Taming Microglia in Alzheimer's Disease: Exploring Potential Implications of Choline Alphoscerate via α7 nAChR Modulation.

Alzheimer's disease (AD), marked by cognitive impairment, predominantly affects the brain regions regulated by cholinergic innervation, such as the cerebral cortex and hippocampus. Cholinergic dysfunction, a key contributor to age-related cognitive decline, has spurred investigations into potential therapeutic interventions. We have previously shown that choline alphoscerate (α-GPC), a cholinergic neurotransmission-enhancing agent, protects from Aß-mediated neurotoxicity. Herein, we investigated the effects of α-GPC on the microglial phenotype in response to Aß via modulation of the nicotinic alpha-7 acetylcholine receptor (α7 nAChR). BV2 microglial cells were pre-treated for 1 h with α-GPC and were treated for 24, 48, and 72 h with Aß1-42 and/or α-BTX, a selective α7nAchR antagonist. Fluorescent immunocytochemistry and Western blot analysis showed that α-GPC was able to antagonize Aß-induced inflammatory effects. Of note, α-GPC exerted its anti-inflammatory effect by directly activating the α7nAChR receptor, as suggested by the induction of an increase in [Ca2+]i and Ach-like currents. Considering that cholinergic transmission appears crucial in regulating the inflammatory profiles of glial cells, its modulation emerges as a potential pharmaco-therapeutic target to improve outcomes in inflammatory neurodegenerative disorders, such as AD.

Ultrafine particulate matter pollution and dysfunction of endoplasmic reticulum Ca2+ store: A pathomechanism shared with amyotrophic lateral sclerosis motor neurons?

Increased risk of neurodegenerative diseases has been envisaged for air pollution exposure. On the other hand, environmental risk factors, including air pollution, have been suggested for Amyotrophic Lateral Sclerosis (ALS) pathomechanism. Therefore, the neurotoxicity of ultrafine particulate matter (PM0.1) (PM < 0.1 µm size) and its sub-20 nm nanoparticle fraction (NP20) has been investigated in motor neuronal-like cells and primary cortical neurons, mainly affected in ALS. The present data showed that PM0.1 and NP20 exposure induced endoplasmic reticulum (ER) stress, as occurred in cortex and spinal cord of ALS mice carrying G93A mutation in SOD1 gene. Furthermore, NSC-34 motor neuronal-like cells exposed to PM0.1 and NP20 shared the same proteomic profile on some apoptotic factors with motor neurons treated with the L-BMAA, a neurotoxin inducing Amyotrophic Lateral Sclerosis/Parkinson-Dementia Complex (ALS/PDC). Of note ER stress induced by PM0.1 and NP20 in motor neurons was associated to pathological changes in ER morphology and dramatic reduction of organellar Ca2+ level through the dysregulation of the Ca2+-pumps SERCA2 and SERCA3, the Ca2+-sensor STIM1, and the Ca2+-release channels RyR3 and IP3R3. Furthermore, the mechanism deputed to ER Ca2+ refilling (e.g. the so called store operated calcium entry-SOCE) and the relative currents ICRAC were also altered by PM0.1 and NP20 exposure. Additionally, these carbonaceous particles caused the exacerbation of L-BMAA-induced ER stress and Caspase-9 activation. In conclusion, this study shows that PM0.1 and NP20 induced the aberrant expression of ER proteins leading to dysmorphic ER, organellar Ca2+ dysfunction, ER stress and neurotoxicity, providing putative correlations with the neurodegenerative process occurring in ALS.

