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Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease currently incurable, in which motor neuron degeneration leads to voluntary skeletal muscle atrophy. Molecularly, ALS is characterized by protein aggregation, synaptic and organellar dysfunction, and Ca 2+ dyshomeostasis. Of interest, autophagy dysfunction is emerging as one of the main putative targets of ALS therapy. A tune regulation of this cleansing process is affordable by a proper stimulation of TRPML1, one of the main lysosomal channels. However, TRPML1 activation by PI(3,5)P 2 has low open probability to remain in an active conformation. To overcome this drawback we developed a lipid-based formulation of PI(3,5)P 2 whose putative therapeutic potential has been tested in in vitro and in vivo ALS models. Pharmacodynamic properties of PI(3,5)P 2 lipid-based formulations (F1 and F2) on TRPML1 activity have been characterized by means of patch-clamp electrophysiology and Fura-2AM video-imaging in motor neuronal cells. Once selected for the ability to stabilize TRPML1 activity, the most effective preparation F1 was studied in vivo to measure neuromuscular function and survival of SOD1 G93A ALS mice, thereby establishing its therapeutic profile. F1, but not PI(3,5)P 2 alone , stabilized the open state of the lysosomal channel TRPML1 and increased the persistence of intracellular calcium concentration ([Ca 2+ ] i ). Then, F1 was effective in delaying motor neuron loss, improving innervated endplants and muscle performance in SOD1 G93A mice, extending overall lifespan by an average of 10 days. Of note F1 prevented gliosis and autophagy dysfunction in ALS mice by restoring PI(3,5)P 2 level. Our novel self-assembling lipidic formulation for PI(3,5)P 2 delivery exerts a neuroprotective effect in preclinical models of ALS mainly regulating dysfunctional autophagy through TRPML1 activity stabilization., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A robust activity of the lysosomal Ca 2+ channel TRPML1 is sufficient to correct cellular defects in neurodegeneration. Importantly, lysosomes are refilled by the endoplasmic reticulum (ER). However, it is unclear how TRPML1 function could be modulated by the ER. Here, we deal with this issue in rat primary cortical neurons exposed to different oxygen conditions affecting neuronal survival. Under normoxic conditions, TRPML1: (1) showed a wide distribution within soma and along neuronal processes; (2) was stimulated by the synthetic agonist ML-SA1 and the analog of its endogenous modulator, PI(3,5)P 2 diC8; (3) its knockdown by siRNA strategy produced an ER Ca 2+ accumulation; (4) co-localized and co-immunoprecipitated with the ER-located Ca 2+ sensor stromal interacting molecule 1 (STIM1). In cortical neurons lacking STIM1, ML-SA1 and PI(3,5)P 2 diC8 failed to induce Ca 2+ release and, more deeply, they induced a negligible Ca 2+ passage through the channel in neurons transfected with the genetically encoded Ca 2+ indicator GCaMP3-ML1. Moreover, TRPML1/STIM1 interplay changed at low-oxygen conditions: both proteins were downregulated during the ischemic preconditioning (IPC) while during IPC followed by 1 hour of normoxia, at which STIM1 is upregulated, TRPML1 protein was reduced. However, during oxygen and glucose deprivation (OGD) followed by reoxygenation, TRPML1 and STIM1 proteins peaked at 8 hours of reoxygenation, when the proteins were co-immunoprecipitated and reactive oxygen species (ROS) hyperproduction was measured in cortical neurons. This may lead to a persistent TRPML1 Ca 2+ release and lysosomal Ca 2+ loss. Collectively, we showed a new modulation exerted by STIM1 on TRPML1 activity that may differently intervene during hypoxia to regulate organellar Ca 2+ homeostasis., (© 2021 Federation of American Societies for Experimental Biology.)

Cellular clearance mechanisms including the autophagy-lysosome pathway are impaired in amyotrophic lateral sclerosis (ALS). One of the most important proteins involved in the regulation of autophagy is the lysosomal Ca 2+ channel Mucolipin TRP channel 1 (TRPML1). Therefore, we investigated the role of TRPML1 in a neuronal model of ALS/Parkinson-dementia complex reproduced by the exposure of motor neurons to the cyanobacterial neurotoxin beta-methylamino-L-alanine (L-BMAA). Under these conditions, L-BMAA induces a dysfunction of the endoplasmic reticulum (ER) leading to ER stress and cell death. Therefore we hypothesized a dysfunctional coupling between lysosomes and ER in L-BMAA-treated motor neurons. Here, we showed that in motor neuronal cells TRPML1 as well as the lysosomal protein LAMP1 co-localized with ER. In addition, TRPML1 co-immunoprecipitated with the ER Ca 2+ sensor STIM1. Functionally, the TRPML1 agonist ML-SA1 induced lysosomal Ca 2+ release in a dose-dependent way in motor neuronal cells. The SERCA inhibitor thapsigargin increased the fluorescent signal associated with lysosomal Ca 2+ efflux in the cells transfected with the genetically encoded Ca 2+ indicator GCaMP3-ML1, thus suggesting an interplay between the two organelles. Moreover, chronic exposure to L-BMAA reduced TRPML1 protein expression and produced an impairment of both lysosomal and ER Ca 2+ homeostasis in primary motor neurons. Interestingly, the preincubation of ML-SA1, by an early activation of AMPK and beclin 1, rescued motor neurons from L-BMAA-induced cell death and reduced the expression of the ER stress marker GRP78. Finally, ML-SA1 reduced the accumulation of the autophagy-related proteins p62/SQSTM1 and LC3-II in L-BMAA-treated motor neurons. Collectively, we propose that the pharmacological stimulation of TRPML1 can rescue motor neurons from L-BMAA-induced toxicity by boosting autophagy and reducing ER stress.