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Through a comprehensive analysis of organellar markers in mouse models of Alzheimer’s disease, we document a massive accumulation of lysosome-like organelles at amyloid plaques and establish that the majority of these organelles reside within swollen axons that contact the amyloid deposits. This close spatial relationship between axonal lysosome accumulation and extracellular amyloid aggregates was observed from the earliest stages of β-amyloid deposition. Notably, we discovered that lysosomes that accumulate in such axons are lacking in multiple soluble luminal proteases and thus are predicted to be unable to efficiently degrade proteinaceous cargos. Of relevance to Alzheimer’s disease, β-secretase (BACE1), the protein that initiates amyloidogenic processing of the amyloid precursor protein and which is a substrate for these proteases, builds up at these sites. Furthermore, through a comparison between the axonal lysosome accumulations at amyloid plaques and neuronal lysosomes of the wild-type brain, we identified a similar, naturally occurring population of lysosome-like organelles in neuronal processes that is also defined by its low luminal protease content. In conjunction with emerging evidence that the lysosomal maturation of endosomes and autophagosomes is coupled to their retrograde transport, our results suggest that extracellular β-amyloid deposits cause a local impairment in the retrograde axonal transport of lysosome precursors, leading to their accumulation and a blockade in their further maturation. This study both advances understanding of Alzheimer’s disease brain pathology and provides new insights into the subcellular organization of neuronal lysosomes that may have broader relevance to other neurodegenerative diseases with a lysosomal component to their pathology.

Ferroptosis is a newly defined programmed cell death process with the hallmark of the accumulation of iron‐dependent lipid peroxides. The term was first coined in 2012 by the Stockwell Lab, who described a unique type of cell death induced by the small molecules erastin or RSL3. Ferroptosis is distinct from other already established programmed cell death and has unique morphological and bioenergetic features. The physiological role of ferroptosis during development has not been well characterized. However, ferroptosis shows great potentials during the cancer therapy. Great progress has been made in exploring the mechanisms of ferroptosis. In this review, we focus on the molecular mechanisms of ferroptosis, the small molecules functioning in ferroptosis initiation and ferroptosis sensitivity in different cancers. We are also concerned with the new arising questions in this particular research area that remains unanswered.

Mitochondrial health is critical for skeletal muscle function and is improved by exercise training through both mitochondrial biogenesis and removal of damaged/dysfunctional mitochondria via mitophagy. The mechanisms underlying exercise-induced mitophagy have not been fully elucidated. Here, we show that acute treadmill running in mice causes mitochondrial oxidative stress at 3–12 h and mitophagy at 6 h post-exercise in skeletal muscle. These changes were monitored using a novel fluorescent reporter gene, pMitoTimer , that allows assessment of mitochondrial oxidative stress and mitophagy in vivo, and were preceded by increased phosphorylation of AMP activated protein kinase (Ampk) at tyrosine 172 and of unc-51 like autophagy activating kinase 1 (Ulk1) at serine 555. Using mice expressing dominant negative and constitutively active Ampk in skeletal muscle, we demonstrate that Ulk1 activation is dependent on Ampk. Furthermore, exercise-induced metabolic adaptation requires Ulk1. These findings provide direct evidence of exercise-induced mitophagy and demonstrate the importance of Ampk-Ulk1 signaling in skeletal muscle. Exercise is associated with biogenesis and removal of dysfunctional mitochondria. Here the authors use a mitochondrial reporter gene to demonstrate the occurrence of mitophagy following exercise in mice, and show this is dependent on AMPK and ULK1 signaling.

Pompe disease, a rare lysosomal storage disease caused by deficiency of the lysosomal acid α-glucosidase (GAA), is characterized by glycogen accumulation, triggering severe secondary cellular damage and resulting in progressive motor handicap and premature death. Numerous disease-causing mutations in the gaa gene have been reported, but the structural effects of the pathological variants were unknown. Here we present the high-resolution crystal structures of recombinant human GAA (rhGAA), the standard care of Pompe disease. These structures portray the unbound form of rhGAA and complexes thereof with active site-directed inhibitors, providing insight into substrate recognition and the molecular framework for the rationalization of the deleterious effects of disease-causing mutations. Furthermore, we report the structure of rhGAA in complex with the allosteric pharmacological chaperone N-acetylcysteine, which reveals the stabilizing function of this chaperone at the structural level. Pompe disease is caused by mutations in lysosomal acid α-glucosidase (GAA) and patients are being treated with recombinant human α-glucosidase (rhGAA). Here the authors present the crystal structures of rhGAA and its complexes with inhibitors and a pharmacological chaperone, which is important for drug development.

