The activation of Mucolipin TRP channel 1 (TRPML1) protects motor neurons from L-BMAA neurotoxicity by promoting autophagic clearance

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 Ca2+ 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 Ca2+ sensor STIM1. Functionally, the TRPML1 agonist ML-SA1 induced lysosomal Ca2+ release in a dose-dependent way in motor neuronal cells. The SERCA inhibitor thapsigargin increased the fluorescent signal associated with lysosomal Ca2+ efflux in the cells transfected with the genetically encoded Ca2+ 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 Ca2+ 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.

Lysosomal Ca 2+ dysregulation is associated with TRPML1 protein downregulation in primary motor neurons exposed to L-BMAA. Due to the functional coupling between ER and lysosomes under physiological conditions, we have investigated the relationship between these organelles in primary motor neurons chronically exposed to the neurotoxin L-BMAA. To this aim, we studied whether TRPML1-mediated lysosomal Ca 2+ release could be altered in the proposed neurotoxic model, by detecting the channel protein expression and activity. Figure 3A,B showed that TRPML1 protein expression was progressively downregulated in primary motor neurons exposed to 24 and 48 h of L-BMAA compared to the untreated controls (Fig. 3A,B). On the other hand, the lysosomal marker LAMP2 was not reduced (Fig. 3A,B). Similar results were obtained in differentiated NSC-34 cells (Supplementary Fig. S2).
Then, we investigated whether TRPML1-mediated lysosomal Ca 2+ release was altered under these conditions. In accordance with the reduced protein expression of TRPML1, ML-SA1 (10 μM) elicited a lower lysosomal Ca 2+ efflux in primary motor neurons exposed to L-BMAA than in control neurons (Fig. 3C,D). That lysosomal Ca 2+ content was reduced after L-BMAA exposure was confirmed by the reduction in lysosomal Ca 2+ discharge elicited by the membrane-permeable dipeptide GPN (300 μM) in motor neurons treated with the toxin (Fig. 3E). Under the same experimental conditions (i.e. 48 h of L-BMAA), a significant ER Ca 2+ depletion occurred in primary motor neurons, as revealed by the low amount of ER Ca 2+ released after the addition of ATP + thapsigargin (Fig. 3F). The same results were obtained in differentiated NSC-34 cells (Supplementary Fig. S2). TRPML1 dysfunction impairs the autophagic flux in primary motor neurons exposed to L-BMAA.
Macroautophagy is defective in TRPML1-deficient neurons 45 whereas TRPML1 overexpression results in a www.nature.com/scientificreports www.nature.com/scientificreports/ significant increase of the autophagic flux 46 . In order to verify the role of TRPML1 in the modulation of autophagy in primary motor neurons, we tested the effect of ML-SA1 alone on some autophagy initiators. The addition of ML-SA1 progressively induced the phosphorylation of AMP-activated protein kinase (p-AMPK), that is thought to activate autophagy through the inactivation of mTOR complex-1, and increased the expression of beclin 1 (Fig. 4A,B), that is involved in the formation of the autophagosomes. Interestingly, under these conditions, ML-SA1 did not affect fluorescence labeling of axonal neurofilament H detected by NF200 in rat primary motor neurons (in arbitrary units: 20.156 ± 5.824 in control conditions vs 19.193 ± 1.546 in ML-SA1-treated neurons) ( Supplementary Fig. S3). On the other hand, to test the hypothesis that TRPML1 reduction could worsen while channel activation could ameliorate autophagy defects in ALS, lysosomal degradation pathway was investigated in this neurotoxic model. L-BMAA (300 μM/48 h) induced a significant increase in the expression of both p62/ SQSTM1 (Fig. 4C) and LC3-II (Fig. 4D) that were significantly reduced in the presence of ML-SA1 (Fig. 4C,D). Since bafilomycin further increased LC3-II expression in L-BMAA-treated motor neurons, the exposure to the toxin impaired the autophagic flux in primary motor neurons (Fig. 4E,F).