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

Amyloid ß 1-42 (Aß1-42) 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 Aß1-42 toxicity. By performing the Thioflavin T fluorescence assay, Western blotting analyses, and Circular Dichroism experiments, we found that ISOAC1 was able to reduce the Aß1-42 aggregation and conformational transition towards ß-sheet structures. Interestingly, in silico studies revealed that ISOAC1 was able to bind to both the monomer and a pentameric protofibril of Aß1-42, establishing a hydrophobic interaction with the PHE19 residue of the Aß1-42 KLVFF motif. In vitro analyses on primary cortical neurons showed that ISOAC1 counteracted the increase of intracellular Ca2+ levels and decreased the Aß1-42-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 Aß1-42 intraneuronal accumulation. Collectively, our results clearly show that ISOAC1 exerts a neuroprotective effect by reducing the Aß1-42 aggregation and toxicity, hence emerging as a promising compound for the development of new Aß-targeting therapeutic strategies for AD treatment.

Size-Based Effects of Anthropogenic Ultrafine Particles on Lysosomal TRPML1 Channel and Autophagy in Motoneuron-like Cells.

Background: An emerging body of evidence indicates an association between anthropogenic particulate matter (PM) and neurodegeneration. Although the historical focus of PM toxicity has been on the cardiopulmonary system, ultrafine PM particles can also exert detrimental effects in the brain. However, only a few studies are available on the harmful interaction between PM and CNS and on the putative pathomechanisms. Methods: Ultrafine PM particles with a diameter < 0.1 µm (PM0.1) and nanoparticles < 20 nm (NP20) were sampled in a lab-scale combustion system. Their effect on cell tracking in the space was studied by time-lapse and high-content microscopy in NSC-34 motor neurons while pHrodo™ Green conjugates were used to detect PM endocytosis. Western blotting analysis was used to quantify protein expression of lysosomal channels (i.e., TRPML1 and TPC2) and autophagy markers. Current-clamp electrophysiology and Fura2-video imaging techniques were used to measure membrane potential, intracellular Ca2+ homeostasis and TRPML1 activity in NSC-34 cells exposed to PM0.1 and NP20. Results: NP20, but not PM0.1, reduced NSC-34 motor neuron movement in the space. Furthermore, NP20 was able to shift membrane potential of motor neurons toward more depolarizing values. PM0.1 and NP20 were able to enter into the cells by endocytosis and exerted mitochondrial toxicity with the consequent stimulation of ROS production. This latter event was sufficient to determine the hyperactivation of the lysosomal channel TRPML1. Consequently, both LC3-II and p62 protein expression increased after 48 h of exposure together with AMPK activation, suggesting an engulfment of autophagy. The antioxidant molecule Trolox restored TRPML1 function and autophagy. Conclusions: Restoring TRPML1 function by an antioxidant agent may be considered a protective mechanism able to reestablish autophagy flux in motor neurons exposed to nanoparticles.

Increased KV2.1 Channel Clustering Underlies the Reduction of Delayed Rectifier K+ Currents in Hippocampal Neurons of the Tg2576 Alzheimer's Disease Mouse.

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.

IN BRAIN POST-ISCHEMIC PLASTICITY, Na+/Ca2+ EXCHANGER 1 AND Ascl1 INTERVENE IN MICROGLIA-DEPENDENT CONVERSION OF ASTROCYTES INTO NEURONAL LINEAGE.

The intricate glia interaction occurring after stroke strongly depend on the maintenance of intraglial ionic homeostasis. Among the several ionic channels and transporters, the plasmamembrane Na+/Ca2+ exchanger (NCX) represents a key player in maintaining astroglial Na+ and Ca2+ homeostasis. Here, using a combined in vitro, in vivo and ex vivo experimental strategy we evaluated whether microglia responding to ischemic injury may influence the morphological and the transcriptional plasticity of post-ischemic astrocytes. Astrocyte plasticity was monitored by the expression of the transcription factor Acheate-scute like 1 (Ascl1), which plays a central role in the commitment of astrocytes towards the neuronal lineage. Furthermore, we explored the implication of NCX1 expression and activity in mediating Ascl1-dependent post-ischemic astrocyte remodeling. We demonstrated that: (a) in astrocytes co-cultured with microglia the exposure to oxygen and glucose deprivation followed by 7 days of reoxygenation induced a prevalence of bipolar astrocytes overexpressing Ascl1 and NCX1, whereas this did not occur in monocultured astrocytes; (b) the reoxygenation of anoxic astrocytes with the conditioned medium derived from IL-4 stimulated microglia strongly elicited the astrocytic co-expression of Ascl1 and NCX1; (c) Ascl1 expression in anoxic astrocytes was dependenton NCX1 since its silencing prevented Ascl1 expression both in in vitro and in post-ischemic ex vivo experimental conditions. Collectively, the results of our study support the idea that, after brain ischemia, astrocyte-microglia crosstalk can influence astrocytic morphology and its Ascl1 expression. This phenomenon is strictly dependent on ischemia-induced increase of NCX1 which in turn induces Ascl1 overexpression possibly through astrocytic Ca2+ elevation.

