 can functionally replace LMD-2 in C. elegans. In mammals, LYSMD1/2 physically interact with the HPS1 subunit of BLOC-3, the GEF of Rab32/38, thus promoting Rab32 activation. Inactivation of both LYSMD1 and LYSMD2 reduces Rab32 activation, causing melanosome enlargement and decreased melanin production in mouse melanoma cells. These findings provide important mechanistic insights into LRO biogenesis and functions. [ABSTRACT FROM AUTHOR]

Lipid droplets (LDs) and lysosomes are vital organelles that play crucial roles in various physiological and pathological processes. However, simultaneous two-color visualization of these two organelles using a single probe for cell homeostasis monitoring remains a challenge due to the lack of rational design strategies. To address this issue, we have developed an aggregation-induced emission (AIE) fluorescent probe named TPE-NDI-Mor with an electron donor (D)–acceptor (A) structure, which can stain both LDs and lysosomes with high selectivity through green and red fluorescence imaging, respectively. A detailed mechanism study revealed that TPE-NDI-Mor, with a twisted intramolecular charge transfer (TICT) effect, shows a high affinity for a polar microenvironment. Additionally, the probe also demonstrates good stability, high anti-interference performance and a large Stokes shift, making it suitable for visualizing cell homeostasis and further disease diagnosis by tracking the dynamic changes of LDs and lysosomes. [ABSTRACT FROM AUTHOR]

Viscosity and hypoxia, as microenvironment parameters, play important roles in maintaining normal biological processes and homeostasis. Therefore, simultaneous and sensitive detection of these elements with simple and effective methods could offer precise information in biology. Here, we report a two-site lysosome-targeting fluorescent probe, NVP, for monitoring viscosity and nitroreductase with dual emission channels (emission shift is 86 nm). The NVP probe has displayed highly sensitive and selective responses towards viscosity and nitroreductase, respectively. Significantly, the fluctuations of viscosity and NTR have been detected in vitro and in vivo. We expect that the dual-responsive fluorescent NVP probe will become a potential molecular tool for the exploration of deeper functions of viscosity and hypoxia. [ABSTRACT FROM AUTHOR]

Neurons are dependent on efficient quality control mechanisms to maintain cellular homeostasis and function due to their polarization and long-life span. Autophagy is a lysosomal degradative pathway that provides nutrients during starvation and recycles damaged and/or aged proteins and organelles. In neurons, autophagosomes constitutively form in distal axons and at synapses and are trafficked retrogradely to the cell soma to fuse with lysosomes for cargo degradation. How the neuronal autophagy pathway is organized and controlled remains poorly understood. Several presynaptic endocytic proteins have been shown to regulate both synaptic vesicle recycling and autophagy. Here, by combining electron, fluorescence, and live imaging microscopy with biochemical analysis, we show that the neuron-specific protein APache, a presynaptic AP-2 interactor, functions in neurons as an important player in the autophagy process, regulating the retrograde transport of autophagosomes. We found that APache colocalizes and co-traffics with autophagosomes in primary cortical neurons and that induction of autophagy by mTOR inhibition increases LC3 and APache protein levels at synaptic boutons. APache silencing causes a blockade of autophagic flux preventing the clearance of p62/SQSTM1, leading to a severe accumulation of autophagosomes and amphisomes at synaptic terminals and along neurites due to defective retrograde transport of TrkB-containing signaling amphisomes along the axons. Together, our data identify APache as a regulator of the autophagic cycle, potentially in cooperation with AP-2, and hypothesize that its dysfunctions contribute to the early synaptic impairments in neurodegenerative conditions associated with impaired autophagy. [ABSTRACT FROM AUTHOR]

Research into the pathophysiology of Parkinson's disease (PD) is a fast-paced pursuit, with new findings about PD and other synucleinopathies being made each year. The involvement of various lysosomal proteins, such as TFEB, TMEM175, GBA, and LAMP1/2, marks the rising awareness about the importance of lysosomes in PD and other neurodegenerative disorders. This, along with recent developments regarding the involvement of microglia and the immune system in neurodegenerative diseases, has brought about a new era in neurodegeneration: the role of proinflammatory cytokines on the nervous system, and their downstream effects on mitochondria, lysosomal degradation, and autophagy. More effort is needed to understand the interplay between neuroimmunology and disease mechanisms, as many of the mechanisms remain enigmatic. a-synuclein, a key protein in PD and the main component of Lewy bodies, sits at the nexus between lysosomal degradation, autophagy, cellular stress, neuroimmunology, PD pathophysiology, and disease progression. This review revisits some fundamental knowledge about PD while capturing some of the latest trends in PD research, specifically as it relates to asynuclein. [ABSTRACT FROM AUTHOR]