TRPML1 activation rescues motor neurons from L-BMAA-induced cell death.
To study the effect of ML-SA1 on L-BMAA-induced cell death, we tested the effect of this toxin in the absence of TRPML1 channel. TRPML1 protein expression was reduced by 70 ± 1.5% (*p < 0.05 vs control + siControl) in primary motor neurons treated with specific siRNA against the channel (Fig. 5A). Functionally, ML-SA1 (10 μM) determined Western blotting and quantification of p62 expression in rat primary motor neurons exposed to L-BMAA (300 μM/48 h) in the absence or presence of ML-SA1 (10 μM). Each bar represents the mean ± S.E. of data obtained from three different sessions. *p < 0.01 vs control; **p < 0.05 vs L-BMAA. (D) Representative Western blotting and quantification of LC3-I and LC3-II expression in rat primary motor neurons exposed to L-BMAA (300 μM/48 h) in the absence or presence of ML-SA1 (10 μM). Each bar represents the mean ± S.E. of data obtained from three different sessions. *p < 0.01 vs control; **p < 0.05 vs L-BMAA. For (C,D), ML-SA1 was preincubated for 1 h before L-BMAA exposure. (E,F) Representative Western blotting and quantification of LC3-I and LC3-II expression in motor neurons exposed to L-BMAA (300 μM/48 h) in the absence or presence of bafilomycin (Bafilo 100 nM/1 h).This latter drug was added 1 h before the end of the experiment. Each bar represents the mean ± S.E. of data obtained from three different sessions. *p < 0.01 vs control; **p < 0.05 vs L-BMAA alone.
www.nature.com/scientificreports www.nature.com/scientificreports/ a significant Ca 2+ release in primary motor neurons that was significantly reduced in the absence of TRPML1 (Fig. 5B).
As a consequence of cell suffering avoidance, the preincubation with ML-SA1 prevented TRPML1 protein downregulation induced by chronic exposure to L-BMAA ( Supplementary Fig. S3).

Discussion
In recent years, the idea of lysosomes as mere degradative organelles has dramatically changed and they are now recognized as critical regulators of cellular homeostasis and survival. In the present study, we have characterized the involvement of one of the main lysosomal channels, TRPML1, in the pathogenesis of L-BMAA-induced ALS/PDC. We found that TRPML1 Ca 2+ channel is an important player in the protective response of lysosomes against L-BMAA in primary motor neurons chronically exposed to the neurotoxin. In fact, we provided evidence that TRPML1 was progressively downregulated in L-BMAA-treated neurons while its early stimulation, by the synthetic compound ML-SA1, efficiently rescued motor neurons from L-BMAA toxicity by counteracting ER stress and autophagy impairment (Fig. 6). Furthermore, under these conditions, TRPML1 protein expression was partially restored as a consequence of ML-SA1-induced neuroprotection.
That ML-SA1 stimulated specifically TRPML1 channel was demonstrated by the absence of the effect on Ca 2+ release in TRPML1-silenced motor neurons. Furthermore, ML-SA1 failed to rescue motor neurons exposed to L-BMAA when TRPML1 was absent. Mechanicistically, lysosomal Ca 2+ efflux through TRPML1 seems to play a www.nature.com/scientificreports www.nature.com/scientificreports/ prominent role in the protective action of ML-SA1. In fact, lysosomal Ca 2+ efflux progressively reduced together with TRPML1 protein expression in motor neurons exposed to the toxin, thus causing lysosomal Ca 2+ dysfunction and cell death. Furthermore, we showed a concomitant dysfunction in ER Ca 2+ homeostasis in neurons exposed to L-BMAA, most likely depending on the disruption of the lysosomal and ER functional interplay. Indeed, the present study suggests that ER store acts as a major source of lysosomal Ca 2+ in motor neurons and that dysfunctional Ca 2+ homeostasis in one of these organelles has a dramatic repercussion on the other store. This is in accordance with the evidence demonstrating the strategic role of ER in promoting lysosomal Ca 2+ refilling in other cellular systems 44 . Accordingly, we showed that the specific and irreversible SERCA inhibitor thapsigargin, by depleting ER Ca 2+ store, influences lysosomal Ca 2+ content measured as Ca 2+ release through TRPML1 by the Ca 2+ indicator GCaMP3-ML1.
