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

nuclear encoded ncx3 and akap121 two novel modulators of mitochondrial calcium efflux in normoxic and hypoxic neurons

Abstract Mitochondria are highly dynamic organelles extremely important for cell survival. Their structure resembles that of prokaryotic cells since they are composed with two membranes, the inner (IMM) and the outer mitochondrial membrane (OMM) delimitating the intermembrane space (IMS) and the matrix which contains mitochondrial DNA (mtDNA). This structure is strictly related to mitochondrial function since they produce the most of the cellular ATP through the oxidative phosphorylation which generate the electrochemical gradient at the two sides of the inner mitochondrial membrane an essential requirement for mitochondrial function. Cells of highly metabolic demand like those composing muscle, liver and brain, are particularly dependent on mitochondria for their activities. Mitochondria undergo to continual changes in morphology since, they fuse and divide, branch and fragment, swell and extend. Importantly, they move throughout the cell to deliver ATP and other metabolites where they are mostly required. Along with the capability to control energy metabolism, mitochondria play a critical role in the regulation of many physiological processes such as programmed cell death, autophagy, redox signalling, and stem cells reprogramming. All these phenomena are regulated by Ca2+ ions within this organelle. This review will discuss the molecular mechanisms regulating mitochondrial calcium cycling in physiological and pathological conditions with particular regard to their impact on mitochondrial dynamics and function during ischemia. Particular emphasis will be devoted to the role played by NCX3 and AKAP121 as new molecular targets for mitochondrial function and dysfunction

molecular mechanisms of NCX1 and NCX3-induced neuroprotection in anoxic brain preconditionoing

Ischemic preconditioning (IPC) represents an important adaptation mechanism of CNS, which results in its increased tolerance to the lethal cerebral ischemia. The molecular mechanisms responsible for the induction and maintenance of ischemic tolerance in the brain are complex and remain undefined. As the expression of the three isoforms of the sodium calcium exchanger (NCX), NCX1, NCX2 and NCX3, was reduced during cerebral ischemia and considering the relevance of nitric oxide (NO) in the IPC modulation, we investigated the functional relationship between NO and NCXs in the regulation of Ca2+ homeostasis, as molecular mechanism responsible for ischemic tolerance. To this aim we set up an in vitro model of Ischemic preconditioning by exposing cortical neurons to a 30-min oxygen and glucose deprivation (OGD, sublethal insult) followed by 3hrs OGD followed by reoxygenation (lethal insult). IPC was able to stimulate NCX activity, an effect blocked by L-NAME and siRNA against NCX1 and NCX3 treatment thus suggesting that NCX1 and NCX3 play a pivotal role in the modulation of calcium homeostasis during IPC in a NO-dependent manner. Interestingly, IPC-induced NCX1 increased activity contributes to ER refilling by counteracting ER stress described in OGD/Reoxygenation. Conversely, NCX3 increased activity promotes mitochondrial calcium handling and improves mitochondrial function during OGD/Reoxygenation. Finally, as concern cell viability our data showed that both siNCX1 and siNCX3 prevented the neuroprotection exerted by IPC in rat cortical neurons exposed to OGD/Reoxygenation. Collectively, these results suggest a possible involvement of NCX1 and NCX3 in the crosstalk between ER and mitochondria as novel mechanism for IPC-induced neuroprotection

Lysosomal calcium is modulated by STIM1/TRPML1 interaction which participates to neuronal survival during ischemic preconditioning

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

Rosuvastatin-induced neuroprotection in cortical neurons exposed to OGD/reoxygenation is due to nitric oxide inhibition and ERK1/2 pathway activation

The aim of the present study was to investigate the molecular mechanisms underlying the neuroprotective effect of the hydrophilic statin rosuvastatin on cortical neurons exposed to oxygen and glucose deprivation (OGD) followed by reoxygenation. Rosuvastatin (RSV), at concentrations ranging from 10 nM to 1μM, was able to ameliorate the survival of cortical neurons exposed to OGD followed by reoxygenation. This effect was observed either if neurons were pretreated with RSV 24 hrs before OGD/reoxygenation exposure or if RSV was added during the OGD or the reoxygenation phase. Moreover, RSV was also able to improve mitochondrial oxidative capacity in basal conditions, an effect that was already observed at 10 nM either after 24 or after 48 hrs of treatment. These neuroprotective actions were not counteracted by mevalonate, an intermediate of cholesterol biosynthesis that bypasses RSV induced blockade of cholesterol synthesis. Furthermore, the hypothesis that RSV might affect neuronal nitric oxide synthase (nNOS) activity during OGD/reoxygenation was explored. RSV was able to reduce the increase of NO occurring during the reoxygenation phase, an effect prevented by NPLA, the selective inhibitor of nNOS. Finally, the possibility that RSV-induced NO reduction during OGD/reoxygenation might involve ERK1/2 activation was also investigated. The treatment of neurons with PD98059, an ERK1/2 kinase inhibitor, abolished the neuroprotective effect exerted by RSV in cortical neurons exposed to OGD/reoxygenation. In conclusion, these results demonstrated that RSV-induced neuroprotection involves an impairment of constitutive and inducible NOS activity which in turn causes the improvement of mitochondrial function and the stimulation of ERK1/2 via H-Ras activation

