Defective lysosomal acidification contributes to virtually all lysosomal storage disorders (LSDs) and to common neurodegenerative diseases like Alzheimer's and Parkinson's. Despite its fundamental importance, the mechanism(s) underlying this defect remains unclear. The v-ATPase, a multisubunit protein complex composed of cytosolic V1-sector and lysosomal membrane-anchored V0-sector, regulates lysosomal acidification. Mutations in the CLN1 gene, encoding PPT1, cause a devastating neurodegenerative LSD, INCL. Here we report that in Cln1−/− mice, which mimic INCL, reduced v-ATPase activity correlates with elevated lysosomal pH. Moreover, v-ATPase subunit a1 of the V0 sector (V0a1) requires palmitoylation for interacting with adaptor protein-2 (AP-2) and AP-3, respectively, for trafficking to the lysosomal membrane. Notably, treatment of Cln1−/− mice with a thioesterase (Ppt1)-mimetic, NtBuHA, ameliorated this defect. Our findings reveal an unanticipated role of Cln1 in regulating lysosomal targeting of V0a1 and suggest that varying factors adversely affecting v-ATPase function dysregulate lysosomal acidification in other LSDs and common neurodegenerative diseases.

Background: How cytosolic thioesterases (APT1 and APT2) depalmitoylate their membrane-anchored substrates remains unclear. Results: APT1 and APT2 require palmitoylation for membrane localization. Although APT1 depalmitoylates itself, H-Ras, and APT2, APT2 depalmitoylates GAP-43. Conclusion: Dynamic palmitoylation regulates steady-state membrane localization and function of cytosolic thioesterases and their substrates. Significance: The findings may provide new insight into the regulation and function of cytosolic thioesterases and their substrates.

Infantile neuronal ceroid lipofuscinosis (INCL) is a devastating childhood neurodegenerative lysosomal storage disease (LSD) that has no effective treatment. It is caused by inactivating mutations in the palmitoyl-protein thioesterase-1 (PPT1) gene. PPT1-deficiency impairs the cleavage of thioester linkage in palmitoylated proteins (constituents of ceroid), preventing degradation by lysosomal hydrolases. Consequently, accumulation of lysosomal ceroid leads to INCL. Thioester linkage is cleaved by nucleophilic attack. Hydroxylamine, a potent nucleophilic cellular metabolite, may have therapeutic potential for INCL but its toxicity precludes clinical application. Here we report that a hydroxylamine-derivative, N-(tert-Butyl) hydroxylamine (NtBuHA), is non-toxic, cleaves thioester linkage in palmitoylated proteins and mediates lysosomal ceroid depletion in cultured cells from INCL patients. Importantly, in Ppt1−/− mice, which mimic INCL, NtBuHA crossed the blood-brain-barrier, depleted lysosomal ceroid, suppressed neuronal apoptosis, slowed neurological deterioration and extended lifespan. Our findings provide the proof of concept that thioesterase-mimetic and antioxidant small molecules like NtBuHA are potential drug-targets for thioesterase deficiency diseases like INCL.

Autosomal recessive mutations in Anoctamin 5 ( ANO5/TMEM16E ), a member of the transmembrane 16 (TMEM16) family of Ca 2+ -activated ion channels and phospholipid scramblases, cause adult-onset muscular dystrophies (limb girdle muscular dystrophy 2L (LGMD2L) and Miyoshi Muscular Dystrophy (MMD3). However, the molecular role of ANO5 is unclear and ANO5 knockout mouse models show conflicting requirements of ANO5 in muscle. To study the role of ANO5 in human muscle cells we generated a myoblast line from a MMD3-patient carrying the c.2272C>T mutation, which we find causes the mutant protein to be degraded. The patient myoblasts exhibit normal myogenesis, but are compromised in their plasma membrane repair (PMR) ability. The repair deficit is linked to the poor ability of the endoplasmic reticulum (ER) to clear cytosolic Ca 2+ increase caused by focal plasma membrane injury. Expression of wild-type ANO5 or pharmacological prevention of injury-triggered cytosolic Ca 2+ overload enable injured patient muscle cells to repair. A homology model of ANO5 shows that several of the known LGMD2L/MMD3 patient mutations line the transmembrane region of the protein implicated in its channel activity. These results point to a role of cytosolic Ca 2+ homeostasis in PMR, indicate a role for ANO5 in ER-mediated cytosolic Ca 2+ uptake and identify normalization of cytosolic Ca 2+ homeostasis as a potential therapeutic approach to treat muscular dystrophies caused by ANO5 deficit.

