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Additional file 1. Fig. S1. Ipatasertib and taselisib determines antitumor effects in MDAMB361 cells. a Ipatasertib and taselisib reduce cell proliferation and ability to prevent clonogenic formation after daily administration for 10 days of IC30 doses for each drug or at fixed drug dose of 1µM, expressed as % of CTL. Each experiment is representative of three independent experiments. b Ipatasertib and taselisib treatment impair 3D tumor spheroid derived from MDAMB361 breast cancer cells. Representative images of first generation 3D tumor spheroid, exposed to ipatasertib and taselisib, administrated to IC30 doses for 72h. Tumor cell growth was reduced. Quantification of ATP was used to measure reduction of cellular growth, expressed as % of CTL. Each experiment is representative of three independent experiments. Statistically significant results are reported (*** indicates P < 0.0005, ** indicates P < 0.005 and * indicates P < 0.05). c The exposure to fixed dose (IC50) of ipatasertib and taselisib induces the reduction of expression of phospho-mTOR, associated with the increase of autophagy signaling, as showed by increase of LC3 II/LC3 I ratio by immunoblot assay by reduction of p62 and increase of ATG5 after 24h of exposure in MDAMB361 cells (*** indicates P < 0.0005, ** indicates P < 0.005 and * indicates P < 0.05). d The addition of CQ 10µM to ipatasertib and taselisib (IC50) induces accumulation of LC3II and p62 protein expression after 24h, due the reduction of autophagic flux, while expression of cleaved parp was not significant increased in taselisib +CQ and in ipatasertib+CQ groups in MDAMB361 cell line. e Representative confocal images of MDAMB361 cell lines immuno-stained with anti-LC3IIb antibody reveals accumulation of autophagosomes (green dots) in treatments with ipatasertib, taselisib, CQ or combinations, due to the induction of autophagy or reduction of autophagic flux exerted by CQ. (*** indicates P < 0.0005, ** indicates P < 0.005 and * indicates P < 0.05). Fig. S2. Ipatasertib and taselisib induce cell cycle alteration as well as apoptosis in Breast cancer cells. a The reduction in proliferation was also associated with perturbation in the cell cycle, after 24h of treatment with ipatasertib and taselisib at IC30 doses, leading to an increase in the G1/G2 phase depending on the cell lines (MCF7, SKBR3, MDAMB361 and MDAMB231) expressed as % of CTL (b) or apoptosis measured by enhancement of Annexin V-FITC and Propidium Iodide, after 24 of treatment at IC30 doses, expressed as % of CTL. Similar representative experiments have yielded similar results. Statistically significant results were reported (*** indicates P < 0.0005, ** indicates P < 0.005 and * indicates P < 0.05). Fig. S3. Ipatasertib and taselisib induce autophagy in MDAMB231 cells. Representative confocal images of MDAMB231 cells overexpressing GFP-LC3 and immuno-stained with anti-p62 ab, reveal accumulation of autophagosomes (green dots) that colocalise with p62 (Merge) in CQ treatments, due to the reduction in autophagic flux exerted by CQ. Ipatasertib, taselisib and CQ were administered at a 10µM dose for 24h. Similar representative experiments yielded similar results. Statistically significant results were reported (*** indicates P < 0.0005, ** indicates P < 0.005 and * indicates P < 0.05). Fig. S4. Chloroquine potentiate antitumor effects induced by ipatasertib and taselisib. a The addition of CQ 1-10-20µM to ipatasertib or taselisib inhibits cell proliferation measured by SRB assay after 48h in MCF7 and SKBR3, and 72h in MDAMB361 on treatments indicated, expressed as % of CTL. Each experiment is representative of three independent experiments. (b) The addition of CQ 10µM to ipatasertib and taselisib (IC30 for MCF7, SKBR3, MDAMB361 and 10µM for MDAMB231) causes an increase of cytotoxicity measured by LDH release after 24h, expressed as % of CTL. Each experiment is representative of three independent experiments. Statistically significant results were reported (*** indicates P < 0.0005, ** indicates P < 0.005 and * indicates P < 0.05). Fig. S5. Taselisib induce anti-proliferative effects via autophagy mediated by ATG5. a ATG-5-siRNA transfection in MDAMB231 cell lines induces a relevant reduction in ATG5 protein expression compared with the control non-transfected or scramble transfected cells within 24 h from transfection b) In the SRB-based proliferation assay, TAS+ATG5-siRNA and TAS+ATG5-siRNA+paclitaxel groups showed a significant reduction in the proliferation rate after 72h of treatment. Ipatasertib and taselisib were administered at a 1µM dose and paclitaxel at a 0.5nM dose. Statistically significant results were reported (* indicates p < 0.05).

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the progressive deterioration of cognitive functions. Cortical and hippocampal hyperexcitability intervenes in the pathological derangement of brain activity leading to cognitive decline. As key regulators of neuronal excitability, the voltage-gated K+ channels (KV) might play a crucial role in the AD pathophysiology. Among them, the KV2.1 channel, the main α subunit mediating the delayed rectifier K+ currents (IDR) and controlling the intrinsic excitability of pyramidal neurons, has been poorly examined in AD. In the present study, we investigated the KV2.1 protein expression and activity in hippocampal neurons from the Tg2576 mouse, a widely used transgenic model of AD. To this aim we performed whole-cell patch-clamp recordings, Western blotting, and immunofluorescence analyses. Our Western blotting results reveal that KV2.1 was overexpressed in the hippocampus of 3-month-old Tg2576 mice and in primary hippocampal neurons from Tg2576 mouse embryos compared with the WT counterparts. Electrophysiological experiments unveiled that the whole IDR were reduced in the Tg2576 primary neurons compared with the WT neurons, and that this reduction was due to the loss of the KV2.1 current component. Moreover, we found that the reduction of the KV2.1-mediated currents was due to increased channel clustering, and that glutamate, a stimulus inducing KV2.1 declustering, was able to restore the IDR to levels comparable to those of the WT neurons. These findings add new information about the dysregulation of ionic homeostasis in the Tg2576 AD mouse model and identify KV2.1 as a possible player in the AD-related alterations of neuronal excitability.

A robust activity of the lysosomal Ca2+ 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)P2 diC8; (3) its knockdown by siRNA strategy produced an ER Ca2+ accumulation; (4) co-localized and co-immunoprecipitated with the ER-located Ca2+ sensor stromal interacting molecule 1 (STIM1). In cortical neurons lacking STIM1, ML-SA1 and PI(3,5)P2 diC8 failed to induce Ca2+ release and, more deeply, they induced a negligible Ca2+ passage through the channel in neurons transfected with the genetically encoded Ca2+ 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 Ca2+ release and lysosomal Ca2+ loss. Collectively, we showed a new modulation exerted by STIM1 on TRPML1 activity that may differently intervene during hypoxia to regulate organellar Ca2+ homeostasis.

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.