Exploring the Therapeutic Potential of Phytochemicals in Alzheimer's Disease: Focus on Polyphenols and Monoterpenes.

Alzheimer's disease (AD) is a chronic, complex neurodegenerative disorder mainly characterized by the irreversible loss of memory and cognitive functions. Different hypotheses have been proposed thus far to explain the etiology of this devastating disorder, including those centered on the Amyloid-ß (Aß) peptide aggregation, Tau hyperphosphorylation, neuroinflammation and oxidative stress. Nonetheless, the therapeutic strategies conceived thus far to treat AD neurodegeneration have proven unsuccessful, probably due to the use of single-target drugs unable to arrest the progressive deterioration of brain functions. For this reason, the theoretical description of the AD etiology has recently switched from over-emphasizing a single deleterious process to considering AD neurodegeneration as the result of different pathogenic mechanisms and their interplay. Moreover, much relevance has recently been conferred to several comorbidities inducing insulin resistance and brain energy hypometabolism, including diabetes and obesity. As consequence, much interest is currently accorded in AD treatment to a multi-target approach interfering with different pathways at the same time, and to life-style interventions aimed at preventing the modifiable risk-factors strictly associated with aging. In this context, phytochemical compounds are emerging as an enormous source to draw on in the search for multi-target agents completing or assisting the traditional pharmacological medicine. Intriguingly, many plant-derived compounds have proven their efficacy in counteracting several pathogenic processes such as the Aß aggregation, neuroinflammation, oxidative stress and insulin resistance. Many strategies have also been conceived to overcome the limitations of some promising phytochemicals related to their poor pharmacokinetic profiles, including nanotechnology and synthetic routes. Considering the emerging therapeutic potential of natural medicine, the aim of the present review is therefore to highlight the most promising phytochemical compounds belonging to two major classes, polyphenols and monoterpenes, and to report the main findings about their mechanisms of action relating to the AD pathogenesis.

Na+/Ca2+ exchanger isoform 1 takes part to the Ca2+-related prosurvival pathway of SOD1 in primary motor neurons exposed to beta-methylamino-L-alanine.

BACKGROUND: The cycad neurotoxin beta-methylamino-L-alanine (L-BMAA), one of the environmental trigger factor for amyotrophic lateral sclerosis/Parkinson-dementia complex (ALS/PDC), may cause neurodegeneration by disrupting organellar Ca2+ homeostasis. Through the activation of Akt/ERK1/2 pathway, the Cu,Zn-superoxide dismutase (SOD1) and its non-metallated form, ApoSOD1, prevent endoplasmic reticulum (ER) stress-induced cell death in motor neurons exposed to L-BMAA. This occurs through the rapid increase of intracellular Ca2+ concentration ([Ca2+]i) in part flowing from the extracellular compartment and in part released from ER. However, the molecular components of this mechanism remain uncharacterized. METHODS: By an integrated approach consisting on the use of siRNA strategy, Western blotting, confocal double- labeling immunofluorescence, patch-clamp electrophysiology, and Fura 2-/SBFI-single-cell imaging, we explored in rat motor neuron-enriched cultures the involvement of the plasma membrane proteins Na+/Ca2+ exchanger (NCX) and purinergic P2X7 receptor as well as that of the intracellular cADP-ribose (cADPR) pathway, in the neuroprotective mechanism of SOD1. RESULTS: We showed that SOD1-induced [Ca2+]i rise was prevented neither by A430879, a P2X7 receptor specific antagonist or 8-bromo-cADPR, a cell permeant antagonist of cADP-ribose, but only by the pan inhibitor of NCX, CB-DMB. The same occurred for the ApoSOD1. Confocal double labeling immunofluorescence showed a huge expression of plasmalemmal NCX1 and intracellular NCX3 isoforms. Furthermore, we identified NCX1 reverse mode as the main mechanism responsible for the neuroprotective ER Ca2+ refilling elicited by SOD1 and ApoSOD1 through which they promoted translocation of active Akt in the nuclei of a subset of primary motor neurons. Finally, the activation of NCX1 by the specific agonist CN-PYB2 protected motor neurons from L-BMAA-induced cell death, mimicking the effect of SOD1. CONCLUSION: Collectively, our data indicate that SOD1 and ApoSOD1 exert their neuroprotective effect by modulating ER Ca2+ content through the activation of NCX1 reverse mode and Akt nuclear translocation in a subset of primary motor neurons. Video Abstract.

