Pharmacological inhibition of lysosomal two-pore channel 2 (TPC2) confers neuroprotection in stroke via autophagy regulation

Lysosomal function and organellar Ca2+ homeostasis become dysfunctional in Stroke causing disturbances in autophagy, the major process for the degradation of abnormal protein aggregates and dysfunctional organelles. However, the role of autophagy in Stroke is controversial since excessive or prolonged autophagy activation exacerbates ischemic brain injury. Of note, glutamate evokes NAADP-dependent Ca2+ release via lysosomal TPC2 channels thus controlling basal autophagy. Considering the massive release of excitotoxins in Stroke, autophagic flux becomes uncontrolled with abnormal formation of autophagosomes causing, in turn, disruption of excitotoxins clearance and neurodegeneration. Here, a fine regulation of autophagy via a proper pharmacological modulation of lysosomal TPC2 channel has been tested in preclinical Stroke models. Primary cortical neurons were subjected to oxygen and glucose deprivation+reoxygenation to reproduce in vitro brain ischemia. Focal brain ischemia was induced in rats by transient middle cerebral artery occlusion (tMCAO). Under these conditions, TPC2 protein expression as well as autophagy and endoplasmic reticulum (ER) stress markers were studied by Western blotting, while TPC2 localization and activity were measured by immunocytochemistry and single-cell video-imaging, respectively. TPC2 protein expression and immunosignal were highly modulated in primary cortical neurons exposed to extreme hypoxic conditions causing dysfunction in organellar Ca2+ homeostasis, ER stress and autophagy-induced cell death. TPC2 knocking down and pharmacological inhibition by Ned-19 during hypoxia induced neuroprotection. The effect of Ned-19 was reversed by the permeable form of TPC2 endogenous agonist, NAADP-AM. Of note, Ned-19 prevented ER stress, as measured by GRP78 (78 kDa glucose-regulated protein) protein reduction and caspase 9 downregulation. In this way Ned-19 restored organellar Ca2+ level. Interestingly, Ned-19 reduced the infarct volume and neurological deficits in rats subjected to tMCAO and prevented hypoxia-induced cell death by blocking autophagic flux. Collectively, the pharmacological inhibition of TPC2 lysosomal channel by Ned-19 protects from focal ischemia by hampering a hyperfunctional autophag

The pharmacological activation of Mucolipin TRP channel 1 (TRPML1) protects motor neurons exposed to the cycad neurotoxin L-BMAA by promoting autophagic clearance

Cellular clearance mechanisms including the autophagy-lysosome pathway are impaired in the central nervous system (CNS) of amyotrophic lateral sclerosis (ALS) patients. However, how the defects in lysosomal function contribute to the pathogenesis of ALS is unclear. Therefore, we investigated the role of lysosomal function in an in vitro model of amyotrophic lateral sclerosis/Parkinson-dementia complex (i.e. motor neurons exposed to the cycad neurotoxin beta-methylamino-L-alanine named L-BMAA). Considering that the lysosomal Ca2+ channel Mucolipin TRP channel 1 (TRPML1) plays a crucial role in lysosomal function, we studied the role of the channel in this neurotoxic model. Under physiological conditions, TRPML1 colocalized with the endoplasmic reticulum (ER), that drives calcium refilling of lysosomes. In fact, the specific and irreversible SERCA inhibitor thapsigargin reduced lysosomal Ca2+ refilling measured by the genetically-encoded Ca2+ indicator GCaMP3 attached to the lysosomal channel (GCaMP3-ML1). However, TRPML1 expression was dramatically reduced in motor neurons exposed to the L-BMAA, a condition characterized by the impairment of ER Ca2+ store. Therefore, lysosomal Ca2+ release and ER Ca2+ content were both dysregulated by L-BMAA. Very interestingly, the specific membrane-permeable synthetic agonist of TRPML1, ML-SA1, rescued motor neurons from death and ER stress induced by chonic exposure to L-BMAA. Furthermore, under the same conditions, the pre-incubation of ML-SA1 prevented the elevation of the ER stress marker GRP78 and of the autophagy-related proteins p62/SQSTM1 and LC3-II. Notably, ML-SA1 per se induced the activation of the autophagy initiators p-AMPK and beclin 1. Collectively, we propose that the pharmacological stimulation of lysosomal Ca2+ channel TRPML1 can efficiently rescue motor neurons from L-BMAA toxicity by improving autophagy-dependent clearance mechanisms

Endoplasmic reticulum refilling and mitochondrial calcium extrusion promoted in neurons by NCX1 and NCX3 in ischemic preconditioning are determinant for neuroprotection