Furthermore, in the present study, ML-SA1 prevented the upregulation of the ER stress marker GRP78 that significantly increases after L-BMAA-induced ER store dysfunction 41 and in sporadic ALS characterized by misfolding of wild-type SOD1 40 . This suggests that TRPML1 stimulation may protect motor neurons from ER stress in different forms of ALS. It could be taken into consideration that, in general, an abundance of ER Ca 2+ is vital for nascent protein folding and cell survival 47 . Loss of ER luminal Ca 2+ is frequently accompanied by ER stress and by the activation of an apoptotic pathway. Therefore, ER Ca 2+ refilling is essential for cell survival under stress conditions 48 . Accordingly, it has been suggested that store-operated calcium entry upon ER calcium depletion is aberrant in SOD1 G93A animals 49 . However, our data showed that, in consideration of the close proximity between the two organelles, TRPML1-dependent lysosomal Ca 2+ release may also contribute to ER Ca 2+ filling state and to ER stress prevention by a continuous refilling of Ca 2+ ions. Therefore, we hypothesize that ER and lysosomes co-localization, together with their functional interdependency, can be crucial in the reduction of the neurotoxic effect of L-BMAA.
The toxicity caused by protein misfolding or aggregation, or proteotoxicity, is a common feature of ALS 50 . Ubiquitin-positive inclusions linked to SOD1, TDP-43, FUS, and C9orf72 have been reported in familial ALS 51 . However, abnormal accumulation of the wild-type form of some proteins, such as TDP-43 and SOD1, has also been observed in sporadic ALS 5,40,52 . This is indicative of defects in cellular cleansing mechanisms occurring in the neurodegenerative disease. Given the important role of autophagy in cleansing the cell of defective organelles and misfolded proteins, our data suggest that the activation of TRPML1 could prevent the engulfment in autophagy provoked by chronic exposure to L-BMAA. In fact, TRPML1 stimulation reduced the accumulation of p62/SQSTM1 and LC3-II in motor neurons exposed to the toxin. In this respect, p62 is known to be involved not only in selective autophagy but also in degradation through the ubiquitin-proteasome system 53 . Interestingly, overexpression of p62/SQSTM1 shortens lifespan in a double transgenic ALS mouse model by compromising the protein degradation pathway 54 . On the other hand, Curcio-Morelli and co-authors showed that mouse TRPML1-deficient neurons show a 2-fold increase in p62/SQSTM1 levels, defect in LC3-II clearance, macroautophagy impairment and increase in polyubiquitin levels 45 . This may suggest that TRPML1 is specifically involved in the impairment of the cleansing machinery in ALS.
Recent studies suggest that the aggregation of ALS-related genes can affect early steps in the autophagic process thus producing a hyperactive induction of autophagy suggestive of AMPK activation and mTOR repression in motor neurons of SOD1 G85R mice 55 . This determines autophagy engulfment. Consistently, upregulation of beclin 1 56 has been found in spinal cord and brainstem fractions of SOD1 G93A animals in which p62 and LC3-II levels were elevated 57 . The engulfment of autophagy was confirmed by the present study in motor neurons exposed to L-BMAA, since the neurotoxin increased the level of the autophagy markers p62 and LC3-II. However, we showed that TRPML1 stimulation by ML-SA1 prevented the elevation of these markers, thus rescuing the intact autophagic flux.