Autophagy is the process by which damaged regions of the cytoplasm and intracellular pathogens are degraded. This mechanism often serves an adaptive role in cells, enhancing their survival. It plays a direct or indirect role in the development of various pathological conditions within the body. This phenomenon is common in various malignant diseases, where autophagy is associated with the resistance of transformed cells to chemotherapy. Conversely, abnormal activation of autophagy can trigger cell death, a process often seen in neurodegenerative conditions. Given that dysregulation of autophagy is associated with the progression of numerous pathological conditions, this is of significant interest to the developers of drugs that can effectively modulate autophagy for both basic research and clinical applications. Here, we provide a brief description of the mechanism of macroautophagy, the most prevalent form of autophagy identified in humans. We also discuss the clinical potential of drugs that can modulate autophagy, highlighting their use in combating diseases associated with direct or indirect dysregulation of this essential process. [ABSTRACT FROM AUTHOR]

Lysosomal compartments control the clearance of cell‐own material (autophagy) or of material that cells endocytose from the external environment (heterophagy) to warrant supply of nutrients, to eliminate macromolecules or parts of organelles present in excess, aged, or containing toxic material. Inherited or sporadic mutations in lysosomal proteins and enzymes may hamper their folding in the endoplasmic reticulum (ER) and their lysosomal transport via the Golgi compartment, resulting in lysosomal dysfunction and storage disorders. Defective cargo delivery to lysosomal compartments is harmful to cells and organs since it causes accumulation of toxic compounds and defective organellar homeostasis. Assessment of resident proteins and cargo fluxes to the lysosomal compartments is crucial for the mechanistic dissection of intracellular transport and catabolic events. It might be combined with high‐throughput screenings to identify cellular, chemical, or pharmacological modulators of these events that may find therapeutic use for autophagy‐related and lysosomal storage disorders. Here, discuss qualitative, quantitative and chronologic monitoring of autophagic, heterophagic and lysosomal protein trafficking in fixed and live cells, which relies on fluorescent single and tandem reporters used in combination with biochemical, flow cytometry, light and electron microscopy approaches implemented by artificial intelligence‐based technology. [ABSTRACT FROM AUTHOR]

Abnormal protein accumulations in the brain are linked to aging and the pathogenesis of dementia of various types, including Alzheimer's disease. These accumulations can be reduced by cell indigenous mechanisms. Among these is autophagy, whereby proteins are transferred to lysosomes for degradation. Autophagic dysfunction hampers the elimination of pathogenic protein aggregations that contribute to cell death. We had observed that the adhesion molecule L1 interacts with microtubule-associated protein 1 light-chain 3 (LC3), which is needed for autophagy substrate selection. L1 increases cell survival in an LC3-dependent manner via its extracellular LC3 interacting region (LIR). L1 also interacts with Aβ and reduces the Aβ plaque load in an AD model mouse. Based on these results, we investigated whether L1 could contribute to autophagy of aggregated Aβ and its clearance. We here show that L1 interacts with autophagy-related protein 12 (ATG12) via its LIR domain, whereas interaction with ubiquitin-binding protein p62/SQSTM1 does not depend on LIR. Aβ, bound to L1, is carried to the autophagosome leading to Aβ elimination. Showing that the mitophagy-related L1-70 fragment is ubiquitinated, we expect that the p62/SQSTM1 pathway also contributes to Aβ elimination. We propose that enhancing L1 functions may contribute to therapy in humans. [ABSTRACT FROM AUTHOR]

In the present study, we demonstrated that the introduction of a 1,4-diethyl-1,2,3,4-tetrahydroquinoxalin moiety into the arylidene part of GFP chromophore-derived compounds results in the formation of environment-sensitive fluorogens. The rationally designed and synthesized compounds exhibit remarkable solvent- and pH-dependence in fluorescence intensity. The solvent-dependent variation in fluorescence quantum yield makes it possible to use some of the proposed compounds as polarity sensors suitable for selective endoplasmic reticulum fluorescent labeling in living cells. Moreover, the pH-dependent emission intensity variation of other fluorogens makes them selective fluorescent labels for the lysosomes in living cells. [ABSTRACT FROM AUTHOR]

Lipofuscin is indigestible garbage that accumulates in the autophagic vesicles and cytosol of postmitotic cells with age. Drs. Brunk and Terman postulated that lipofuscin accumulation is the main or at least a major driving factor in aging. They even posited that the evolution of memory is the reason why we get lipofuscin at all, as stable synaptic connections must be maintained over time, meaning that the somas of neurons must also remain in the same locale. In other words, they cannot dilute out their garbage over time through cell division. Mechanistically, their position certainly makes sense given that rendering a large percentage of a postmitotic cell's lysosomes useless must almost certainly negatively affect that cell and the surrounding microenvironment. It may be the case that lipofuscin accumulation is the main issue with regard to current age-related disease. Degradation in situ may be an insurmountable task currently. However, a method of systemic lipofuscin removal is discussed herein. [ABSTRACT FROM AUTHOR]