L-Ornithine L-Aspartate Restores Mitochondrial Function and Modulates Intracellular Calcium Homeostasis in Parkinson's Disease Models

The altered crosstalk between mitochondrial dysfunction, intracellular Ca2+ homeostasis, and oxidative stress has a central role in the dopaminergic neurodegeneration. In the present study, we investigated the hypothesis that pharmacological strategies able to improve mitochondrial functions might prevent neuronal dysfunction in in vitro models of Parkinson's disease. To this aim, the attention was focused on the amino acid ornithine due to its ability to cross the blood-brain barrier, to selectively reach and penetrate the mitochondria through the ornithine transporter 1, and to control mitochondrial function. To pursue this issue, experiments were performed in human neuroblastoma cells SH-SY5Y treated with rotenone and 6-hydroxydopamine to investigate the pharmacological profile of the compound L-Ornithine-L-Aspartate (LOLA) as a new potential therapeutic strategy to prevent dopaminergic neurons' death. In these models, confocal microscopy experiments with fluorescent dyes measuring mitochondrial calcium content, mitochondrial membrane potential, and mitochondrial ROS production, demonstrated that LOLA improved mitochondrial functions. Moreover, by increasing NCXs expression and activity, LOLA also reduced cytosolic [Ca2+] thanks to its ability to modulate NO production. Collectively, these results indicate that LOLA, by interfering with those mitochondrial mechanisms related to ROS and RNS production, promotes mitochondrial functional recovery, thus confirming the tight relationship existing between cytosolic ionic homeostasis and cellular metabolism depending on the type of insult applied

Expression and function of Kv7.4 channels in rat cardiac mitochondria:Possible targets for cardioprotection

AIMS: Plasmalemmal Kv7.1 (KCNQ1) channels are critical players in cardiac excitability; however, little is known on the functional role of additional Kv7 family members (Kv7.2-5) in cardiac cells. In this work, the expression, function, cellular and subcellular localization, and potential cardioprotective role against anoxic-ischaemic cardiac injury of Kv7.4 channels have been investigated. METHODS AND RESULTS: Expression of Kv7.1 and Kv7.4 transcripts was found in rat heart tissue by quantitative polymerase chain reaction. Western blots detected Kv7.4 subunits in mitochondria from Kv7.4-transfected cells, H9c2 cardiomyoblasts, freshly isolated adult cardiomyocytes, and whole hearts. Immunofluorescence experiments revealed that Kv7.4 subunits co-localized with mitochondrial markers in cardiac cells, with ∼30-40% of cardiac mitochondria being labelled by Kv7.4 antibodies, a result also confirmed by immunogold electron microscopy experiments. In isolated cardiac (but not liver) mitochondria, retigabine (1-30 µM) and flupirtine (30 µM), two selective Kv7 activators, increased Tl+ influx, depolarized the membrane potential, and inhibited calcium uptake; all these effects were antagonized by the Kv7 blocker XE991. In intact H9c2 cells, reducing Kv7.4 expression by RNA interference blunted retigabine-induced mitochondrial membrane depolarization; in these cells, retigabine decreased mitochondrial Ca2+ levels and increased radical oxygen species production, both effects prevented by XE991. Finally, retigabine reduced cellular damage in H9c2 cells exposed to anoxia/re-oxygenation and largely prevented the functional and morphological changes triggered by global ischaemia/reperfusion (I/R) in Langendorff-perfused rat hearts. CONCLUSION: Kv7.4 channels are present and functional in cardiac mitochondria; their activation exerts a significant cardioprotective role, making them potential therapeutic targets against I/R-induced cardiac injury

The 3-(3-oxoisoindolin-1-yl)pentane-2,4-dione (ISOAC1) as a new molecule able to inhibit Amyloid β aggregation and neurotoxicity

: Amyloid β 1-42 (Aβ1-42) protein aggregation is considered one of the main triggers of Alzheimer's disease (AD). In this study, we examined the in vitro anti-amyloidogenic activity of the isoindolinone derivative 3-(3-oxoisoindolin-1-yl)pentane-2,4-dione (ISOAC1) and its neuroprotective potential against the Aβ1-42 toxicity. By performing the Thioflavin T fluorescence assay, Western blotting analyses, and Circular Dichroism experiments, we found that ISOAC1 was able to reduce the Aβ1-42 aggregation and conformational transition towards β-sheet structures. Interestingly, in silico studies revealed that ISOAC1 was able to bind to both the monomer and a pentameric protofibril of Aβ1-42, establishing a hydrophobic interaction with the PHE19 residue of the Aβ1-42 KLVFF motif. In vitro analyses on primary cortical neurons showed that ISOAC1 counteracted the increase of intracellular Ca2+ levels and decreased the Aβ1-42-induced toxicity, in terms of mitochondrial activity reduction and increase of reactive oxygen species production. In addition, confocal microscopy analyses showed that ISOAC1 was able to reduce the Aβ1-42 intraneuronal accumulation. Collectively, our results clearly show that ISOAC1 exerts a neuroprotective effect by reducing the Aβ1-42 aggregation and toxicity, hence emerging as a promising compound for the development of new Aβ-targeting therapeutic strategies for AD treatment