Na+/Ca2+ exchanger isoform 1 (NCX1) and canonical transient receptor potential channel 6 (TRPC6) are recruited by STIM1 to mediate Store-Operated Calcium Entry in primary cortical neurons.

Excessive calcium (Ca2+) release from the endoplasmic reticulum (ER) represents an important hallmark of several neurodegenerative diseases. ER is recharged from Ca2+ through the so-called Store-Operated Calcium Entry (SOCE) thus providing Ca2+ signals to regulate critical cell functions. Single transmembrane-spanning domain protein stromal interacting molecule 1 (STIM1), mainly residing in the ER, and plasmalemmal channel Orai1 represent the SOCE key components at neuronal level. However, many other proteins participate to ER Ca2+ refilling including the Na+/Ca2+ exchanger isoform 1 (NCX1), whose regulation by ER remains unknown. In this study, we tested the possibility that neuronal NCX1 may take part to SOCE through the interaction with STIM1. In rat primary cortical neurons and in nerve growth factor (NGF)-differentiated PC12 cells NCX1 knocking down by siRNA strategy significantly prevented SOCE as well as SOCE pharmacological inhibition by SKF-96365 and 2-APB. A significant reduction of SOCE was recorded also in synaptosomes from ncx1-/- mice brain compared with ncx1+/+ mice. Double labeling confocal experiments showed a large co-localization between NCX1 and STIM1 in rat primary cortical neurons. Accordingly, NCX1 and STIM1 co-immunoprecipitated and functionally interacted each other during ischemic preconditioning, a phenomenon inducing ischemic tolerance. However, STIM1 knocking down reduced NCX1 activity recorded by either patch-clamp electrophysiology or Fura-2 single-cell microfluorimetry. Furthermore, canonical transient receptor potential channel 6 (TRPC6) was identified as the mechanism mediating local increase of sodium (Na+) useful to drive NCX1 reverse mode and, therefore, NCX1-mediated Ca2+ refilling. In fact, TRPC6 not only interacted with STIM1, as shown by the co-localization and co-immunoprecipitation with the ER Ca2+ sensor, but it also mediated 1,3-Benzenedicarboxylic acid, 4,4'-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,12-benzofurandiyl)]bis-, tetrakis[(acetyloxy)methyl] ester (SBFI)-monitored Na+ increase elicited by thapsigargin in primary cortical neurons. Accordingly, efficient TRPC6 knockdown prevented thapsigargin-induced intracellular Na+ elevation and SOCE. Collectively, we identify NCX1 as a new partner of STIM1 in mediating SOCE, whose activation in the reverse mode may be facilitated by the local increase of Na+ concentration due to the interaction between STIM1 and TRPC6 in primary cortical neurons.

