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, 9 (1), 10743

The Activation of Mucolipin TRP Channel 1 (TRPML1) Protects Motor Neurons From L-BMAA Neurotoxicity by Promoting Autophagic Clearance

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The Activation of Mucolipin TRP Channel 1 (TRPML1) Protects Motor Neurons From L-BMAA Neurotoxicity by Promoting Autophagic Clearance

Valentina Tedeschi et al. Sci Rep.

Abstract

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.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
TRPML1 expression and co-localization with the ER Ca2+ sensor STIM1 in differentiated NSC-34 cells. (A) TRPML1 and STIM1 immunosignals in differentiated NSC-34 cells (a–e). Higher magnifications of the frame “e” illustrate TRPML1/STIM1 staining in cell body (e’–e”). Scale bars: 10 μm (a–d), 5 μm (e), 2 μm (e”) and 1 μm (e’). Bar graph depicts % of TRPML1 and STIM1 co-localization. (B) Lysates from differentiated NSC-34 cells subjected to immunoprecipitation (IP) using anti-STIM1 (top row) or anti-TRPML1 (bottom row). (C) LAMP1 and STIM1 immunosignals in differentiated NSC-34 cells. Bar graph depicts % of LAMP1 and STIM1 co-localization. Scale bars: 10 μm (a–d), 2 μm (e). (D) Representative Western blotting of TRPML1 and α-tubulin expression in NSC-34 cells treated with siControl or siRNA #2 against TRPML1. *p < 0.05 vs siControl. (E) TRPML1 and STIM1 immunosignals after transfection with siControl (a–d) or siRNA #2 against TRPML1 (a’–d’). Bar graph on the top depicts the quantification of TRPML1 as fluorescent signal; bar graph on the bottom depicts % of TRPML1 and STIM1 co-localization after siRNAs treatment. Scale bars: 5 μm (a–d), 10 μm (a’–d’). *p < 0.05 vs respective controls.
Figure 2
Figure 2
TRPML1 activity in differentiated NSC-34 cells. (A) Effect of ML-SA1 (1–30 μM) on [Ca2+]i in differentiated NSC-34 motor neurons. Representative trace of the effect of ML-SA1 at 10 μM on [Ca2+]i is reported below the images depicting Fura-2-loaded NSC-34 cells as 340 nm/380 nm. Bar graph depicts the dose/effect curve of ML-SA1. Each bar represents the mean ± S.E. (n = 30 cells studied in three different experimental sessions). *p < 0.05 vs control and 1 μM; **p < 0.05 vs 10 μM. (B) Superimposed traces representative of the effect of ML-SA1 (10 μM) on [Ca2+]i in NSC-34 cells transfected with siControl or siRNA #2 against TRPML1. Inset: bar graph depicts the effect of ML-SA1 expressed as Δ% of [Ca2+]i increase. Each bar represents the mean ± S.E. (n = 20 cells studied in three different experimental sessions). *p < 0.05 vs siControl. (C) Representative traces of differentiated NSC-34 cells transfected with GCaMP3-ML1 construct and treated with ML-SA1 (10 μM) (left) or thapsigargin (1 μM) (right). Insets: bar graphs depict the quantification of the effect of each drug as % of fluorescence signal increase. Each bar represents drug effects reported as mean ± S.E. (n = 10 cells studied in three different experimental sessions). *p < 0.01 vs respective basal values.
Figure 3
Figure 3
TRPML1 expression and activity in rat primary motor neurons exposed to L-BMAA. (A,B) Representative Western blotting and quantification of TRPML1 and LAMP2 expression in rat primary motor neurons exposed to L-BMAA (300 μM) for 24 and 48 h. All experiments were repeated at least three times and expressed as mean ± S.E. *p < 0.05 vs control; **p < 0.05 vs 24 h L-BMAA. (C,D) Representative traces and quantification of the effect of ML-SA1 (10 μM) on [Ca2+]i in neurons under control conditions (black trace, n = 20 cells) or after exposure to 48 h L-BMAA (red trace, n = 15 cells). *p < 0.05 vs control. All the experiments were repeated in three different experimental sessions. (E,F) Representative traces depicting the effect of GPN and ATP + Tg on [Ca2+]i in cells treated with the toxin for 48 h. Lysosomal Ca2+ content was determined in indirect way by the membrane-permeable dipeptide GPN (300 μM) in a Ca2+-containing solution (E). ER Ca2+ content was determined in indirect way by thapsigargin (Tg, 1 μM) and ATP (100 μM) in 0 Ca2+ (F). Insets: each bar graph depicts the effect of the treatments in E and F expressed as Δ% of [Ca2+]i increase (n = 40 cells in three different experimental sessions). *p < 0.05 vs its respective control. Baseline calcium values in control conditions and in L-BMAA-treated neurons were statistically different (106.9 ± 5 vs 79 ± 6, respectively, *p < 0.001).
Figure 4
Figure 4
Effect of ML-SA1 on autophagic markers in rat primary motor neurons. (A,B) Representative Western blotting and quantification of p-AMPKα, AMPKα, and beclin 1 expression in rat primary motor neurons treated for 8 and 24 h with 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 control and 8 h ML-SA1. (C) Representative 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.
Figure 5
Figure 5
Effect of ML-SA1 on cell death and ER stress in rat primary motor neurons exposed to L-BMAA. (A) Representative Western blotting of TRPML1 and α-tubulin expression in rat primary motor neurons treated with siControl or siRNA #2 against TRPML1. *p < 0.05 vs siControl. (B) Superimposed traces representative of the effect of ML-SA1 (10 μM) on [Ca2+]i in rat primary motor neurons transfected with siControl or siRNA #2 against TRPML1. Inset: bar graph depicts the effect of ML-SA1 expressed as Δ% of [Ca2+]i increase. Each bar represents the mean ± S.E. (n = 20 cells studied in three different experimental sessions). *p < 0.05 vs basal values **p < 0.05 vs siControl. (C) Bar graph depicting the effect of ML-SA1 on cell viability rate of rat primary motor neurons exposed to L-BMAA (300 μM/48 h) in the presence or absence of siRNA against TRPML1. Data are expressed as mean ± S.E. of three different experimental sessions. *p < 0.05 vs control; **p < 0.05 vs L-BMAA alone; ***p < 0.05 vs ML-SA1 + L-BMAA. (DF) Representative Western blotting and quantification of GRP78 (E) and caspase 9 (F) 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 all, ML-SA1 was preincubated for 1 h before L-BMAA exposure.
Figure 6
Figure 6
Effect of ML-SA1 on the autophagic flux in L-BMAA-treated motor neurons. Schematic representation of the prosurvival pathway elicited by ML-SA1 involving TRPML1 activation, lysosomal Ca2+ release and autophagic flux induction in a neurotoxic model of ALS/PDC.

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