Ischemic preconditioning (IPC), an important endogenous adaptive mechanism of the CNS, renders the brain more tolerant to lethal cerebral ischemia. The molecular mechanisms responsible for the induction and maintenance of ischemic tolerance in the brain are complex and still remain undefined. Considering the increased expression of the two sodium calcium exchanger (NCX) isoforms, NCX1 and NCX3, during cerebral ischemia and the relevance of nitric oxide (NO) in IPC modulation, we investigated whether the activation of the NO/PI3K/Akt pathway induced by IPC could regulate calcium homeostasis through changes in NCX1 and NCX3 expression and activity, thus contributing to ischemic tolerance. To this aim, we set up an in vitro model of IPC by exposing cortical neurons to a 30-min oxygen and glucose deprivation (OGD) followed by 3-h OGD plus reoxygenation. IPC was able to stimulate NCX activity, as revealed by Fura-2AM single-cell microfluorimetry. This effect was mediated by the NO/PI3K/ Akt pathway since it was blocked by the following: (a) the NOS inhibitors L-NAME and 7-Nitroindazole, (b) the IP3K/Akt inhibitors LY294002, wortmannin and the Akt-negative dominant, (c) the NCX1 and NCX3 siRNA. Intriguingly, this IPC-mediated upregulation of NCX1 and NCX3 activity may control calcium level within endoplasimc reticulum (ER) and mitochondria, respectively. In fact, IPC-induced NCX1 upregulation produced an increase in ER calcium refilling since this increase was prevented by siNCX1. Moreover, by increasing NCX3 activity, IPC reduced mitochondrial calcium concentration. Accordingly, the inhibition of NCX by CGP37157 reverted this effect, thus suggesting that IPC-induced NCX3-increased activity may improve mitochondrial function during OGD/reoxygenation. Collectively, these results indicate that IPC-induced neuroprotection may occur through the modulation of calcium homeostasis in ER and mitochondria through NO/PI3K/Akt-mediated NCX1 and NCX3 upregulations

New insights in mitochondrial calcium handling by sodium/calcium exchanger

Mitochondria are now recognized as one of the main intracellular calcium-storing organelles which play a key role in the intracellular calcium signalling. Indeed, besides performing oxidative phosphorylation, mitochondria are able to sense and shape calcium (Ca(2+)) transients, thus controlling cytosolic Ca(2+) signals and Ca(2+)-dependent protein activity. It has been well established for many years that mitochondria have a huge capacity to accumulate calcium. While the physiological significance of this pathway was hotly debated until relatively recently, it is now clear that the ability of mitochondria in calcium handling is a ubiquitous phenomenon described in every cell system in which the issue has been addressed.In this chapter, we will review the molecular mechanisms involved in the regulation of mitochondrial calcium cycling in physiological conditions with particular regard to the role played by the mitochondrial Na(+)/Ca(2+) exchange

Transcriptional Regulation of ncx1 Gene in the Brain

The ubiquitous sodium-calcium exchanger isoform 1 (NCX1) is a -bidirectional transporter that plays a relevant role under physiological and pathophysiological conditions including brain ischemia by regulating intraneuronal Ca(2+) and Na(+) homeostasis. Although changes in ncx1 protein and transcript expression have been detected during stroke, its transcriptional regulation is still largely unexplored. Here, we reviewed our recent findings on several transcription factors including cAMP response element-binding protein (CREB), nuclear factor kappa B (NF-κB), and hypoxia-inducible factor-1 (HIF-1) in the control of the ncx1 gene expression in neuronal cells

NCX1 Is a Novel Target Gene for Hypoxia-Inducible Factor-1 in Ischemic Brain Preconditioning

BACKGROUND AND PURPOSE: The sodium-calcium exchanger-1 (NCX1) represents a key mediator for maintaining [Na(+)](i) and [Ca(2+)](i) homeostasis. Although changes in NCX1 protein and transcript expression have been detected during stroke, its transcriptional regulation is still unknown. Thus far, however, there is evidence that hypoxia-inducible factor-1 (HIF-1) is a nuclear factor required for transcriptional activation of several genes implicated in stroke. The main objective of this study was to investigate whether NCX1 gene might be a novel target of HIF-1 in the brain. METHODS: Here we report that: (1) in neuronal cells, NCX1 increased expression after oxygen and glucose deprivation or cobalt-induced HIF-1 activation was prevented by silencing HIF-1; (2) the brain NCX1 promoter cloned upstream of the firefly-luciferase gene contained 2 regions of HIF-1 target genes called hypoxia-responsive elements that are sensitive to oxygen and glucose deprivation or cobalt chloride; (3) HIF-1 specifically bound hypoxia-responsive elements on brain NCX1, as demonstrated by band-shift and chromatin immunoprecipitation assays; (4) HIF-1α silencing prevented NCX1 upregulation and neuroprotection induced by ischemic preconditioning; and (5) NCX1 silencing partially reverted the preconditioning-induced neuroprotection in rats. CONCLUSIONS: NCX1 gene is a novel HIF-1 target, and HIF-1 exerts its prosurvival role through NCX1 upregulation during brain preconditioning

NCX3 regulates mitochondrial calcium handling through AKAP121-anchored signaling complex and prevents hypoxia-induced cell death.

