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The downstream regulatory element antagonist modulator (DREAM) is a multifunctional Ca2+-sensitive protein exerting a dual mechanism of action to regulate several Ca2+-dependent processes. Upon sumoylation, DREAM enters in nucleus where it downregulates the expression of several genes provided with a consensus sequence named dream regulatory element (DRE). On the other hand, DREAM could also directly modulate the activity or the localization of several cytosolic and plasma membrane proteins. In this review, we summarize recent advances in the knowledge of DREAM dysregulation and DREAM-dependent epigenetic remodeling as a central mechanism in the progression of several diseases affecting central nervous system, including stroke, Alzheimer's and Huntington's diseases, amyotrophic lateral sclerosis, and neuropathic pain. Interestingly, DREAM seems to exert a common detrimental role in these diseases by inhibiting the transcription of several neuroprotective genes, including the sodium/calcium exchanger isoform 3 (NCX3), brain-derived neurotrophic factor (BDNF), pro-dynorphin, and c-fos. These findings lead to the concept that DREAM might represent a pharmacological target to ameliorate symptoms and reduce neurodegenerative processes in several pathological conditions affecting central nervous system. [ABSTRACT FROM AUTHOR]

Intrathecal delivery of Nusinersen–an antisense oligonucleotide that promotes survival motor neuron (SMN) protein induction–is an approved therapy for spinal muscular atrophy (SMA). Here, we employed nuclear magnetic resonance (NMR) spectroscopy to longitudinally characterize the unknown metabolic effects of Nusinersen in the cerebrospinal fluid (CSF) of SMA patients across disease severity. Modulation of amino acid metabolism is a common denominator of biochemical changes induced by Nusinersen, with distinct downstream metabolic effects according to disease severity. In severe SMA1 patients, Nusinersen stimulates energy-related glucose metabolism. In intermediate SMA2 patients, Nusinersen effects are also related to energy homeostasis but involve ketone body and fatty acid biosynthesis. In milder SMA3 patients, Nusinersen mainly modulates amino acid metabolism. Moreover, Nusinersen modifies the CSF metabolome of a more severe clinical group towards the profile of untreated SMA patients with milder disease. These findings reveal disease severity-specific neurometabolic signatures of Nusinersen treatment, suggesting a selective modulation of peripheral organ metabolism by this CNS-directed therapy in severe SMA patients. [ABSTRACT FROM AUTHOR]

L-Tryptophan (TRP) metabolites and related biomarkers play crucial roles in physiological functions, and their imbalances are implicated in central nervous system pathologies and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, schizophrenia and depression. The measurement of TRP metabolites and related biomarkers possesses great potential to elucidate the disease mechanisms, aid preclinical drug development, highlight potential therapeutic targets and evaluate the outcomes of therapeutic interventions. An effective, straightforward, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of 24 TRP-related compounds in miniaturised murine whole blood samples. Sampling and sample pretreatment miniaturisation were achieved thanks to the development of a volumetric dried blood microsampling approach. Volumetric absorptive microsampling (VAMS) allows the accurate sampling of microvolumes of blood with advantages including, but not limited to, minimal sampling invasiveness, logistical improvements, method sustainability in terms of solvents and energy consumption, and improvement of animal studies in the framework of the 3Rs (Replacement, Reduction and Refinement) principles on animal welfare. The VAMS-LC-MS/MS method exhibited good selectivity, and correlation coefficient values for the calibration curves of each analyte were >0.9987. The limits of quantitation ranged from 0.1 to 25 ng/mL. The intra- and inter-day precisions in terms of RSD were <9.6%. All analytes were stable in whole blood VAMS samples stored at room temperature for at least 30 days with analyte losses < 14%. The developed method was successfully applied to the analysis of biological samples from mice, leading to the unambiguous determination of all the considered target analytes. This method can therefore be applied to analyse TRP metabolites and related biomarkers levels to monitor disease states, perform mechanistic studies and investigate the outcomes of therapeutic interventions. [ABSTRACT FROM AUTHOR]

