A CLN6-CLN8 complex recruits lysosomal enzymes at the ER for Golgi transfer.

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.

Combination of whole exome sequencing and animal modeling identifies TMPRSS9 as a candidate gene for autism spectrum disorder.

Autism spectrum disorders are associated with some degree of developmental regression in up to 30% of all cases. Rarely, however, is the regression so extreme that a developmentally advanced young child would lose almost all ability to communicate and interact with her surroundings. We applied trio whole exome sequencing to a young woman who experienced extreme developmental regression starting at 2.5 years of age and identified compound heterozygous nonsense mutations in TMPRSS9, which encodes for polyserase-1, a transmembrane serine protease of poorly understood physiological function. Using semiquantitative polymerase chain reaction, we showed that Tmprss9 is expressed in various mouse tissues, including the brain. To study the consequences of TMPRSS9 loss of function on the mammalian brain, we generated a knockout mouse model. Through a battery of behavioral assays, we found that Tmprss9-/- mice showed decreased social interest and social recognition. We observed a borderline recognition memory deficit by novel object recognition in aged Tmprss9-/- female mice, but not in aged Tmprss9-/- male mice or younger adult Tmprss9-/- mice in both sexes. This study provides evidence to suggest that loss of function variants in TMPRSS9 are related to an autism spectrum disorder. However, the identification of more individuals with similar phenotypes and TMPRSS9 loss of function variants is required to establish a robust gene-disease relationship.

mTOR and autophagy pathways are dysregulated in murine and human models of Schaaf-Yang syndrome.

MAGEL2 is a maternally imprinted, paternally expressed gene, located in the Prader-Willi region of human chromosome 15. Pathogenic variants in the paternal copy of MAGEL2 cause Schaaf-Yang syndrome (SHFYNG), a neurodevelopmental disorder related to Prader-Willi syndrome (PWS). Patients with SHFYNG, like PWS, manifest neonatal hypotonia, feeding difficulties, hypogonadism, intellectual disability and sleep apnea. However, individuals with SHFYNG have joint contractures, greater cognitive impairment, and higher prevalence of autism than seen in PWS. Additionally, SHFYNG is associated with a lower prevalence of hyperphagia and obesity than PWS. Previous studies have shown that truncating variants in MAGEL2 lead to SHFYNG. However, the molecular pathways involved in manifestation of the SHFYNG disease phenotype are still unknown. Here we show that a Magel2 null mouse model and fibroblast cell lines from individuals with SHFYNG exhibit increased expression of mammalian target of rapamycin (mTOR) and decreased autophagy. Additionally, we show that SHFYNG induced pluripotent stem cell (iPSC)-derived neurons exhibit impaired dendrite formation. Alterations in SHFYNG patient fibroblast lines and iPSC-derived neurons are rescued by treatment with the mTOR inhibitor rapamycin. Collectively, our findings identify mTOR as a potential target for the development of pharmacological treatments for SHFYNG.

Abnormal glycogen storage in tuberous sclerosis complex caused by impairment of mTORC1-dependent and -independent signaling pathways.

Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome that causes tumor formation in multiple organs. TSC is caused by inactivating mutations in the genes encoding TSC1/2, negative regulators of the mammalian target of rapamycin complex 1 (mTORC1). Diminished TSC function is associated with excess glycogen storage, but the causative mechanism is unknown. By studying human and mouse cells with defective or absent TSC2, we show that complete loss of TSC2 causes an increase in glycogen synthesis through mTORC1 hyperactivation and subsequent inactivation of glycogen synthase kinase 3ß (GSK3ß), a negative regulator of glycogen synthesis. Specific TSC2 pathogenic mutations, however, result in elevated glycogen levels with no changes in mTORC1 or GSK3ß activities. We identify mTORC1-independent lysosomal depletion and impairment of autophagy as the driving causes underlying abnormal glycogen storage in TSC irrespective of the underlying mutation. The defective autophagic degradation of glycogen is associated with abnormal ubiquitination and degradation of essential proteins of the autophagy-lysosome pathway, such as LC3 and lysosomal associated membrane protein 1 and 2 (LAMP1/2) and is restored by the combined use of mTORC1 and Akt pharmacological inhibitors. In complementation to current models that place mTORC1 as the central therapeutic target for TSC pathogenesis, our findings identify mTORC1-independent pathways that are dysregulated in TSC and that should therefore be taken into account in the development of a therapeutic treatment.