Lysosomes are the main proteolytic compartments of mammalian cells comprising of a battery of hydrolases. Lysosomes dispose and recycle extracellular or intracellular macromolecules by fusing with endosomes or autophagosomes through specific waste clearance processes such as chaperone-mediated autophagy or microautophagy. The proteolytic end product is transported out of lysosomes via transporters or vesicular membrane trafficking. Recent studies have demonstrated lysosomes as a signaling node which sense, adapt and respond to changes in substrate metabolism to maintain cellular function. Lysosomal dysfunction not only influence pathways mediating membrane trafficking that culminate in the lysosome but also govern metabolic and signaling processes regulating protein sorting and targeting. In this review, we describe the current knowledge of lysosome in influencing sorting and nutrient signaling. We further present a mechanistic overview of intra-lysosomal processes, along with extra-lysosomal processes, governing lysosomal fusion and fission, exocytosis, positioning and membrane contact site formation. This review compiles existing knowledge in the field of lysosomal biology by describing various lysosomal events necessary to maintain cellular homeostasis facilitating development of therapies maintaining lysosomal function.

The polypeptide SPAR is encoded by a long non-coding RNA, localizes to the late endosome and lysosome, and regulates muscle regeneration by inhibiting mTORC1. SPAR polypeptide is a product of a 'non-coding' RNA By definition, long non-coding RNAs (lncRNAs) are not translated into proteins. But there are a few exceptions to this rule, raising interest in the biological function of the curious polypeptides that emerge from lncRNAs. Pier Paolo Pandolfi and co-workers discover and functionally characterize one such polypeptide, which they name SPAR ('small regulatory polypeptide of amino acid response'). They find that, within cells, SPAR localizes to the late endosome/lysosome and interacts with the lysosomal v-ATPase to negatively regulate mTORC1 activation by amino acids. At an organismal level, SPAR is highly expressed in a subset of tissues. But on examining a SPAR-knockout mouse, the authors find that, upon injury, SPAR specifically regulates muscle regeneration in an mTORC1-dependent manner. Although long non-coding RNAs (lncRNAs) are non-protein-coding transcripts by definition, recent studies have shown that a fraction of putative small open reading frames within lncRNAs are translated 1 , 2 , 3 . However, the biological significance of these hidden polypeptides is still unclear. Here we identify and functionally characterize a novel polypeptide encoded by the lncRNA LINC00961. This polypeptide is conserved between human and mouse, is localized to the late endosome/lysosome and interacts with the lysosomal v-ATPase to negatively regulate mTORC1 activation. This regulation of mTORC1 is specific to activation of mTORC1 by amino acid stimulation, rather than by growth factors. Hence, we termed this polypeptide ‘small regulatory polypeptide of amino acid response’ (SPAR). We show that the SPAR-encoding lncRNA is highly expressed in a subset of tissues and use CRISPR/Cas9 engineering to develop a SPAR-polypeptide-specific knockout mouse while maintaining expression of the host lncRNA. We find that the SPAR-encoding lncRNA is downregulated in skeletal muscle upon acute injury, and using this in vivo model we establish that SPAR downregulation enables efficient activation of mTORC1 and promotes muscle regeneration. Our data provide a mechanism by which mTORC1 activation may be finely regulated in a tissue-specific manner in response to injury, and a paradigm by which lncRNAs encoding small polypeptides can modulate general biological pathways and processes to facilitate tissue-specific requirements, consistent with their restricted and highly regulated expression profile.

Lysosomes are acidic organelles essential for degradation and cellular homoeostasis and recently lysosomes have been shown as signaling hub to respond to the intra and extracellular changes (e.g. amino acid availability). Compounds including pharmaceutical drugs that are basic and lipophilic will become sequestered inside lysosomes (lysosomotropic). How cells respond to the lysosomal stress associated with lysosomotropism is not well characterized. Our goal is to assess the lysosomal changes and identify the signaling pathways that involve in the lysosomal changes. Eight chemically diverse lysosomotropic drugs from different therapeutic areas were subjected to the evaluation using the human adult retinal pigmented epithelium cell line, ARPE-19. All lysosomotropic drugs tested triggered lysosomal activation demonstrated by increased lysosotracker red (LTR) and lysosensor green staining, increased cathepsin activity, and increased LAMP2 staining. However, tested lysosomotropic drugs also prompted lysosomal dysfunction exemplified by intracellular and extracellular substrate accumulation including phospholipid, SQSTM1/p62, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) and opsin. Lysosomal activation observed was likely attributed to lysosomal dysfunction, leading to compensatory responses including nuclear translocation of transcriptional factors TFEB, TFE3 and MITF. The adaptive changes are protective to the cells under lysosomal stress. Mechanistic studies implicate calcium and mTORC1 modulation involvement in the adaptive changes. These results indicate that lysosomotropic compounds could evoke a compensatory lysosomal biogenic response but with the ultimate consequence of lysosomal functional impairment. This work also highlights a pathway of response to lysosomal stress and evidences the role of TFEB, TFE3 and MITF in the stress response.