Abstract Background Current tools for analgesia are often only partially successful, thus investigations of new targets for pain therapy stimulate great interest. Consequent to peripheral nerve injury, c-Jun N-terminal kinase (JNK) activity in cells of the dorsal root ganglia (DRGs) and spinal cord is involved in triggering neuropathic pain. However, the relative contribution of distinct JNK isoforms is unclear. Using knockout mice for single isoforms, and blockade of JNK activity by a peptide inhibitor, we have used behavioral tests to analyze the contribution of JNK in the development of neuropathic pain after unilateral sciatic nerve transection. In addition, immunohistochemical labelling for the growth associated protein (GAP)-43 and Calcitonin Gene Related Peptide (CGRP) in DRGs was used to relate injury related compensatory growth to altered sensory function. Results Peripheral nerve injury produced pain–related behavior on the ipsilateral hindpaw, accompanied by an increase in the percentage of GAP43-immunoreactive (IR) neurons and a decrease in the percentage of CGRP-IR neurons in the lumbar DRGs. The JNK inhibitor, D-JNKI-1, successfully modulated the effects of the sciatic nerve transection. The onset of neuropathic pain was not prevented by the deletion of a single JNK isoform, leading us to conclude that all JNK isoforms collectively contribute to maintain neuropathy. Autotomy behavior, typically induced by sciatic nerve axotomy, was absent in both the JNK1 and JNK3 knockout mice. Conclusions JNK signaling plays an important role in regulating pain threshold: the inhibition of all of the JNK isoforms prevents the onset of neuropathic pain, while the deletion of a single splice JNK isoform mitigates established sensory abnormalities. JNK inactivation also has an effect on axonal sprouting following peripheral nerve injury.

Amyotrophic lateral sclerosis (ALS) is one of the most threatening neurodegenerative disease since it causes muscular paralysis for the loss of Motor Neurons in the spinal cord, brainstem and motor cortex. Up until now, no effective pharmacological treatment is available. Two forms of ALS have been described so far: 90% of the cases presents the sporadic form (sALS) whereas the remaining 10% of the cases displays the familiar form (fALS). Approximately 20% of fALS is associated with inherited mutations in the Cu, Zn-superoxide dismutase 1 (SOD1) gene. In the last decade, ionic homeostasis dysregulation has been proposed as the main trigger of the pathological cascade that brings to motor-neurons loss. In the light of these premises, the present review will analyze the involvement in ALS pathophysiology of the most well studied metal ions, i.e., calcium, sodium, iron, copper and zinc, with particular focus to the role of ionic channels and transporters able to contribute in the regulation of ionic homeostasis, in order to propose new putative molecular targets for future therapeutic strategies to ameliorate the progression of this devastating neurodegenerative disease. [ABSTRACT FROM AUTHOR]

Methylmercury (MeHg) causes neuronal death through different pathways. Particularly, we found that in cortical neurons it increased the expression of Repressor Element-1 Silencing Transcription Factor (REST), histone deacetylase (HDAC)4, Specificity Protein (Sp)1, Sp4, and reduced the levels of brain-derived neurotrophic factor (BDNF). Herein, in rat cortical neurons we investigated whether microRNA (miR)206 can modulate MeHg-induced cell death by regulating REST/HDAC4/Sp1/Sp4/BDNF axis. MeHg (1 µM) reduced miR206 expression after both 12 and 24 h and miR206 transfection prevented MeHg-induced neuronal death. Furthermore, miR206 reverted MeHg-induced REST and Sp4 increase and BDNF reduction at gene and protein level, and reverted HDAC4 protein increase, but not HDAC4 mRNA upregulation. Moreover, since no miR206 seed sequences were identified in the 3'-untranslated regions (3'-UTRs) of REST and SP4, we investigated the role of JunD, that presents a consensus motif on REST, Sp4, and BDNF promoters. Indeed, MeHg increased JunD mRNA and protein levels, and JunD knockdown counteracted MeHg-induced REST, Sp4 increase, but not BDNF reduction. Furthermore, we identified a miR206 binding site in the 3'-UTR of JunD mRNA (miR206/JunD) and mutagenesis of miR206/JunD site reverted JunD luciferase activity reduction induced by miR206. Finally, miR206 prevented MeHg-increased JunD binding to REST and Sp4 promoters, and MeHg-reduced BDNF expression was determined by the increase of HDAC4 binding on BDNF promoter IV. Collectively, these results suggest that miR206 downregulation induced by MeHg exposure determines an upregulation of HDAC4, that in turn downregulated BDNF, and the activation of JunD that, by binding REST and Sp4 gene promoters, increased their expression. [ABSTRACT FROM AUTHOR]