The Na+/Ca2+ Exchanger 3 Is Functionally Coupled With the NaV1.6 Voltage-Gated Channel and Promotes an Endoplasmic Reticulum Ca2+ Refilling in a Transgenic Model of Alzheimer's Disease.

The remodelling of neuronal ionic homeostasis by altered channels and transporters is a critical feature of the Alzheimer's disease (AD) pathogenesis. Different reports converge on the concept that the Na+/Ca2+ exchanger (NCX), as one of the main regulators of Na+ and Ca2+ concentrations and signalling, could exert a neuroprotective role in AD. The activity of NCX has been found to be increased in AD brains, where it seemed to correlate with an increased neuronal survival. Moreover, the enhancement of the NCX3 currents (INCX) in primary neurons treated with the neurotoxic amyloid ß 1-42 (Aß1-42) oligomers prevented the endoplasmic reticulum (ER) stress and neuronal death. The present study has been designed to investigate any possible modulation of the INCX, the functional interaction between NCX and the NaV1.6 channel, and their impact on the Ca2+ homeostasis in a transgenic in vitro model of AD, the primary hippocampal neurons from the Tg2576 mouse, which overproduce the Aß1-42 peptide. Electrophysiological studies, carried in the presence of siRNA and the isoform-selective NCX inhibitor KB-R7943, showed that the activity of a specific NCX isoform, NCX3, was upregulated in its reverse, Ca2+ influx mode of operation in the Tg2576 neurons. The enhanced NCX activity contributed, in turn, to increase the ER Ca2+ content, without affecting the cytosolic Ca2+ concentrations of the Tg2576 neurons. Interestingly, our experiments have also uncovered a functional coupling between NCX3 and the voltage-gated NaV1.6 channels. In particular, the increased NaV1.6 currents appeared to be responsible for the upregulation of the reverse mode of NCX3, since both TTX and the Streptomyces griseolus antibiotic anisomycin, by reducing the NaV1.6 currents, counteracted the increase of the INCX in the Tg2576 neurons. In agreement, our immunofluorescence analyses revealed that the NCX3/NaV1.6 co-expression was increased in the Tg2576 hippocampal neurons in comparison with the WT neurons. Collectively, these findings indicate that NCX3 might intervene in the Ca2+ remodelling occurring in the Tg2576 primary neurons thus emerging as a molecular target with a neuroprotective potential, and provide a new outcome of the NaV1.6 upregulation related to the modulation of the intracellular Ca2+ concentrations in AD neurons.

New Insights into the Structure-Activity Relationship and Neuroprotective Profile of Benzodiazepinone Derivatives of Neurounina-1 as Modulators of the Na+/Ca2+ Exchanger Isoforms.

Due to the neuroprotective role of the Na+/Ca2+ exchanger (NCX) isoforms NCX1 and NCX3, we synthesized novel benzodiazepinone derivatives of the unique NCX activator Neurounina-1, named compounds 1-19. The derivatives are characterized by a benzodiazepinonic nucleus linked to five- or six-membered cyclic amines via a methylene, ethylene, or acetyl spacer. The compounds have been screened on NCX1/NCX3 isoform activities by a high-throughput screening approach, and the most promising were characterized by patch-clamp electrophysiology and Fura-2AM video imaging. We identified two novel modulators of NCX: compound 4, inhibiting NCX1 reverse mode, and compound 14, enhancing NCX1 and NCX3 activity. Compound 1 displayed neuroprotection in two preclinical models of brain ischemia. The analysis of the conformational and steric features led to the identification of the molecular volume required for selective NCX1 activation for mixed NCX1/NCX3 activation or for NCX1 inhibition, providing the first prototypal model for the design of optimized isoform modulators.

Rebound effects of NCX3 pharmacological inhibition: A novel strategy to accelerate myelin formation in oligodendrocytes.