Lysosome biogenesis in health and disease.

This review focuses on the pathways that regulate lysosome biogenesis and that are implicated in numerous degenerative storage diseases, including lysosomal storage disorders and late-onset neurodegenerative diseases. Lysosomal proteins are synthesized in the endoplasmic reticulum and trafficked to the endolysosomal system through the secretory route. Several receptors have been characterized that execute post-Golgi trafficking of lysosomal proteins. Some of them recognize their cargo proteins based on specific amino acid signatures, others based on a particular glycan modification that is exclusively found on lysosomal proteins. Nearly all receptors serving lysosome biogenesis are under the transcriptional control of transcription factor EB (TFEB), a master regulator of the lysosomal system. TFEB coordinates the expression of lysosomal hydrolases, lysosomal membrane proteins, and autophagy proteins in response to pathways sensing lysosomal stress and the nutritional conditions of the cell among other stimuli. TFEB is primed for activation in lysosomal storage disorders but surprisingly its function is impaired in some late-onset neurodegenerative storage diseases like Alzheimer's and Parkinson's, because of specific detrimental interactions that limit TFEB expression or activation. Thus, disrupted TFEB function presumably plays a role in the pathogenesis of these diseases. Multiple studies in animal models of degenerative storage diseases have shown that exogenous expression of TFEB and pharmacological activation of endogenous TFEB attenuate disease phenotypes. These results highlight TFEB-mediated enhancement of lysosomal biogenesis and function as a candidate strategy to counteract the progression of these diseases. This article is part of the Special Issue "Lysosomal Storage Disorders".

Arachidonic Acid Induces the Migration of MDA-MB-231 Cells by Activating Raft-associated Leukotriene B4 Receptors.

BACKGROUND: The migration of tumor cells is critical in spreading cancers through the lymphatic nodes and circulatory systems. Although arachidonic acid (AA) and its soluble metabolites have been shown to induce the migration of breast and colon cancer cells, the mechanism by which it induces such migration has not been fully understood. OBJECTIVE: The effect of AA on migratory responses of the MDA-MB-231 cell line (a triple-negative breast cancer cell) was examined and compared with MCF-7 (estrogen-receptor positive) breast cancer cells to elucidate the mechanism of AA-induced migration. METHODS: Migrations of breast cancer cells were examined with the help of wound-healing assays. AA-induced eicosanoid synthesis was monitored by RP-HPLC. Cellular localizations of lipoxygenase and lipid rafts were assessed by immunoblot and confocal microscopy. RESULTS: AA treatment stimulated the synthesis of leukotriene B4 (LTB4) and HETE-8, but lowered the levels of prostaglandin E2 (PGE2), prostaglandin D2 (PGD2), and HETE-5 in MDA-MB-231 cells. Further analysis indicated that AA increased the expression of 5-lipoxygenase (5-LOX) in this cell line and inhibiting its expression by small molecule inhibitors lowered the production of LTB4 and reduced migration. In contrast, MCF-7 cells did not show any appreciable changes in eicosanoid synthesis, 5-LOX expression, or cellular migration. CONCLUSION: Our results suggest that AA treatment activates the BLT1 receptor (present in membrane microdomains) and stimulates the synthesis of LTB4 production, which is likely to be associated with the migration of MDA-MB-231 cells.

Src regulates amino acid-mediated mTORC1 activation by disrupting GATOR1-Rag GTPase interaction.