Spinal muscular atrophy (SMA) is a pediatric genetic disease, characterized by motor neuron (MN) death, leading to progressive muscle weakness, respiratory failure, and, in the most severe cases, to death. Abnormalities at the neuromuscular junction (NMJ) have been reported in SMA, including neurofilament (NF) accumulation at presynaptic terminals, immature and smaller than normal endplates, reduced transmitter release, and, finally, muscle denervation. Here we have studied the role of agrin in SMAΔ7 mice, the experimental model of SMAII. We observed a 50% reduction in agrin expression levels in quadriceps of P10 SMA mice compared to age-matched WT controls. To counteract such condition, we treated SMA mice from birth onwards with therapeutic agrin biological NT-1654, an active splice variant of agrin retaining synaptogenic properties, which is also resistant to proteolytic cleavage by neurotrypsin. Mice were analyzed for behavior, muscle and NMJ histology, and survival. Motor behavior was significantly improved and survival was extended by treatment of SMA mice with NT-1654. At P10, H/E-stained sections of the quadriceps, a proximal muscle early involved in SMA, showed that NT-1654 treatment strongly prevented the size decrease of muscle fibers. Studies of NMJ morphology on whole-mount diaphragm preparations revealed that NT-1654-treated SMA mice had more mature NMJs and reduced NF accumulation, compared to vehicle-treated SMA mice. We conclude that increasing agrin function in SMA has beneficial outcomes on muscle fibers and NMJs as the agrin biological NT-1654 restores the crosstalk between muscle and MNs, delaying muscular atrophy, improving motor performance and extending survival. [ABSTRACT FROM AUTHOR]

The molecular pathways involved in methylmercury (MeHg)-induced neurotoxicity are not fully understood. Since pan-Histone deacetylases (HDACs) inhibition has been found to revert the neurodetrimental effect of MeHg, it appeared of interest to investigate whether the pattern of HDACs isoform protein expression is modified during MeHg-induced neurotoxicity and the transcriptional/transductional mechanisms involved. SH-SY5Y neuroblastoma cells treated with MeHg 1 μM for 12 and 24 h showed a significant increase of HDAC4 protein and gene expression, whereas the HDACs isoforms 1-3, 5, and 6 were unmodified. Furthermore, MeHg-induced HDAC4 increase was reverted when cells were transfected with siRNAs against specificity protein 1 (Sp1) and Sp4, that were both increased during MeHg exposure. Next we studied the role of extracellular-signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK), and p38 mitogen-activated protein kinases (MAPKs) in MeHg-induced increase of Sp1, Sp4, and HDAC4 expression. As shown by Western Blot analysis MeHg exposure increased the phosphorylation of p38, but not of ERK and JNK. Notably, when p38 was pharmacologically blocked, MeHg-induced Sp1, Sp4 protein expression, and HDAC4 protein and gene expression was reverted. In addition, MeHg exposure increased the binding of HDAC4 to the promoter IV of the Brain-derived neurotrophic factor (BDNF) gene, determining its mRNA reduction, that was significantly counteracted by HDAC4 knocking down. Furthermore, rat cortical neurons exposed to MeHg (1 μM/24 h) showed an increased phosphorylation of p38, in parallel with an up-regulation of Sp1, Sp4, and HDAC4 and a down-regulation of BDNF proteins. Importantly, transfection of siRNAs against p38, Sp1, Sp4, and HDAC4 or transfection of vector overexpressing BDNF significantly blocked MeHg-induced cell death in cortical neurons. All these results suggest that p38/Sp1-Sp4/HDAC4/BDNF may represent a new pathway involved in MeHg-induced neurotoxicity. [ABSTRACT FROM AUTHOR]