The Na+/Ca2+ exchanger NCX3 is an important regulator of sodium and calcium homeostasis in oligodendrocyte lineage. To date, no information is available on the effects resulting from prolonged exposure to NCX3 blockers and subsequent drug washout in oligodendroglia. Here, we investigated, by means of biochemical, morphological and functional analyses, the pharmacological effects of the NCX3 inhibitor, the 5-amino-N-butyl-2-(4-ethoxyphenoxy)-benzamide hydrochloride (BED), on NCXs expression and activity, as well as intracellular [Na+]i and [Ca2+]i levels, during treatment and following drug washout both in human MO3.13 oligodendrocytes and rat primary oligodendrocyte precursor cells (OPCs). BED exposure antagonized NCX activity, induced OPCs proliferation and [Na+]i accumulation. By contrast, 2 days of BED washout after 4 days of treatment significantly upregulated low molecular weight NCX3 proteins, reversed NCX activity, and increased intracellular [Ca2+]i. This BED-free effect was accompanied by an upregulation of NCX3 expression in oligodendrocyte processes and accelerated expression of myelin markers in rat primary oligodendrocytes. Collectively, our findings show that the pharmacological inhibition of the NCX3 exchanger with BED blocker maybe followed by a rebound increase in NCX3 expression and reversal activity that accelerate myelin sheet formation in oligodendrocytes. In addition, they indicate that a particular attention should be paid to the use of NCX inhibitors for possible rebound effects, and suggest that further studies will be necessary to investigate whether selective pharmacological modulation of NCX3 exchanger may be exploited to benefit demyelination and remyelination in demyelinating diseases.

The Antioxidant Activity of Limonene Counteracts Neurotoxicity Triggered byAß1-42 Oligomers in Primary Cortical Neurons.

Many natural-derived compounds, including the essential oils from plants, are investigated to find new potential protective agents in several neurodegenerative disorders such as Alzheimer's disease (AD). In the present study, we tested the neuroprotective effect of limonene, one of the main components of the genus Citrus, against the neurotoxicity elicited by Aß1-42 oligomers, currently considered a triggering factor in AD. To this aim, we assessed the acetylcholinesterase activity by Ellman's colorimetric method, the mitochondrial dehydrogenase activity by MTT assay, the nuclear morphology by Hoechst 33258, the generation of reactive oxygen species (ROS) by DCFH-DA fluorescent dye, and the electrophysiological activity of KV3.4 potassium channel subunits by patch-clamp electrophysiology. Interestingly, the monoterpene limonene showed a specific activity against acetylcholinesterase with an IC50 almost comparable to that of galantamine, used as positive control. Moreover, at the concentration of 10 µg/mL, limonene counteracted the increase of ROS production triggered by Aß1-42 oligomers, thus preventing the upregulation of KV3.4 activity. This, in turn, prevented cell death in primary cortical neurons, showing an interesting neuroprotective profile against Aß1-42-induced toxicity. Collectively, the present results showed that the antioxidant properties of the main component of the genus Citrus, limonene, may be useful to prevent neuronal suffering induced by Aß1-42 oligomers preventing the hyperactivity of KV3.4.

Synthesis and Characterization of Novel Mono- and Bis-Guanyl Hydrazones as Potent and Selective ASIC1 Inhibitors Able to Reduce Brain Ischemic Insult.

Acid-sensitive ion channels (ASICs) are sodium channels partially permeable to Ca2+ ions, listed among putative targets in central nervous system (CNS) diseases in which a pH modification occurs. We targeted novel compounds able to modulate ASIC1 and to reduce the progression of ischemic brain injury. We rationally designed and synthesized several diminazene-inspired diaryl mono- and bis-guanyl hydrazones. A correlation between their predicted docking affinities for the acidic pocket (AcP site) in chicken ASIC1 and their inhibition of homo- and heteromeric hASIC1 channels in HEK-293 cells was found. Their activity on murine ASIC1a currents and their selectivity vs mASIC2a were assessed in engineered CHO-K1 cells, highlighting a limited isoform selectivity. Neuroprotective effects were confirmed in vitro, on primary rat cortical neurons exposed to oxygen-glucose deprivation followed by reoxygenation, and in vivo, in ischemic mice. Early lead 3b, showing a good selectivity for hASIC1 in human neurons, was neuroprotective against focal ischemia induced in mice.