The mechanistic target of rapamycin complex 1 (mTORC1) regulates cell survival and autophagy, and its activity is regulated by amino acid availability. Rag GTPase-GATOR1 interactions inhibit mTORC1 in the absence of amino acids, and GATOR1 release and activation of RagA/B promotes mTORC1 activity in the presence of amino acids. However, the factors that play a role in Rag-GATOR1 interaction are still poorly characterized. Here, we show that the tyrosine kinase Src is crucial for amino acid-mediated activation of mTORC1. Src acts upstream of the Rag GTPases by promoting dissociation of GATOR1 from the Rags, thereby determining mTORC1 recruitment and activation at the lysosomal surface. Accordingly, amino acid-mediated regulation of Src/mTORC1 modulates autophagy and cell size expansion. Finally, Src hyperactivation overrides amino acid signaling in the activation of mTORC1. These results shed light on the mechanisms underlying pathway dysregulation in many cancer types.

NADPH oxidase mediates microtubule alterations and diaphragm dysfunction in dystrophic mice.

Skeletal muscle from mdx mice is characterized by increased Nox2 ROS, altered microtubule network, increased muscle stiffness, and decreased muscle/respiratory function. While microtubule de-tyrosination has been suggested to increase stiffness and Nox2 ROS production in isolated single myofibers, its role in altering tissue stiffness and muscle function has not been established. Because Nox2 ROS production is upregulated prior to microtubule network alterations and ROS affect microtubule formation, we investigated the role of Nox2 ROS in diaphragm tissue microtubule organization, stiffness and muscle/respiratory function. Eliminating Nox2 ROS prevents microtubule disorganization and reduces fibrosis and muscle stiffness in mdx diaphragm. Fibrosis accounts for the majority of variance in diaphragm stiffness and decreased function, implicating altered extracellular matrix and not microtubule de-tyrosination as a modulator of diaphragm tissue function. Ultimately, inhibiting Nox2 ROS production increased force and respiratory function in dystrophic diaphragm, establishing Nox2 as a potential therapeutic target in Duchenne muscular dystrophy.

Corrigendum: mTORC1-independent TFEB activation via Akt inhibition promotes cellular clearance in neurodegenerative storage diseases.

This corrects the article DOI: 10.1038/ncomms14338.

Inhibition of ERK1/2 Restores GSK3ß Activity and Protein Synthesis Levels in a Model of Tuberous Sclerosis.

Tuberous sclerosis (TS) is a multi-organ autosomal dominant disorder that is best characterized by neurodevelopmental deficits and the presence of benign tumors. TS pathology is caused by mutations in tuberous sclerosis complex (TSC) genes and is associated with insulin resistance, decreased glycogen synthase kinase 3ß (GSK3ß) activity, activation of the mammalian target of rapamycin complex 1 (mTORC1), and subsequent increase in protein synthesis. Here, we show that extracellular signal-regulated kinases (ERK1/2) respond to insulin stimulation and integrate insulin signaling to phosphorylate and thus inactivate GSK3ß, resulting in increased protein synthesis that is independent of Akt/mTORC1 activity. Inhibition of ERK1/2 in Tsc2 -/- cells-a model of TS-rescues GSK3ß activity and protein synthesis levels, thus highlighting ERK1/2 as a potential therapeutic target for the treatment of TS.

mTORC1-independent TFEB activation via Akt inhibition promotes cellular clearance in neurodegenerative storage diseases.

Neurodegenerative diseases characterized by aberrant accumulation of undigested cellular components represent unmet medical conditions for which the identification of actionable targets is urgently needed. Here we identify a pharmacologically actionable pathway that controls cellular clearance via Akt modulation of transcription factor EB (TFEB), a master regulator of lysosomal pathways. We show that Akt phosphorylates TFEB at Ser467 and represses TFEB nuclear translocation independently of mechanistic target of rapamycin complex 1 (mTORC1), a known TFEB inhibitor. The autophagy enhancer trehalose activates TFEB by diminishing Akt activity. Administration of trehalose to a mouse model of Batten disease, a prototypical neurodegenerative disease presenting with intralysosomal storage, enhances clearance of proteolipid aggregates, reduces neuropathology and prolongs survival of diseased mice. Pharmacological inhibition of Akt promotes cellular clearance in cells from patients with a variety of lysosomal diseases, thus suggesting broad applicability of this approach. These findings open new perspectives for the clinical translation of TFEB-mediated enhancement of cellular clearance in neurodegenerative storage diseases.