Spinal muscular atrophy (SMA) is a severe neuromuscular disease, the most common in infancy, and the third one among young people under 18 years. The major pathological landmark of SMA is a selective degeneration of lower motor neurons, resulting in progressive skeletal muscle denervation, atrophy, and paralysis. Recently, it has been shown that specific or general changes in the activity of ribonucleoprotein containing micro RNAs (miRNAs) play a role in the development of SMA. Additionally miRNA-206 has been shown to be required for efficient regeneration of neuromuscular synapses after acute nerve injury in an ALS mouse model. Therefore, we correlated the morphology and the architecture of the neuromuscular junctions (NMJs) of quadriceps, a muscle affected in the early stage of the disease, with the expression levels of miRNA-206 in a mouse model of intermediate SMA (SMAII), one of the most frequently used experimental model. Our results showed a decrease in the percentage of type II fibers, an increase in atrophic muscle fibers and a remarkable accumulation of neurofilament (NF) in the pre-synaptic terminal of the NMJs in the quadriceps of SMAII mice. Furthermore, molecular investigation showed a direct link between miRNA-206-HDAC4-FGFBP1, and in particular, a strong up-regulation of this pathway in the late phase of the disease. We propose that miRNA-206 is activated as survival endogenous mechanism, although not sufficient to rescue the integrity of motor neurons. We speculate that early modulation of miRNA-206 expression might delay SMA neurodegenerative pathway and that miRNA-206 could be an innovative, still relatively unexplored, therapeutic target for SMA. [ABSTRACT FROM AUTHOR]

The Na+-Ca2+ exchanger 1 (NCX1) is reduced in stroke by the RE1-silencing transcription factor (REST), whereas it is increased in ischemic brain preconditioning (PC) by hypoxia-inducible factor 1 (HIF-1). Because ncx1 brain promoter (ncx1-Br) has five putative consensus sequences, named Sp1A-E, for the specificity protein (Sp) family of transcription factors (Sp1-4), we investigated the role of this family in regulating ncx1 transcription in rat cortical neurons. Here we found that Sp1 is a transcriptional activator, whereas Sp3 is a transcriptional repressor of ncx1, and that both bind ncx1-Br in a sequence-specific manner, modulating ncx1 transcription through the Sp1 sites C-E. Furthermore, by transient middle cerebral artery occlusion (tMCAO) in rats, the transcriptional repressors Sp3 and REST colocalized with the two histone-deacetylases (HDACs) HDAC1 and HDAC2 on the ncx1-Br, with a consequent hypoacetylation. Contrarily, in PC+tMCAO the transcriptional activators Sp1 and HIF-1 colocalized with histone acetyltransferase p300 on ncx1-Br with a consequent hyperacetylation. In addition, in neurons silenced with siRNA of NCX1 and subjected to oxygen and glucose deprivation (OGD) (3 h) plus reoxygenation (RX) (24 h), the neuroprotection of Class I HDAC inhibitor MS-275 was counteracted, whereas in neurons overexpressing NCX1 and subjected to ischemic preconditioning (PC+OGD/RX), the neurotoxic effect of p300 inhibitor C646 was prevented. Collectively, these results demonstrate that NCX1 expression is regulated by the Sp3/REST/HDAC1/HDAC2 complex in tMCAO and by the Sp1/HIF-1/p300 complex in PC+tMCAO and that epigenetic intervention, by modulating the acetylation of ncx1-Br, may be a strategy for the development of innovative therapeutic intervention in stroke. [ABSTRACT FROM AUTHOR]