mTOR complex 1 (mTORC1) regulates cell growth and metabolism in response to multiple environmental cues. Nutrients signal via the Rag guanosine triphosphatases (GTPases) to promote the localization of mTORC1 to the lysosomal surface, its site of activation. We identified SAMTOR, a previously uncharacterized protein, which inhibits mTORC1 signaling by interacting with GATOR1, the GTPase activating protein (GAP) for RagA/B. We found that the methyl donor -adenosylmethionine (SAM) disrupts the SAMTOR-GATOR1 complex by binding directly to SAMTOR with a dissociation constant of approximately 7 μM. In cells, methionine starvation reduces SAM levels below this dissociation constant and promotes the association of SAMTOR with GATOR1, thereby inhibiting mTORC1 signaling in a SAMTOR-dependent fashion. Methionine-induced activation of mTORC1 requires the SAM binding capacity of SAMTOR. Thus, SAMTOR is a SAM sensor that links methionine and one-carbon metabolism to mTORC1 signaling.

Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and transferred to the Golgi complex by interaction with the Batten disease protein CLN8 (ceroid lipofuscinosis, neuronal, 8). Here we investigated the relationship of this pathway with CLN6, an ER-associated protein of unknown function that is defective in a different Batten disease subtype. Experiments focused on protein interaction and trafficking identified CLN6 as an obligate component of a CLN6-CLN8 complex (herein referred to as EGRESS: ER-to-Golgi relaying of enzymes of the lysosomal system), which recruits lysosomal enzymes at the ER to promote their Golgi transfer. Mutagenesis experiments showed that the second luminal loop of CLN6 is required for the interaction of CLN6 with the enzymes but dispensable for interaction with CLN8. In vitro and in vivo studies showed that CLN6 deficiency results in inefficient ER export of lysosomal enzymes and diminished levels of the enzymes at the lysosome. Mice lacking both CLN6 and CLN8 did not display aggravated pathology compared with the single deficiencies, indicating that the EGRESS complex works as a functional unit. These results identify CLN6 and the EGRESS complex as key players in lysosome biogenesis and shed light on the molecular etiology of Batten disease caused by defects in CLN6.

Key Points Mast cell secretory granules are lysosome-like organelles that contain a large panel of preformed bioactive constituents, including lysosomal hydrolases, amines, cytokines, proteases and proteoglycans. Mast cell granule biogenesis is initiated in the trans -Golgi and is followed by extensive maturation processes, which are strongly dependent on proteoglycans of the serglycin type. When mast cells are activated — for example, in the context of an allergic reaction — degranulation occurs, whereby the bioactive granule compounds are expelled to the cell exterior and can cause a powerful inflammatory reaction. Mast cell degranulation is a highly complex process that involves a large number of kinases, adaptor molecules and second messengers, as well as extensive membrane fusion events, which are mediated by numerous factors. Mast cells are implicated in many pathological conditions and their effects in such settings are often mediated by the compounds that are secreted from the mast cell granules. Recent research has indicated that the mast cell-specific proteases — chymases, tryptases and carboxypeptidase A3 — account for many of the functions that are ascribed to mast cells. Mast cell granules contain numerous immunomodulatory compounds that are rapidly released in various inflammatory settings. Here, the authors describe the composition, biogenesis and maturation of mast cell granules, and they focus on the biological effects of the mast cell proteases that are released. Mast cells are important effector cells of the immune system and recent studies show that they have immunomodulatory roles in diverse processes in both health and disease. Mast cells are distinguished by their high content of electron-dense secretory granules, which are filled with large amounts of preformed and pre-activated immunomodulatory compounds. When appropriately activated, mast cells undergo degranulation, a process by which these preformed granule compounds are rapidly released into the surroundings. In many cases, the effects that mast cells have on an immune response are closely associated with the biological actions of the granule compounds that they release, as exemplified by the recent studies showing that mast cell granule proteases account for many of the protective and detrimental effects of mast cells in various inflammatory settings. In this Review, we discuss the current knowledge of mast cell secretory granules.