Neuronal Intracellular Calcium Research Articles

Biallelic ADGRV1 variants are associated with Rolandic epilepsy.

Rolandic epilepsy (RE) is among the most common focal epilepsies in childhood. For the majority of patients with RE and atypical RE (ARE), the etiology remains elusive. We thus screened patients with RE/ARE in order to detect disease-causing variants.. A trios-based whole-exome sequencing approach was performed in a cohort of 28 patients with RE/ARE. Clinical data and EEGs were reviewed. Variants were validated by Sanger sequencing. Two compound heterozygous missense variants p.Val272Ile/p.Asn3028Ser and p.Ala3657Val/p.Met4419Val of ADGRV1 were identified in two unrelated familial cases of RE/ARE. All the variants were in the calcium exchanger β domain and were suggested to be damaging by at least one web-based prediction tool. These variants are not present or are present at a very low minor allele frequency in the gnomAD database. Previously, biallelic ADGRV1 variants (p.Gly2756Arg and p.Glu4410Lys) have been observed in RE, consistent with the observation in this study and supporting the association between ADGRV1 variants and RE. Additionally, a de novo mutation, p.Asp668Asn, in GRIN2B was identified in a sporadic case of ARE, and a missense variant, p.Asn1551Ser, in RyR2 was identified in a family with RE with incomplete penetrance. These genes are all calcium homeostasis associated genes, suggesting the potential effect of calcium homeostasis in RE/ARE. The results from the present study suggest that the genes ADGRV1, GRIN2B, and RyR2 are associated with RE/ARE. These data link defects in neuronal intracellular calcium homeostasis to RE/ARE pathogenesis implicating that these defects plays an important role in the development of these conditions.

GPR68 Contributes to Persistent Acidosis-Induced Activation of AGC Kinases and Tyrosine Phosphorylation in Organotypic Hippocampal Slices.

Persistent acidosis occurs in ischemia and multiple neurological diseases. In previous studies, acidic stimulation leads to rapid increase in intracellular calcium in neurons. However, it remains largely unclear how a prolonged acidosis alters neuronal signaling. In our previous study, we found that GPR68-mediated PKC activities are protective against acidosis-induced injury in cortical slices. Here, we first asked whether the same principle holds true in organotypic hippocampal slices. Our data showed that 1-h pH 6 induced PKC phosphorylation in a GPR68-dependent manner. Go6983, a PKC inhibitor worsened acidosis-induced neuronal injury in wild type (WT) but had no effect in GPR68−/− slices. Next, to gain greater insights into acid signaling in brain tissue, we treated organotypic hippocampal slices with pH 6 for 1-h and performed a kinome profiling analysis by Western blot. Acidosis had little effect on cyclin-dependent kinase (CDK) or casein kinase 2 activity, two members of the CMGC family, or Ataxia telangiectasia mutated (ATM)/ATM and RAD3-related (ATR) activity, but reduced the phosphorylation of MAPK/CDK substrates. In contrast, acidosis induced the activation of CaMKIIα, PKA, and Akt. Besides these serine/threonine kinases, acidosis also induced tyrosine phosphorylation. Since GPR68 is widely expressed in brain neurons, we asked whether GPR68 contributes to acidosis-induced signaling. Deleting GPR68 had no effect on acidosis-induced CaMKII phosphorylation, attenuated that of phospho-Akt and phospho-PKA substrates, while abolishing acidosis-induced tyrosine phosphorylation. These data demonstrate that prolonged acidosis activates a network of signaling cascades, mediated by AGC kinases, CaMKII, and tyrosine kinases. GPR68 is the primary mediator for acidosis-induced activation of PKC and tyrosine phosphorylation, while both GPR68-dependent and -independent mechanisms contribute to the activation of PKA and Akt.