• NCX2 promoter is up-regulated by CREB1, Sp1 and Sp4 in PC12 cells. • NCX2 promoter has many putative binding sites for TFs involved in neuronal development and plasticity, regulation of processes related to metabolism, cell proliferation and oncogenesis. The isoform 2 of sodium-calcium exchanger family (NCX2) is selectively expressed in neuronal and glial cells where it participates in Ca2+-clearance following neuronal depolarization, synaptic plasticity, hippocampal-dependent learning and memory consolidation processes. On the other hand, NCX2 is also involved in a neuroprotective effect following stroke. Despite the relevance of this antiporter under physiological and pathophysiological conditions, no studies have been reported on its genetic/epigenetic regulation. Therefore, we identified, cloned, and characterized a transcriptional regulatory region (R3) of rat Slc8a2 gene encoding for NCX2. In particular, R3 sequence displayed a promoter activity in PC12, SH-SY5Y and U87MG cell lines consistent with their endogenous NCX2 expression levels. On the other hand, R3 failed to induce detectable luciferase activity in BHK cell line that does not express NCX2 under control conditions. These data support the hypothesis that R3 represents the promoter region of NCX2. Moreover, among several conserved binding sequences for transcription factors identified by in-silico analysis, we evaluated the transcriptional regulation and the binding sites of Sp1, Sp4, NFkB1, GATA2 and CREB1 on R3 sequence by using site-direct mutagenesis and ChIP assays. In particular, transfection of Sp1, Sp4, and CREB1 enhanced both R3 promoter activity and NCX2 transcription in PC12 cell line. More important, CREB1 transfection also enhanced NCX2 protein levels and NCX reverse mode activity in PC12 cells. Altogether, these data suggested that: (i) the identified region contained the regulatory promoter of the antiporter; (ii) NCX2 might represent a downstream effector of transcription factors involved in synaptic plasticity and neuronal survival. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract: The specific role played by NCX1, NCX2, and NCX3, the three isoforms of the Na+/Ca2+ exchanger (NCX), has been explored during hypoxic conditions in BHK cells stably transfected with each of these isoforms. Six major findings emerged from the present study: (1) all the three isoforms were highly expressed on the plasma membranes of BHK cells; (2) under physiological conditions, the three NCX isoforms showed similar functional activity; (3) hypoxia plus reoxygenation induced a lower increase of [Ca2+]i in BHK-NCX3-transfected cells than in BHK-NCX1- and BHK-NCX2-transfected cells; (4) NCX3-transfected cells were more resistant to chemical hypoxia plus reoxygenation than NCX1- and NCX2-transfected cells. Interestingly, such augmented resistance was eliminated by CBDMD (10μM), an inhibitor of NCX and by the specific silencing of the NCX3 isoform; (5) chemical hypoxia plus reoxygenation produced a loss of mitochondrial membrane potential in NCX1- and NCX2-transfected cells, but not in NCX3-transfected cells; (6) the forward mode of operation in NCX3-transfected cells was not affected by ATP depletion, as it occurred in NCX1- and NCX2-transfected cells. Altogether, these results indicate that the brain specifically expressed NCX3 isoform more significantly contributes to the maintenance of [Ca2+]i homeostasis during experimental conditions mimicking ischemia, thereby preventing mitochondrial Δψ collapses and cell death. [Copyright &y& Elsevier]

Imbalance in cellular ionic homeostasis is a hallmark of several neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS). Sodium-calcium exchanger (NCX) is a membrane antiporter that, operating in a bidirectional way, couples the exchange of Ca2+ and Na + ions in neurons and glial cells, thus controlling the intracellular homeostasis of these ions. Among the three NCX genes, NCX1 and NCX2 are widely expressed within the CNS, while NCX3 is present only in skeletal muscles and at lower levels of expression in selected brain regions. ALS mice showed a reduction in the expression and activity of NCX1 and NCX2 consistent with disease progression, therefore we aimed to investigate their role in ALS pathophysiology. Notably, we demonstrated that the pharmacological activation of NCX1 and NCX2 by the prolonged treatment of SOD1G93A mice with the newly synthesized compound neurounina: (1) prevented the reduction in NCX activity observed in spinal cord; (2) preserved motor neurons survival in the ventral spinal horn of SOD1G93A mice; (3) prevented the spinal cord accumulation of misfolded SOD1; (4) reduced astroglia and microglia activation and spared the resident microglia cells in the spinal cord; (5) improved the lifespan and mitigated motor symptoms of ALS mice. The present study highlights the significant role of NCX1 and NCX2 in the pathophysiology of this neurodegenerative disorder and paves the way for the design of a new pharmacological approach for ALS. • The pharmacological activation of NCX1 and NCX2 prevented the reduction in NCX activity observed in spinal cord of ALS mice. • NCX activation preserved motor neurons survival in the ventral spinal horn of SOD1G93A mice. • NCX activation prevented the spinal cord accumulation of misfolded SOD1. • NCX activation reduced astroglia and microglia activation and spared the resident microglia cells in the spinal cord. • NCX activation improved the lifespan and mitigated motor symptoms of ALS mice. [ABSTRACT FROM AUTHOR]

