Mucolipidosis type IV (MLIV) is caused by loss of function mutations in the TRPML1 ion channel. We previously reported that tissue zinc levels in MLIV were abnormally elevated; however, the mechanism behind this pathologic accumulation remains unknown. Here, we identify transmembrane (TMEM)-163 protein, a putative zinc transporter, as a novel interacting partner for TRPML1. Evidence from yeast two-hybrid, tissue expression pattern, co-immunoprecipitation, mass spectrometry, and confocal microscopy studies confirmed the physical association of TMEM163 with TRPML1. This interaction is disrupted when a part of TMEM163's N-terminus was deleted. Further studies to define the relevance of their interaction revealed that the plasma membrane (PM) levels of TMEM163 significantly decrease when TRPML1 is co-expressed in HEK-293 cells, while it mostly localizes within the PM when co-expressed with a mutant TRPML1 that distributes mostly in the PM. Meanwhile, co-expression of TMEM163 does not alter TRPML1 channel activity, but its expression levels in MLIV patient fibroblasts are reduced, which correlate with marked accumulation of zinc in lysosomes when these cells are acutely exposed to exogenous zinc (100 μM). When TMEM163 is knocked down or when TMEM163 and TRPML1 are co-knocked down in HEK-293 cells treated overnight with 100 nM zinc, the cells have significantly higher intracellular zinc levels than untreated control. Overall, these findings suggest that TMEM163 and TRPML1 proteins play a critical role in cellular zinc homeostasis, and thus possibly explain a novel mechanism for the pathological overload of zinc in MLIV disease.

The discovery of the TRPML subfamily of ion channels has created an exciting niche in the fields of membrane trafficking, signal transduction, autophagy, and metal homeostasis. The TRPML protein subfamily consist three members, TRPML1, -2, and -3, which are encoded by MCOLN1, -2, and -3 genes, respectively. They are non-selective cation channels with six predicted transmembrane domains, and intracellular amino- and carboxyl-terminus regions. They localize to the plasma membrane, endosomes, and lysosomes of cells. TRPML1 is associated with the human lysosomal storage disease known as Mucolipidosis type IV (MLIV), but TRPML2 and TRPML3 have not been linked with a human disease. Although TRPML1 is expressed in many tissues, TRPML3 is expressed in a varied but limited set of tissues, while TRPML2 has a more limited expression pattern where it is mostly detected in lymphoid and myeloid tissues. This review focuses on TRPML2 because it appears to play an important, yet unrecognized role in the immune system. While the evidence has been mostly indirect, we present and discuss relevant data that strengthen the connection of TRPML2 with cellular immunity. We also discuss the functional redundancy between the TRPML proteins, and how such features could be exploited as a potential therapeutic strategy for MLIV disease. We present evidence that TRPML2 expression may complement certain phenotypic alterations in MLIV cells, and briefly examine the challenges of functional complementation. In conclusion, the function of TRPML2 still remains obscure, but emerging data show that it may serve a critical role in immune cell development and inflammatory responses.

Background Excess alcohol (ethanol, EtOH) consumption is a significant cause of chronic liver disease, accounting for nearly half of the cirrhosis‐associated deaths in the United States. EtOH‐induced liver toxicity is linked to EtOH metabolism and its associated increase in proinflammatory cytokines, oxidative stress, and the subsequent activation of Kupffer cells. Dihydromyricetin (DHM), a bioflavonoid isolated from Hovenia dulcis , can reduce EtOH intoxication and potentially protect against chemical‐induced liver injuries. But there remains a paucity of information regarding the effects of DHM on EtOH metabolism and liver protection. As such, the current study tests the hypothesis that DHM supplementation enhances EtOH metabolism and reduces EtOH‐mediated lipid dysregulation, thus promoting hepatocellular health. Methods The hepatoprotective effect of DHM (5 and 10 mg/kg; intraperitoneal injection) was evaluated using male C57BL/6J mice and a forced drinking ad libitum EtOH feeding model and HepG2/VL‐17A hepatoblastoma cell models. EtOH‐mediated lipid accumulation and DHM effects against lipid deposits were determined via H&E stains, triglyceride measurements, and intracellular lipid dyes. Protein expression of phosphorylated/total proteins and serum and hepatic cytokines was determined via Western blot and protein array. Total NAD + /NADH Assay of liver homogenates was used to detect NAD + levels. Results DHM reduced liver steatosis, liver triglycerides, and liver injury markers in mice chronically fed EtOH. DHM treatment resulted in increased activation of AMPK and downstream targets, carnitine palmitoyltransferase (CPT)‐1a, and acetyl CoA carboxylase (ACC)‐1. DHM induced expression of EtOH‐metabolizing enzymes and reduced EtOH and acetaldehyde concentrations, effects that may be partly explained by changes in NAD + . Furthermore, DHM reduced the expression of proinflammatory cytokines and chemokines in sera and cell models. Conclusion In total, these findings support the utility of DHM as a dietary supplement to reduce EtOH‐induced liver injury via changes in lipid metabolism, enhancement of EtOH metabolism, and suppressing inflammation responses to promote liver health.

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Anxiety disorders are the most common mental illness in the U.S. and are estimated to consume one-third of the country's mental health spending. Although anxiolytic therapies are available, many patients exhibit treatment-resistance, relapse, or substantial side effects. An urgent need exists to explore the underlying mechanisms of chronic anxiety and to develop alternative therapies. Presently, we identified dihydromyricetin (DHM), a flavonoid that has anxiolytic properties in a mouse model of isolation-induced anxiety. Socially isolated mice demonstrated increased anxiety levels and reduced exploratory behavior measured by elevated plus-maze and open-field tests. Socially isolated mice showed impaired GABAergic neurotransmission, including reduction in GABA A receptormediated extrasynaptic tonic currents, as well as amplitude and frequency of miniature inhibitory postsynaptic currents measured by whole-cell patch-clamp recordings from hippocampal slices. Furthermore, intracellular ATP levels and gephyrin expression decreased in anxious animals. DHM treatment restored ATP and gephyrin expression, GABAergic transmission and synaptic function, as well as decreased anxiety-like behavior. Our findings indicate broader roles for DHM in anxiolysis, GABAergic neurotransmission, and synaptic function. Collectively, our data suggest that reduction in intracellular ATP and gephyrin contribute to the development of anxiety, and represent novel treatment targets. DHM is a potential candidate for pharmacotherapy for anxiety disorders.

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