Acetaldehyde within e‐cigarette aerosol and cigarette smoke triggers intracellular calcium release in primary adult cardiomyocytes that is exacerbated in ALDH2*2 mice

Background Cigarette smoke can trigger calcium influx in cardiac myocytes that over time can lead to cardiovascular diseases linked to calcium handling such as atrial fibrillation and heart failure. E-cigarette vapor, similar to cigarette smoke, contains aldehydes that trigger intracellular calcium in neurons. Further, ~560 million people carry an inactivating genetic variant in aldehyde dehydrogenase 2 (ALDH2), known as ALDH2*2, the primary enzyme responsible for metabolizing acetaldehyde. However, little is known how acetaldehyde present in cigarette smoke and e-cigarette aerosol, when coupled with genetics, influence intracellular calcium release within the cardiac myocyte. Hypothesis: Acetaldehyde in e-cigarette aerosol and cigarette smoke can trigger calcium influx in cardiac myocytes. Methods: ALDH2 expression and activity were quantified from wild type ALDH2 and ALDH2*2 male mice heart homogenates. Further, ALDH2 and ALDH2*2 primary adult cardiac myocytes were treated with a short burst of acetaldehyde (0.1-100 μM for 30 seconds, to recapitulate vaping or smoking) and intracellular calcium levels were assessed by fura-2 (5 μM). A Student's t-test was used to compare responses between rodents with significance *p<0.05. Results: The enzymatic activity to metabolize aldehydes within the heart was 3-fold lower for the ALDH2*2 relative to ALDH2 heart homogenates (0.14±0.003* versus 0.45±0.02, ALDH2/GAPDH relative densitometry units, n=3/group). Further, acetaldehyde (0.1 μM) triggered intracellular calcium influx that was 5-fold higher for ALDH2*2 myocytes compared to ALDH2 myocytes (8.32±0.72 %* versus 1.46±0.31%, percent of maximal response, n=4-6 cells from 4 biological replicates). Peak intracellular calcium influx was also ~40% higher for ALDH2*2 myocytes when compared to wild type ALDH2 myocytes over a range of additional acetaldehyde doses (0.1-100 μM) tested. Conclusion: Acetaldehyde, the primary aldehyde in e-cigarette aerosol, triggers intracellular calcium influx that is ~40% greater for rodents carrying an inactivating ALDH2 variant, ALDH2*2. People carrying an inactive ALDH2*2 genetic variant may be more susceptible to acetaldehyde present within e-cigarette vapor and cigarette smoke which over time and continued use lead to developing cardiovascular diseases linked to altered calcium handling.

GPR18 drives FAAH inhibition-induced neuroprotection against HIV-1 Tat-induced neurodegeneration

Human immunodeficiency virus type 1 (HIV-1) is known to provoke microglial immune responses which likely play a paramount role in the development of chronic neuroinflammatory conditions and neuronal damage related to HIV-1 associated neurocognitive disorders (HAND). In particular, HIV-1 Tat protein is a proinflammatory neurotoxin which predisposes neurons to synaptodendritic injury. Drugs targeting the degradative enzymes of endogenous cannabinoids have shown promise in reducing inflammation with minimal side effects in rodent models. Considering that markers of neuroinflammation can predict the extent of neuronal injury in HAND patients, we evaluated the neurotoxic effect of HIV-1 Tat-exposed microglia following blockade of fatty acid amid hydrolyze (FAAH), a catabolic enzyme responsible for degradation of endocannabinoids, e.g. anandamide (AEA). In the present study, cultured murine microglia were incubated with Tat and/or a FAAH inhibitor (PF3845). After 24 h, cells were imaged for morphological analysis and microglial conditioned media (MCM) was collected. Frontal cortex neuron cultures (DIV 7–11) were then exposed to MCM, and neurotoxicity was assessed via live cell calcium imaging and staining of actin positive dendritic structures. Results demonstrate a strong attenuation of microglial responses to Tat by PF3845 pretreatment, which is indicated by 1) microglial changes in morphology to a less proinflammatory phenotype using fractal analysis, 2) a decrease in release of neurotoxic cytokines/chemokines (MCP-1/CCL2) and matrix metalloproteinases (MMPs; MMP-9) using ELISA/multiplex assays, and 3) enhanced production of endocannabinoids (AEA) using LC/MS/MS. Additionally, PF3845's effects on Tat-induced microglial-mediated neurotoxicity, decreased dysregulation of neuronal intracellular calcium and prevented the loss of actin-positive staining and punctate structure in frontal cortex neuron cultures. Interestingly, these observed neuroprotective effects appeared to be independent of cannabinoid receptor activity (CB1R & CB2R). We found that a purported GPR18 antagonist, CID-85469571, blocked the neuroprotective effects of PF3845 in all experiments. Collectively, these experiments increase understanding of the role of FAAH inhibition and Tat in mediating microglial neurotoxicity in the HAND condition.