• Ion channels and transporters represent an important class of SUMO targets. • NCX3 represents a target of SUMO-1 conjugation. • SUMOylation regulates the stability of NCX3 during ischemic preconditioning. • NCX3 SUMOylation is a neuroprotective mechanism during ischemic preconditioning. • SUMO-1 silencing prevents neuroprotection through NCX3 downregulation. The small ubiquitin-like modifier (SUMO) conjugation (or SUMOylation) is a post-translational protein modification mechanism activated by different stress conditions that has been recently investigated in experimental models of cerebral ischemia. The expression of SUMOylation enzymes and substrates is not restricted to the nucleus, since they are present also in the cytoplasm and on plasma membrane and are involved in several physiological and pathological conditions. In the last decades, convincing evidence have supported the idea that the increased levels of SUMOylated proteins may induce tolerance to ischemic stress. In particular, it has been established that protein SUMOylation may confer neuroprotection during ischemic preconditioning. Considering the increasing evidence that SUMO can modify stability and expression of ion channels and transporters and the relevance of controlling ionic homeostasis in ischemic conditions, the present review will resume the main aspects of SUMO pathways related to the key molecules involved in maintenance of ionic homeostasis during cerebral ischemia and ischemic preconditioning, with a particular focus on the on Na+/Ca2+ exchangers. [ABSTRACT FROM AUTHOR]

Loss of the survival motor neuron gene (SMN1) is responsible for spinal muscular atrophy (SMA), the most common inherited cause of infant mortality. Even though the SMA phenotype is traditionally considered as related to spinal motor neuron loss, it remains debated whether the specific targeting of motor neurons could represent the best therapeutic option for the disease. We here investigated, using stereological quantification methods, the spinal cord and cerebral motor cortex of ∆7 SMA mice during development, to verify extent and selectivity of motor neuron loss. We found progressive post-natal loss of spinal motor neurons, already at pre-symptomatic stages, and a higher vulnerability of motor neurons innervating proximal and axial muscles. Larger motor neurons decreased in the course of disease, either for selective loss or specific developmental impairment. We also found a selective reduction of layer V pyramidal neurons associated with layer V gliosis in the cerebral motor cortex. Our data indicate that in the ∆7 SMA model SMN loss is critical for the spinal cord, particularly for specific motor neuron pools. Neuronal loss, however, is not selective for lower motor neurons. These data further suggest that SMA pathogenesis is likely more complex than previously anticipated. The better knowledge of SMA models might be instrumental in shaping better therapeutic options for affected patients. [ABSTRACT FROM AUTHOR]

Abstract: The Na+–Ca2+ exchanger 1 (NCX1), a bidirectional transporter that mediates the electrogenic exchange of one calcium ion for three sodium ions across the plasmamembrane, 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 NCX1overexpression 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. [Copyright &y& Elsevier]

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by reduced expression of the survival motor neuron (SMN) protein. In addition to motor neuron survival, SMN deficiency affects the integrity and function of afferent synapses that provide glutamatergic excitatory drive essential for motor neuron firing and muscle contraction. However, it is unknown whether deficits in the metabolism of excitatory amino acids and their precursors contribute to neuronal dysfunction in SMA. To address this issue, we measured the levels of the main neuroactive D- and L-amino acids acting on glutamatergic receptors in the central nervous system of SMNΔ7 mice as well as the cerebrospinal fluid (CSF) of SMA patients of varying severity before and after treatment with the SMN-inducing drug Nusinersen. Our findings reveal that SMN deficiency disrupts glutamate and serine metabolism in the CSF of severe SMA patients, including decreased concentration of L-glutamate, which is partially corrected by Nusinersen therapy. Moreover, we identify dysregulated L-glutamine to L-glutamate conversion as a shared neurochemical signature of altered glutamatergic synapse metabolism that implicates astrocyte dysfunction in both severe SMA patients and mouse models. Lastly, consistent with a correlation of higher CSF levels of D-serine with better motor function in severe SMA patients, we show that daily supplementation with the NMDA receptor co-agonist D-serine improves neurological deficits in SMNΔ7 mice. Altogether, these findings provide direct evidence for dysregulation of D- and L-amino acid metabolism linked to glutamatergic neurotransmission in severe SMA and have potential implications for treating this neurological disorder., Competing Interests: Declaration of Competing Interest C.B. received advisory board honoraria from Avexis, Biogen, Novartis and Roche. The other authors declare no competing interests.