The mitochondrial influx and efflux calcium pathways play a relevant role in cytosolic and mitochondrial calcium homeostasis and contribute to the regulation of mitochondrial functions in neurons. The mitochondrial Na+/Ca2+ exchanger, although hypothesized in 1974, has been primarily investigated only from a functional point of view and its identity and localization in the mitochondria have been a matter of debate over the last three decades. Recently, a lithium-dependent sodium/calcium exchanger extruding calcium from the matrix has been found in the inner mitochondrial membrane of neuronal cells. However, evidence has been provided that the outer membrane is impermeable to calcium efflux into the cytoplasm. In this study, we have demonstrated for the first time that the nuclear encoded NCX3 isoform (a) is localized on the outer mitochondrial membrane (OMM) of neurons, (b) co-localizes and immunoprecipitates with AKAP121, a member of the protein kinase A anchoring proteins (AKAPs) present on the outer membrane, (c) extrudes calcium from mitochondria through AKAP121 interaction in a PKA-mediated manner, both under normoxia and hypoxia, and (d) improves cell survival when it works in the Ca2+ efflux mode at the level of the OMM. Collectively, these results suggest that, in neurons, NCX3 regulates mitochondrial calcium handling from the OMM through an AKAP121-anchored signalling complex, thus promoting cell survival during hypoxia

NCX1 is a new rest target gene: role in cerebral ischemia

The Na(+)-Ca(2+) exchanger 1 (NCX1), a bidirectional transporter that mediates the electrogenic exchange of one calcium ion for three sodium ions across the plasma membrane, is known to be involved in brain ischemia. Since the RE1-silencing transcription factor (REST) is a key modulator of neuronal gene expression in several neurological conditions, we studied the possible involvement of REST in regulating NCX1 gene expression and activity in stroke. We found that: (1) REST binds in a sequence specific manner and represses through H4 deacetylation, ncx1 gene in neuronal cells by recruting CoREST, but not mSin3A. (2) In neurons and in SH-SY5Y cells REST silencing by siRNA and site-direct mutagenesis of REST consensus sequence on NCX1 brain promoter determined an increase in NCX1 promoter activity. (3) By contrast, REST overexpression caused a reduction in NCX1 protein expression and activity. (4) Interestingly, in rats subjected to transient middle cerebral artery occlusion (tMCAO) and in organotypic hippocampal slices or SH-SY5Y cells exposed to oxygen and glucose deprivation (OGD) plus reoxygenation (RX), the increase in REST was associated with a decrease in NCX1. However, this reduction was reverted by REST silencing. (5) REST knocking down, along with the deriving NCX1 overexpression in the deep V and VIb cortical layers caused a marked reduction in infarct volume after tMCAO. Double silencing of REST and NCX1 completely abolished neuroprotection induced by siREST administration. Collectively, these results demonstrate that REST, by regulating NCX1 expression, may represent a potential druggable target for the treatment of brain ischemia

Neurounina-1, a Novel Compound that Increases Na+/Ca2+ Exchanger Activity, Effectively Protects Against Stroke Damage

Previous studies have demonstrated that the knock-down or knock-out of the three Na(+)/Ca(2+) exchanger (NCX) isoforms, NCX1, NCX2 and NCX3, worsens ischemic brain damage. This suggests that the activation of these antiporters exerts a neuroprotective action against stroke damage. However, drugs able to increase the activity of NCXs are not yet available. We have here succeeded in synthesizing a new compound, named neurounina-1 (7-nitro-5-phenyl-1-(pyrrolidin-1-ylmethyl)-1H-benzo[e][1,4]diazepin-2(3H)-one), provided with an high lipophilicity index and able to increase NCX activity. Ca(2+)-radiotracer, Fura-2-microfluorimetry, and patch-clamp techniques revealed that neurounina-1 stimulated NCX1 and NCX2 activities with an EC(50) in the picomolar-low-nanomolar range, whereas it did not affect NCX3 activity. Furthermore, by using chimera strategy and site-directed mutagenesis, three specific molecular determinants of NCX1 responsible for neurounina-1 activity were identified in the α-repeats. Interestingly, NCX3 became responsive to neurounina-1 when both α-repeats were replaced with the corresponding regions of NCX1. In vitro studies showed that 10nM neurounina-1 reduced cell death of primary cortical neurons exposed to oxygen-glucose deprivation followed by reoxygenation. Moreover, in vitro, neurounina-1 also reduced GABA release, enhanced GABA(A)-currents, and inhibited both glutamate release and NMDA receptors. More important, neurounina-1 proved to have a wide therapeutic window in vivo. Indeed, when administered i.p. at doses ranging 0.003-30 μg/kg, it was able to reduce the infarct volume of mice subjected to transient middle cerebral artery occlusion even up to 3-5h after stroke onset. Collectively, the present study shows that neurounina-1 exerts a remarkable neuroprotective effect during stroke and increases NCX1 and NCX2 activities