Genetic Ablation of Neuronal Mitochondrial Calcium Uptake Halts Alzheimer's Disease Progression

Alzheimer’s disease (AD) is characterized by the extracellular deposition of amyloid beta, intracellular neurofibrillary tangles, synaptic dysfunction, and neuronal cell death. These phenotypes correlate with and are linked to elevated neuronal intracellular calcium (iCa2+) levels. Recently, our group reported that mitochondrial calcium (mCa2+) overload, due to loss of mCa2+ efflux capacity, contributes to AD development and progression. We also noted proteomic remodeling of the mitochondrial calcium uniporter channel (mtCU) in sporadic AD brain samples, suggestive of altered mCa2+ uptake in AD. Since the mtCU is the primary mechanism for Ca2+ uptake into the mitochondrial matrix, inhibition of the mtCU has the potential to reduce or prevent mCa2+ overload in AD. Here, we report that neuronal-specific loss of mtCU-dependent mCa2+ uptake in the 3xTg-AD mouse model of AD reduced Aβ and tau-pathology, synaptic dysfunction, and cognitive decline. Knockdown of Mcu in a cellular model of AD significantly decreased matrix Ca2+ content, oxidative stress and cell death. These results suggest that inhibition of neuronal mCa2+ uptake is a novel therapeutic target to impede AD progression.

Temporal Progression of Excitotoxic Calcium Following Distal Middle Cerebral Artery Occlusion in Freely Moving Mice.

Ischemic stroke is recognized as one of the leading causes of adult disability, morbidity, and death worldwide. Following stroke, acute neuronal excitotoxicity can lead to many deleterious consequences, one of which is the dysregulation of intracellular calcium ultimately culminating in cell death. However, to develop neuroprotective treatments that target neuronal excitotoxicity, it is essential to know the therapeutic time window for intervention following an ischemic event. To address this question, the current study aimed to characterize the magnitude and temporal progression of neuronal intracellular calcium observed following distal middle cerebral artery occlusion (dMCAO) in mice. Using the calcium fluorescence indicator, GCaMP, we tracked neuronal population response in freely moving animals immediately following dMCAO in both the core infarct and peri-infarct regions. Our results demonstrate that calcium excitotoxicity following artery occlusion can be generally characterized by two phases: a transient increase in activity that lasts tens of minutes, followed by a long, slow sustained increase in fluorescence signal. The first phase is primarily thought to represent neuronal hyperexcitability, defining our therapeutic window, while the second may represent gradual cell death. Importantly, we show that the level of intracellular calcium following artery occlusion correlated with the infarct size at 24 h demonstrating a direct connection between excitotoxicity and cell death in our stroke model. In addition, we show that administration of the NMDA antagonist MK-801 resulted in both a decrease in calcium signal and a subsequent reduction in the infarct size. Altogether, this study represents the first demonstration in freely moving animals characterizing the temporal progression of toxic calcium signaling following artery occlusion. In addition, these results define a critical time window for neuroprotective therapeutic intervention in mice.

Surviving nutritional deprivation during development: neuronal intracellular calcium signaling is critical.

Developing cells and tissues in a growing animal need to sense food quality and integrate this information with on-going time-bound developmental programs. The integration of metabolism with development requires cellular and systemic coordination. Work in our laboratory has focused on Ca2+ signaling arising from the release of Ca2+ stored in the endoplasmic reticulum (ER), which triggers store-operated Ca2+ entry. We describe a role for ER-store Ca2+ that operates at the cellular level in various classes of neurons, and eventually drives the systemic coordination required to survive and complete development under conditions of nutritional deprivation. In the model system Drosophila melanogaster, we have developed a paradigm to induce nutritional stress during the larval stage and used pupariation as a read-out for development. Applying the vast genetic tool kit available in Drosophila to this paradigm, we have uncovered novel roles for intracellular Ca 2+ signaling in regulating neuronal activity, at the level of transcription in glutamatergic neurons, and translation in neuropeptidergic neurons. We find that such regulation of cellular processes is critical for integrating information across a neural circuit at multiple levels, starting from the point of sensing systemic and environmental levels of amino acids to finally connecting with neuropeptide secreting neurons, that communicate with the prothoracic gland, an organ that makes the key developmental hormone, ecdysone. This work underscores the importance of ER-store Ca2+ for neuronal health, with consequences for animal development.