Remote limb ischemic postconditioning (RLIP) is a well-established neuroprotective strategy able to protect the brain from a previous harmful ischemic insult through a sub-lethal occlusion of the femoral artery. Neural and humoral mechanisms have been proposed as mediators required to transmit the peripheral signal from limb to brain. Moreover, different studies suggest that protection observed at brain level is associated to a general genetic reprogramming involving also microRNAs (miRNAs) intervention. Methods: Brain ischemia was induced in male rats by transient occlusion of the middle cerebral artery (tMCAO), whereas RLIP was achieved by one cycle of temporary occlusion of the ipsilateral femoral artery after tMCAO. The expression profile of 810 miRNAs was evaluated in ischemic brain samples from rats subjected either to tMCAO or to RLIP. Among all analyzed miRNAs, there were four whose expression were upregulated after stroke and returned to basal level after RLIP, thus suggesting a possible involvement in RLIP-induced neuroprotection. These selected miRNAs were intracerebroventricularly infused in rats subjected to remote ischemic postconditioning, and their effect was evaluated in terms of brain damage, neurological deficit scores and expression of putative targets. Results: Twenty-one miRNAs, whose expression was significantly affected by tMCAO and by tMCAO plus RLIP, were selected based on microarray microfluidic profiling. Our data showed that: (1) stroke induced an up-regulation of let-7a and miR-143 (2) these two miRNAs were involved in the protective effects induced by RLIP and (3) HIF1-α contributes to their protective effect. Indeed, their expression was reduced after RLIP and the exogenous intracerebroventricularly infusion of let-7a and miR-143 mimics prevented neuroprotection and HIF1-α overexpression induced by RLIP. Conclusions: Prevention of cerebral let-7a and miR-143 overexpression induced by brain ischemia emerges as new potential strategy in stroke intervention., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects both lower and upper motor neurons (MNs) in the central nervous system. ALS etiology is highly multifactorial and multifarious, and an effective treatment is still lacking. Neuroinflammation is a hallmark of ALS and could be targeted to develop new therapeutic approaches. Interestingly, the transcription factor Nurr1 has been demonstrated to have an important role in the inflammatory process in several neurological disorders, such as Parkinson's disease and multiple sclerosis. In the present paper, we demonstrate for the first time that Nurr1 expression levels are upregulated in the peripheral blood of ALS patients. Moreover, we investigated Nurr1 function in the SOD1-G93A mouse model of ALS. Nurr1 was strongly upregulated in the spinal cord during the asymptomatic and early symptomatic phases of the disease, where it promoted the expression of brain-derived neurotrophic factor mRNA and the repression of NFκB pro-inflammatory targets, such as inducible nitric oxide synthase. Therefore, we hypothesize that Nurr1 is activated in an early phase of the disease as a protective endogenous anti-inflammatory mechanism, although not sufficient to reverse disease progression. On the basis of these observations, Nurr1 could represent a potential biomarker for ALS and a promising target for future therapies., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Since the discovery of the three isoforms of the Na + /Ca 2+ 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., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Spinal muscular atrophy (SMA) is a severe neuromuscular disease affecting infants caused by alterations of the survival motor neuron gene, which results in progressive degeneration of motor neurons (MNs). Although an effective treatment for SMA patients has been recently developed, the molecular pathway involved in selective MN degeneration has not been yet elucidated. In particular, miR-206 has been demonstrated to play a relevant role in the regeneration of neuromuscular junction in several MN diseases, and particularly it is upregulated in the quadriceps, tibialis anterior, spinal cord, and serum of SMA mice. In the present paper, we demonstrated that miR-206 was transiently upregulated also in the brainstem of the mouse model of SMA, SMAΔ7, in the early phase of the disease paralleling MN degeneration and was down-regulated in the late symptomatic phase. To prevent this downregulation, we intracerebroventricularly injected miR-206 in SMA pups, demonstrating that miR-206 reduced the severity of SMA pathology, slowing down disease progression, increasing survival rate, and improving behavioral performance of mice. Interestingly, exogenous miRNA-206-induced upregulation caused a reduction of the predicted target sodium calcium exchanger isoform 2, NCX2, one of the main regulators of intracellular [Ca 2+ ] and [Na + ]. Therefore, we hypothesized that miR-206 might exert part of its neuroprotective effect modulating NCX2 expression in SMA disease., (Copyright © 2020. Published by Elsevier Inc.)