Redox regulation of autophagy in skeletal muscle.

Autophagy is a cellular degradative pathway that involves the delivery of cytoplasmic components, including proteins and organelles, to the lysosome for degradation. Autophagy is implicated in the maintenance of skeletal muscle; increased autophagy leads to muscle atrophy while decreased autophagy leads to degeneration and weakness. A growing body of work suggests that reactive oxygen species (ROS) are important cellular signal transducers controlling autophagy. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and mitochondria are major sources of ROS generation in skeletal muscle that are likely regulating autophagy through different signaling cascades based on localization of the ROS signals. This review aims to provide insight into the redox control of autophagy in skeletal muscle. Understanding the mechanisms by which ROS regulate autophagy will provide novel therapeutic targets for skeletal muscle diseases.

NADPH oxidase promotes Parkinsonian phenotypes by impairing autophagic flux in an mTORC1-independent fashion in a cellular model of Parkinson's disease.

Oxidative stress and aberrant accumulation of misfolded proteins in the cytosol are key pathological features associated with Parkinson's disease (PD). NADPH oxidase (Nox2) is upregulated in the pathogenesis of PD; however, the underlying mechanism(s) of Nox2-mediated oxidative stress in PD pathogenesis are still unknown. Using a rotenone-inducible cellular model of PD, we observed that a short exposure to rotenone (0.5 µM) resulted in impaired autophagic flux through activation of a Nox2 dependent Src/PI3K/Akt axis, with a consequent disruption of a Beclin1-VPS34 interaction that was independent of mTORC1 activity. Sustained exposure to rotenone at a higher dose (10 µM) decreased mTORC1 activity; however, autophagic flux was still impaired due to dysregulation of lysosomal activity with subsequent induction of the apoptotic machinery. Cumulatively, our results highlight a complex pathogenic mechanism for PD where short- and long-term oxidative stress alters different signaling pathways, ultimately resulting in anomalous autophagic activity and disease phenotype. Inhibition of Nox2-dependent oxidative stress attenuated the impaired autophagy and cell death, highlighting the importance and therapeutic potential of these pathways for treating patients with PD.

Nuclear glutaredoxin 3 is critical for protection against oxidative stress-induced cell death.

Mammalian glutaredoxin 3 (Grx3) has been shown to be critical in maintaining redox homeostasis and regulating cell survival pathways in cancer cells. However, the regulation of Grx3 is not fully understood. In the present study, we investigate the subcellular localization of Grx3 under normal growth and oxidative stress conditions. Both fluorescence imaging of Grx3-RFP fusion and Western blot analysis of cellular fractionation indicate that Grx3 is predominantly localized in the cytoplasm under normal growth conditions, whereas under oxidizing conditions, Grx3 is translocated into and accumulated in the nucleus. Grx3 nuclear accumulation was reversible in a redox-dependent fashion. Further analysis indicates that neither the N-terminal Trx-like domain nor the two catalytic cysteine residues in the active CGFS motif of Grx3 are involved in its nuclear translocation. Decreased levels of Grx3 render cells susceptible to cellular oxidative stress, whereas overexpression of nuclear-targeted Grx3 is sufficient to suppress cells' sensitivity to oxidant treatments and reduce reactive oxygen species production. These findings provide novel insights into the regulation of Grx3, which is crucial for cell survival against environmental insults.