Lavender and coriander essential oils and their main component linalool exert a protective effect against amyloid-ß neurotoxicity.

Alzheimer's disease (AD) is a neurodegenerative disorder leading to cognitive deficits and cognitive decline. Since no cure or preventing therapy is currently available to counteract AD, natural-derived compounds are investigated to find new potential neuroprotective agents for its treatment. In the present study, we tested the neuroprotective effect of lavender and coriander essential oils (EOs) and their main active constituent linalool, against the neurotoxicity elicited by Aß1-42 oligomers, a key molecular factor in the neurodegeneration of AD. Importantly, our findings on neuronally differentiated PC12 cells exposed to Aß1-42 oligomers are in accordance with previous in vivo studies reporting the neuroprotective potential of lavender and coriander EOs and linalool. We found that lavender and coriander EOs at the concentration of 10 µg/mL as well as linalool at the same concentration were able to improve viability and to reduce nuclear morphological abnormalities in cells treated with Aß1-42 oligomers for 24 hours. Lavender and coriander EOs and linalool also showed to counteract the increase of intracellular reactive oxygen species production and the activation of the pro-apoptotic enzyme caspase-3 induced by Aß1-42 oligomers. Our findings provide further evidence that these EOs and their main constituent linalool could be natural agents of therapeutic interest against Aß1-42 -induced neurotoxicity.

Genetically modified mice to unravel physiological and pathophysiological roles played by NCX isoforms.

Since the discovery of the three isoforms of the Na+/Ca2+ exchanger, NCX1, NCX2 and NCX3 in 1990s, many studies have been devoted to identifying their specific roles in different tissues under several physiological or pathophysiological conditions. In particular, several seminal experimental works laid the foundation for better understanding gene and protein structures, tissue distribution, and regulatory functions of each antiporter isoform. On the other hand, despite the efforts in the development of specific compounds selectively targeting NCX1, NCX2 or NCX3 to test their physiological or pathophysiological roles, several drawbacks hampered the achievement of these goals. In fact, at present no isoform-specific compounds have been yet identified. Moreover, these compounds, despite their potency, possess some nonspecific actions against other ion antiporters, ion channels, and channel receptors. As a result, it is difficult to discriminate direct effects of inhibition/activation of NCX isoforms from the inhibitory or stimulatory effects exerted on other antiporters, channels, receptors, or enzymes. To overcome these difficulties, some research groups used transgenic, knock-out and knock-in mice for NCX isoforms as the most straightforward and fruitful strategy to characterize the biological role exerted by each antiporter isoform. The present review will survey the techniques used to study the roles of NCXs and the current knowledge obtained from these genetic modified mice focusing on the advantages obtained with these strategies in understanding the contribution exerted by each isoform.

The Na+/Ca2+exchanger in Alzheimer's disease.

As a pivotal player in regulating sodium (Na+) and calcium (Ca2+) homeostasis and signalling in excitable cells, the Na+/Ca2+ exchanger (NCX) is involved in many neurodegenerative disorders in which an imbalance of intracellular Ca2+ and/or Na+ concentrations occurs, including Alzheimer's disease (AD). Although NCX has been mainly implicated in neuroprotective mechanisms counteracting Ca2+ dysregulation, several studies highlighted its role in the neuronal responses to intracellular Na+ elevation occurring in several pathophysiological conditions. Since the alteration of Na+ and Ca2+ homeostasis significantly contributes to synaptic dysfunction and neuronal loss in AD, it is of crucial importance to analyze the contribution of NCX isoforms in the homeostatic responses at neuronal and synaptic levels. Some studies found that an increase of NCX activity in brains of AD patients was correlated with neuronal survival, while other research groups found that protein levels of two NCX subtypes, NCX2 and NCX3, were modulated in parietal cortex of late stage AD brains. In particular, NCX2 positive synaptic terminals were increased in AD cohort while the number of NCX3 positive terminals were reduced. In addition, NCX1, NCX2 and NCX3 isoforms were up-regulated in those synaptic terminals accumulating amyloid-beta (Aß), the neurotoxic peptide responsible for AD neurodegeneration. More recently, the hyperfunction of a specific NCX subtype, NCX3, has been shown to delay endoplasmic reticulum stress and apoptotic neuronal death in hippocampal neurons exposed to Aß insult. Despite some issues about the functional role of NCX in synaptic failure and neuronal loss require further studies, these findings highlight the putative neuroprotective role of NCX in AD and open new strategies to develop new druggable targets for AD therapy.