Loss of the survival motor neuron gene (SMN1) is responsible for spinal muscular atrophy (SMA), the most common inherited cause of infant mortality. Even though the SMA phenotype is traditionally considered as related to spinal motor neuron loss, it remains debated whether the specific targeting of motor neurons could represent the best therapeutic option for the disease. We here investigated, using stereological quantification methods, the spinal cord and cerebral motor cortex of ∆7 SMA mice during development, to verify extent and selectivity of motor neuron loss. We found progressive post-natal loss of spinal motor neurons, already at pre-symptomatic stages, and a higher vulnerability of motor neurons innervating proximal and axial muscles. Larger motor neurons decreased in the course of disease, either for selective loss or specific developmental impairment. We also found a selective reduction of layer V pyramidal neurons associated with layer V gliosis in the cerebral motor cortex. Our data indicate that in the ∆7 SMA model SMN loss is critical for the spinal cord, particularly for specific motor neuron pools. Neuronal loss, however, is not selective for lower motor neurons. These data further suggest that SMA pathogenesis is likely more complex than previously anticipated. The better knowledge of SMA models might be instrumental in shaping better therapeutic options for affected patients.

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.

The proteins NCX1, NCX2, and NCX3 expressed on the plasma membrane of neurons play a crucial role in ionic regulation because they are the major bidirectional system promoting the efflux and influx of Na(+) and Ca(2+) ions. Here, we demonstrate that NCX1 and NCX3 proteins are novel additional targets for the survival action of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. Indeed, the doxycycline-dependent overexpression of constitutively active Akt1 in tetracycline (Tet)-Off PC-12 positive mutants and the exposure of Tet-Off PC-12 wild type to nerve growth factor induced an up-regulation of NCX1 and NCX3 proteins. NCX1 up-regulation induced by Akt1 activation occurred at the transcriptional level because NCX1 mRNA increased, and it was counteracted by cAMP response element-binding protein 1 inhibition through small interfering RNA strategy. In contrast, Akt1-induced NCX3 up-regulation recognized a post-transcriptional mechanism occurring at the proteasome level because 1) NCX3 transcript did not increase and 2) the proteasome inhibitor N-benzyloxycarbonyl (Z)-Leu-Leu-leucinal (MG-132) did not further enhance NCX3 protein levels in Akt1 active mutants as it would be expected if the ubiquitin-proteasome complex was not already blocked by Akt1 pathway. As expected, in PC-12 Tet-Off wild-type cells MG-132 enhanced NCX3 protein levels. This up-regulation produced an increased activity of NCX function. Furthermore, NCX1 and NCX3 up-regulation contributed to the survival action of Akt1 during chemical hypoxia because both the silencing of NCX1 or NCX3 and the pharmacological paninhibition of NCX isoforms reduced the prosurvival property of Akt1. Together, these results indicated that NCX1 and NCX3 represent novel additional molecular targets for the prosurvival action of PI3-K/Akt pathway.

K(+)-dependent Na(+)/Ca(2+) exchangers (NCKX) catalyze cytosolic Ca(2+) extrusion and are particularly important for neuronal Ca(2+) signaling. Of the five mammalian isoforms, the detailed functional characteristics have only been reported for NCKX1 and -2. In the current study, the functional characteristics of recombinant NCKX3 and -4 expressed in HEK293 cells were determined and compared with those of NCKX2. Although the apparent affinities of the three isoforms for Ca(2+) and Na(+) were similar, NCKX3 and -4 displayed approximately 40-fold higher affinities for K(+) ions than NCKX2. Functional analysis of various NCKX2 mutants revealed that mutation of Thr-551 to Ala, the corresponding residue in NCKX4, resulted in an apparent K(+) affinity shift to one similar to that of NCKX4 without a parallel shift in apparent Ca(2+) affinity. In the converse situation, when Gln-476 of NCKX4 was converted to Lys, the corresponding residue in NCKX2, both the K(+) and Ca(2+) affinities were reduced. These results indicate that the apparently low K(+) affinity of NCKX2 requires a Thr residue at position 551 that may reduce the conformational flexibility and/or K(+) liganding strength of side-chain moieties on critical neighboring residues. This interaction appears to be specific to the structural context of the NCKX2 K(+) binding pocket, because it was not possible to recreate the K(+)-specific low affinity phenotype with reciprocal mutations in NCKX4. The results of this study provide important information about the structure and function of NCKX proteins and will be critical to understanding their roles in neuronal Ca(2+) signaling.