In vitro cellular uptake and neuroprotective efficacy of poly-arginine-18 (R18) and poly-ornithine-18 (O18) peptides: critical role of arginine guanidinium head groups for neuroprotection.

We have previously demonstrated that Cationic Arginine-Rich Peptides (CARPs) and in particular poly-arginine-18 (R18; 18-mer of arginine) exhibit potent neuroprotective properties in both in vitro and in vivo neuronal injury models. Based on the current literature, there is a consensus that arginine residues by virtue of their positive charge and guanidinium head group is the critical element for imparting CARP neuroprotective properties and their ability to traverse cell membranes. This study examined the importance of guanidinium head groups in R18 for peptide cellular uptake, localization, and neuroprotection. This was achieved by using poly-ornithine-18 (O18; 18-mer of ornithine) as a control, which is structurally identical to R18, but possesses amino head groups rather than guanidino head groups. Epifluorescence and confocal fluorescence microscopy was used to examine the cellular uptake and localization of the FITC-conjugated R18 and O18 in primary rat cortical neurons and SH-SY5Y human neuroblastoma cell cultures. An in vitro cortical neuronal glutamic acid excitotoxicity model was used to compare the effectiveness of R18 and O18 to inhibit cell death and intracellular calcium influx, as well as caspase and calpain activation. Fluorescence imaging studies revealed cellular uptake of both FITC-R18 and FITC-O18 in neuronal and SH-SY5Y cells; however, intracellular localization of the peptides differed in neurons. Following glutamic acid excitotoxicity, only R18 was neuroprotective, prevented caspases and calpain activation, and was more effective at reducing neuronal intracellular calcium influx. Overall, this study demonstrated that for long chain cationic poly-arginine peptides, the guanidinium head groups provided by arginine residues are an essential requirement for neuroprotection but are not required for entry into neurons.

RyRs mediate lead-induced neurodegenerative disorders through calcium signaling pathways

Heavy metal lead (Pb) is widely distributed in the environment and can induce neurodegeneration. Accumulating evidence has shown that ryanodine receptors (RyRs) play vital roles in neurodegenerative brain. However, whether aberrant RyRs levels contribute to Pb-induced neurodegeneration has largely remained unknown. In the present study, we report the important role of elevated levels of RyRs in Pb-induced neurodegeneration. Pb was found to upregulate the levels of RyRs in the rat hippocampal tissues and rat pheochromocytoma (PC12) cells. Furthermore, exposure to Pb induced neurodegenerative cognitive impairment in rats, depressed the long-term potentiation (LTP) in the rat brain slices, increased the neuronal intracellular free calcium concentration ([Ca2+]i), inhibited the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and cyclic adenosine 3′,5′-monophosphate (cAMP) response element binding protein (CREB) as well as the expression of anti-apoptotic protein B-cell lymphoma 2 (Bcl2), and activated the phosphorylation of extracellular regulated protein kinases (Erk) protein both in vitro and in vivo. In addition, the knockdown of RyR3 in PC12 cells significantly decreased the [Ca2+]i levels, increased the CaMKIIα and CREB phosphorylation, decrease the phosphorylation of Erk, and elongated the cognitive function-related neurite outgrowth after exposure to Pb. Moreover, treatment with a RyRs agonist showed the involvement of RyRs in Pb-induced depression in LTP in the rat brain slices. In summary, we determined that Pb-mediated upregulation of RyRs led to neurodegeneration via high levels of free calcium, depression of the calcium-dependent CaMKIIα/CREB mnemonic signaling pathway, and activation of the calcium-dependent Erk/Bcl2 apoptotic signaling pathway. These findings on the impact of Pb on the levels of RyRs could further improve our understanding of Pb-induced neurotoxicity and provide a promising molecular target to antagonize Pb-induced neurodegenerative diseases.

Impaired mitochondrial calcium efflux contributes to disease progression in models of Alzheimer\u2019s disease

Impairments in neuronal intracellular calcium (iCa2+) handling may contribute to Alzheimer’s disease (AD) development. Metabolic dysfunction and progressive neuronal loss are associated with AD progression, and mitochondrial calcium (mCa2+) signaling is a key regulator of both of these processes. Here, we report remodeling of the mCa2+ exchange machinery in the prefrontal cortex of individuals with AD. In the 3xTg-AD mouse model impaired mCa2+ efflux capacity precedes neuropathology. Neuronal deletion of the mitochondrial Na+/Ca2+ exchanger (NCLX, Slc8b1 gene) accelerated memory decline and increased amyloidosis and tau pathology. Further, genetic rescue of neuronal NCLX in 3xTg-AD mice is sufficient to impede AD-associated pathology and memory loss. We show that mCa2+ overload contributes to AD progression by promoting superoxide generation, metabolic dysfunction and neuronal cell death. These results provide a link between the calcium dysregulation and metabolic dysfunction hypotheses of AD and suggest mCa2+ exchange as potential therapeutic target in AD.