Genetic Up-Regulation or Pharmacological Activation of the Na+/Ca2+ Exchanger 1 (NCX1) Enhances Hippocampal-Dependent Contextual and Spatial Learning and Memory.

The Na+/Ca2+ exchanger 1 (NCX1) participates in the maintenance of neuronal Na+ and Ca2+ homeostasis, and it is highly expressed at synapse level of some brain areas involved in learning and memory processes, including the hippocampus, cortex, and amygdala. Furthermore, NCX1 increases Akt1 phosphorylation and enhances glutamate-mediated Ca2+ influx during depolarization in hippocampal and cortical neurons, two processes involved in learning and memory mechanisms. We investigated whether the modulation of NCX1 expression/activity might influence learning and memory processes. To this aim, we used a knock-in mouse overexpressing NCX1 in hippocampal, cortical, and amygdala neurons (ncx1.4over) and a newly synthesized selective NCX1 stimulating compound, named CN-PYB2. Both ncx1.4over and CN-PYB2-treated mice showed an amelioration in spatial learning performance in Barnes maze task, and in context-dependent memory consolidation after trace fear conditioning. On the other hand, these mice showed no improvement in novel object recognition task which is mainly dependent on non-spatial memory and displayed an increase in the active phosphorylated CaMKIIα levels in the hippocampus. Interestingly, both of these mice showed an increased level of context-dependent anxiety.Altogether, these results demonstrate that neuronal NCX1 participates in spatial-dependent hippocampal learning and memory processes.

The Anemonia sulcata Toxin BDS-I Protects Astrocytes Exposed to Aß1-42 Oligomers by Restoring [Ca2+]i Transients and ER Ca2+ Signaling.

Intracellular calcium concentration ([Ca2+]i) transients in astrocytes represent a highly plastic signaling pathway underlying the communication between neurons and glial cells. However, how this important phenomenon may be compromised in Alzheimer's disease (AD) remains unexplored. Moreover, the involvement of several K+ channels, including KV3.4 underlying the fast-inactivating currents, has been demonstrated in several AD models. Here, the effect of KV3.4 modulation by the marine toxin blood depressing substance-I (BDS-I) extracted from Anemonia sulcata has been studied on [Ca2+]i transients in rat primary cortical astrocytes exposed to Aß1-42 oligomers. We showed that: (1) primary cortical astrocytes expressing KV3.4 channels displayed [Ca2+]i transients depending on the occurrence of membrane potential spikes, (2) BDS-I restored, in a dose-dependent way, [Ca2+]i transients in astrocytes exposed to Aß1-42 oligomers (5 µM/48 h) by inhibiting hyperfunctional KV3.4 channels, (3) BDS-I counteracted Ca2+ overload into the endoplasmic reticulum (ER) induced by Aß1-42 oligomers, (4) BDS-I prevented the expression of the ER stress markers including active caspase 12 and GRP78/BiP in astrocytes treated with Aß1-42 oligomers, and (5) BDS-I prevented Aß1-42-induced reactive oxygen species (ROS) production and cell suffering measured as mitochondrial activity and lactate dehydrogenase (LDH) release. Collectively, we proposed that the marine toxin BDS-I, by inhibiting the hyperfunctional KV3.4 channels and restoring [Ca2+]i oscillation frequency, prevented Aß1-42-induced ER stress and cell suffering in astrocytes.