The therapeutic utility of pharmacological upregulation of autophagy in some neurodegenerative diseases 58 and in ALS models 59 is supported by a wealth of data. Nonetheless, enhanced autophagy seems to further exacerbate axonal degeneration and disease phenotype 50,60 . This is indicative of an urgent need for other strategies aimed at finely regulating autophagy to promote neuronal protection and survival. To date, it has been reported that www.nature.com/scientificreports www.nature.com/scientificreports/ autophagy disturbances vary depending on the phases of disease. In fact, at the early stage of disease, autophagy fulfills a neuroprotective role in SOD G93A mice 61 . The present study suggests that, at least in vitro, the initial triggering of lysosomal Ca 2+ release by TRPML1 stimulation could promote a sort of autophagy reprogramming leading to a long-lasting effect. Altogether, this study shows that the specific membrane-permeable synthetic agonist of TRPML1, ML-SA1, rescued motor neurons from death induced by chronic exposure to L-BMAA. Furthermore, ML-SA1 prevented the elevation of the ER stress marker GRP78 and of the autophagy-related proteins p62/SQSTM1 and LC3-II. Collectively, we propose that an early pharmacological stimulation of lysosomal Ca 2+ channel TRPML1 can efficiently rescue motor neurons from L-BMAA toxicity through the restoration of the autophagic flux.

Materials and Methods
Reagents. Rabbit  Primary cultures of rat motor neurons. Rat primary motor neurons were isolated from the spinal cord of 12-14-day old Wistar embryos and cultured as previously described 41,63 . To prevent non-neuronal cell growth, cytosine β-D-arabinofuranoside hydrochloride (Ara-C; 10 μM) was added to the cell culture medium at 4 and 8 DIV. These primary cultures were used for experiments at 10-12 DIV.
ML-SA1 (10 μM) was added to the cell culture medium 1 h before L-BMAA treatment. The chronic exposure to this non-proteic amino acid has been associated to ALS/PDC, a Guamanian form of ALS affecting Chamorro people 19 . Immunocytochemistry and confocal microscopy. Twenty-four hours after transfection with ERdsRED and siTRPML1, NSC-34 cells were washed twice in cold 0.01 M PBS (pH 7.4) and fixed at room temperature in 4% paraformaldehyde for 20 min. Following three washes in PBS, cells were blocked in PBS containing 3% BSA for 30 min and then incubated overnight at 4 °C with anti-TRPML1 antibody (1:1000). In another set of experiments, NSC-34 cells were incubated with anti-TRPML1 (1:1000) or anti-LAMP1 (1:500) antibody and STIM1-ATTO-550 (1:200) (overnight at 4 °C). Then, cells were washed with PBS and incubated with anti-rabbit Cy2-conjugated antibody (1:200, Jackson Immuno Research Laboratories, Inc. PA, USA) for 1 h at room temperature under dark conditions. Cells were finally incubated for 5 min with Hoechst. Controls of the method in the double immunofluorescence experiments included the replacement of primary antibodies against TRPML1 or LAMP1 with normal serum. In addition, the anti-rabbit Cy2-conjugated antibody was highly preadsorbed to the IgGs of numerous species. For immunocytochemical analysis of the image shown in Supplementary Fig. S3, rat primary motor neurons were incubated overnight at 4 °C with anti-neurofilament 200 (NF200) antibody (1:1000). Finally, cover glasses were mounted with a SlowFade TM Antifade Kit (Molecular Probes, Life Technologies, Milan, Italy) and acquired by a 63X oil immersion objective using a Zeiss inverted 700 confocal microscope 64 . Co-localization between TRPML1 or LAMP1 and STIM1-ATTO-550 was analyzed by using the 'co-localization highlighter' plug-in for ImageJ Software (NIH, Bethesda, MA, USA). Before the analysis of co-localization, threshold settings for each image were determined, and quantification was accomplished by counting the number of TRPML1 or LAMP1 and STIM1 co-localized points per microscope field. Results were expressed as a percentage of co-localization 64 . Fluorescence intensity of TRPML1 was quantified as pixel intensity value with the NIH