Src-dependent impairment of autophagy by oxidative stress in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal degenerative muscle disease resulting from mutations in the dystrophin gene. Increased oxidative stress and altered Ca(2+) homeostasis are hallmarks of dystrophic muscle. While impaired autophagy has recently been implicated in the disease process, the mechanisms underlying the impairment have not been elucidated. Here we show that nicotinamide adenine dinucleotide phosphatase (Nox2)-induced oxidative stress impairs both autophagy and lysosome formation in mdx mice. Persistent activation of Src kinase leads to activation of the autophagy repressor mammalian target of rapamycin (mTOR) via PI3K/Akt phosphorylation. Inhibition of Nox2 or Src kinase reduces oxidative stress and partially rescues the defective autophagy and lysosome biogenesis. Genetic downregulation of Nox2 activity in the mdx mouse decreases reactive oxygen species (ROS) production, abrogates defective autophagy and rescues histological abnormalities and contractile impairment. Our data highlight mechanisms underlying the pathogenesis of DMD and identify NADPH oxidase and Src kinase as potential therapeutic targets.

Rotenone induces neurotoxicity through Rac1-dependent activation of NADPH oxidase in SHSY-5Y cells.

Neurodegenerative diseases are attributed to impairment of the ubiquitin-proteasome system (UPS). Oxidative stress has been considered a contributing factor in the pathology of impaired UPS by promoting protein misfolding and subsequent protein aggregate formation. Increasing evidence suggests that NADPH oxidase is a likely source of excessive oxidative stress in neurodegenerative disorders. However, the mechanism of activation and its role in impaired UPS is not understood. We show that activation of NADPH oxidase in a neuroblastoma cell line (SHSY-5Y) resulted in increased oxidative and nitrosative stress, elevated cytosolic calcium, ER-stress, impaired UPS, and apoptosis. Rac1 inhibition mitigated the oxidative/nitrosative stress, prevented calcium-dependent ER-stress, and partially rescued UPS function. These findings demonstrate that Rac1 and NADPH oxidase play an important role in rotenone neurotoxicity.

EF24 prevents rotenone-induced estrogenic status alteration in breast cancer.

Protein disulfide isomerase (PDI), an important endoplasmic reticulum-resident oxidoreductase chaperone can bind to estrogens as well as intact with its receptor proteins [i.e. estrogen receptors (ER) α and ß]. It has been postulated that PDI also acts as an intracellular 17ß-estradiol (E2)-binding protein that transports and accumulates E2 in live cells. Drop in E2 level promotes dissociation of E2 from PDI and released in cytosol; the released E2 can augment estrogen receptor-mediated transcriptional activity and mitogenic action in cultured cells by modulating the ERß/ERα ratio. In this study, we observed rotenone-induced damage to PDI leads to significant increase in ERß/ERα ratio by down-regulating ERα and up-regulating ERß. We demonstrated that nitrosative stress induced disruption of the cellular estrogenic status can be prevented through diphenyl difluoroketone (EF24, curcumin analog) intervention by protecting PDI from reactive oxygen species (ROS)-induced damage. Together, our study suggests that both PDI and EF24 can play a vital role in maintaining cellular estrogenic homeostasis.

Sphingomyelinase promotes oxidant production and skeletal muscle contractile dysfunction through activation of NADPH oxidase.

Elevated concentrations of sphingomyelinase (SMase) have been detected in a variety of diseases. SMase has been shown to increase muscle derived oxidants and decrease skeletal muscle force; however, the sub-cellular site of oxidant production has not been elucidated. Using redox sensitive biosensors targeted to the mitochondria and NADPH oxidase (Nox2), we demonstrate that SMase increased Nox2-dependent ROS and had no effect on mitochondrial ROS in isolated FDB fibers. Pharmacological inhibition and genetic knockdown of Nox2 activity prevented SMase induced ROS production and provided protection against decreased force production in the diaphragm. In contrast, genetic overexpression of superoxide dismutase within the mitochondria did not prevent increased ROS production and offered no protection against decreased diaphragm function in response to SMase. Our study shows that SMase induced ROS production occurs in specific sub-cellular regions of skeletal muscle; however, the increased ROS does not completely account for the decrease in muscle function.