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Mutations of the DYSF gene leading to reduced dysferlin protein level causes limb girdle muscular dystrophy type 2B (LGMD2B). Dysferlin facilitates sarcolemmal membrane repair in healthy myofibers, thus its deficit compromises myofiber repair and leads to chronic muscle inflammation. An experimental therapeutic approach for LGMD2B is to protect damage or improve repair of myofiber sarcolemma. Here, we compared the effects of prednisolone and vamorolone (a dissociative steroid; VBP15) on dysferlin-deficient myofiber repair. Vamorolone, but not prednisolone, stabilized dysferlin-deficient muscle cell membrane and improved repair of dysferlin-deficient mouse (B6A/J) myofibers injured by focal sarcolemmal damage, eccentric contraction-induced injury or injury due to spontaneous in vivo activity. Vamorolone decreased sarcolemmal lipid mobility, increased muscle strength, and decreased late-stage myofiber loss due to adipogenic infiltration. In contrast, the conventional glucocorticoid prednisolone failed to stabilize dysferlin deficient muscle cell membrane or improve repair of dysferlinopathic patient myoblasts and mouse myofibers. Instead, prednisolone treatment increased muscle weakness and myofiber atrophy in B6A/J mice-findings that correlate with reports of prednisolone worsening symptoms of LGMD2B patients. Our findings showing improved cellular and pre-clinical efficacy of vamorolone compared to prednisolone and better safety profile of vamorolone indicates the suitability of vamorolone for clinical trials in LGMD2B.

Objective A hitherto undescribed phenotype of early onset muscular dystrophy associated with sensorineural hearing loss and primary ovarian insufficiency was initially identified in 2 siblings and in subsequent patients with a similar constellation of findings. The goal of this study was to understand the genetic and molecular etiology of this condition. Methods We applied whole exome sequencing (WES) superimposed on shared haplotype regions to identify the initial biallelic variants in GGPS1 followed by GGPS1 Sanger sequencing or WES in 5 additional families with the same phenotype. Molecular modeling, biochemical analysis, laser membrane injury assay, and the generation of a Y259C knock‐in mouse were done. Results A total of 11 patients in 6 families carrying 5 different biallelic pathogenic variants in specific domains of GGPS1 were identified. GGPS1 encodes geranylgeranyl diphosphate synthase in the mevalonate/isoprenoid pathway, which catalyzes the synthesis of geranylgeranyl pyrophosphate, the lipid precursor of geranylgeranylated proteins including small guanosine triphosphatases. In addition to proximal weakness, all but one patient presented with congenital sensorineural hearing loss, and all postpubertal females had primary ovarian insufficiency. Muscle histology was dystrophic, with ultrastructural evidence of autophagic material and large mitochondria in the most severe cases. There was delayed membrane healing after laser injury in patient‐derived myogenic cells, and a knock‐in mouse of one of the mutations (Y259C) resulted in prenatal lethality. Interpretation The identification of specific GGPS1 mutations defines the cause of a unique form of muscular dystrophy with hearing loss and ovarian insufficiency and points to a novel pathway for this clinical constellation. ANN NEUROL 2020;88:332–347.

Emerging evidence indicates a link between inflammation and cancer metastasis, but the molecular mechanism(s) remains unclear. Uteroglobin (UG), a potent anti-inflammatory protein, is constitutively expressed in the lungs of virtually all mammals. UG-knock-out (UG-KO) mice, which are susceptible to pulmonary inflammation, and B16F10 melanoma cells, which preferentially metastasize to the lungs, provide the components of a model system to determine how inflammation and metastasis are linked. We report here that B16F10 cells, injected into the tail vein of UG-KO mice, form markedly elevated numbers of tumor colonies in the lungs compared with their wild type littermates. Remarkably, UG-KO mouse lungs overexpress two calcium-binding proteins, S100A8 and S100A9, whereas B16F10 cells express the receptor for advanced glycation end products (RAGE), which is a known receptor for these proteins. Moreover, S100A8 and S100A9 are potent chemoattractants for RAGE-expressing B16F10 cells, and pretreatment of these cells with a blocking antibody to RAGE suppressed migration and invasion. Interestingly, in UG-KO mice S100A8/S100A9 concentrations in blood are lowest in tail vein and highest in the lungs, which most likely guide B16F10 cells to migrate to the lungs. Further, B16F10 cells treated with S100A8 or S100A9 overexpress matrix metalloproteinases, which are known to promote tumor invasion. Most notably, the metastasized B16F10 cells in UG-KO mouse lungs express MMP-2, MMP-9, and MMP-14 as well as furin, a pro-protein convertase that activates MMPs. Taken together, our results suggest that a lack of an anti-inflammatory protein leads to increased pulmonary colonization of melanoma cells and identify RAGE as a potential anti-metastatic drug target.It is estimated that more than 90% of human cancer deaths result from metastasis (1-8), a complex process in which the cells from a primary tumor successfully invade and colonize a distant organ (6 -9). It has long been suspected that a functional relationship exists between inflammation and cancer. Indeed, in 1863, Virchow observed that cancer develops at locations where chronic inflammation is present. This hypothesis is partly based on his assumption that some substances that cause tissue injury and inflammation also enhance cell proliferation (10). Although it is clear now that proliferation alone does not account for cancer, the cause and effect relationship between inflammation and cancer is widely accepted. Nonetheless, many aspects of the molecular and cellular mechanisms that link inflammation, migration, and establishment of tumor colonies in a distant organ remain unresolved (5). Recently, it has been reported that a tumor microenvironment actively contributes to cancer initiation, progression, and metastasis (11, 12). It has also been suggested that reductions in the rates of cancer morbidity and mortality may be achieved by gaining greater understanding of the molecular mechanism(s) of tumor cell migration, invasion, and establishment of colonies (13). In this regard...