Lysophosphatidic Acid and Glutamatergic Transmission.

Signaling through bioactive lipids regulates nervous system development and functions. Lysophosphatidic acid (LPA), a membrane-derived lipid mediator particularly enriched in brain, is able to induce many responses in neurons and glial cells by affecting key processes like synaptic plasticity, neurogenesis, differentiation and proliferation. Early studies noted sustained elevations of neuronal intracellular calcium, a primary response to LPA exposure, suggesting functional modifications of NMDA and AMPA glutamate receptors. However, the crosstalk between LPA signaling and glutamatergic transmission has only recently been shown. For example, stimulation of presynaptic LPA receptors in hippocampal neurons regulates glutamate release from the presynaptic terminal, and excess of LPA induce seizures. Further evidence indicating a role of LPA in the modulation of neuronal transmission has been inferred from animal models with deficits on LPA receptors, mainly LPA1 which is the most prevalent receptor in human and mouse brain tissue. LPA1 null-mice exhibit cognitive and attention deficits characteristic of schizophrenia which are related with altered glutamatergic transmission and reduced neuropathic pain. Furthermore, silencing of LPA1 receptor in mice induced a severe down-regulation of the main glutaminase isoform (GLS) in cerebral cortex and hippocampus, along with a parallel sharp decrease on active matrix-metalloproteinase 9. The downregulation of both enzymes correlated with an altered morphology of glutamatergic pyramidal cells dendritic spines towards a less mature phenotype, indicating important implications of LPA in synaptic excitatory plasticity which may contribute to the cognitive and memory deficits shown by LPA1-deficient mice. In this review, we present an updated account of current evidence pointing to important implications of LPA in the modulation of synaptic excitatory transmission.

Transcription Factor 2I Regulates Neuronal Development via TRPC3 in 7q11.23 Disorder Models

Williams syndrome (WS) and 7q11.23 duplication syndrome (Dup7q11.23) are neurodevelopmental disorders caused by the deletion and duplication, respectively, of ~ 25 protein-coding genes on chromosome 7q11.23. The general transcription factor 2I (GTF2I, protein TFII-I) is one of these proteins and has been implicated in the neurodevelopmental phenotypes of WS and Dup7q11.23. Here, we investigated the effect of copy number alterations in Gtf2i on neuronal maturation and intracellular calcium entry mechanisms known to be associated with this process. Mice with a single copy of Gtf2i (Gtf2i+/Del) had increased axonal outgrowth and increased TRPC3-mediated calcium entry upon carbachol stimulation. In contrast, mice with 3 copies of Gtf2i (Gtf2i+/Dup) had decreases in axon outgrowth and in TRPC3-mediated calcium entry. The underlying mechanism was that TFII-I did not affect TRPC3 protein expression, while it regulated TRPC3 membrane translocation. Together, our results provide novel functional insight into the cellular mechanisms that underlie neuronal maturation in the context of the 7q11.23 disorders.

Human carbonic anhydrase-8 AAV8 gene therapy inhibits nerve growth factor signaling producing prolonged analgesia and anti-hyperalgesia in mice.

Carbonic anhydrase-8 (Car8; murine gene symbol) is an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1), which regulates neuronal intracellular calcium release. We previously reported that wildtype Car8 overexpression corrects the baseline allodynia and hyperalgesia associated with calcium dysregulation in the waddle (wdl) mouse due to a 19 bp deletion in exon 8 of the Car8 gene. In this report, we provide preliminary evidence that overexpression of the human wildtype ortholog of Car8 (CA8WT), but not the reported CA8 S100P loss-of-function mutation (CA8MT); inhibits nerve growth factor (NGF)-induced phosphorylation of ITPR1, TrkA (NGF high affinity receptor); and ITPR1-mediated cytoplasmic free calcium release in vitro. Additionally, we show that gene-transfer using AAV8-V5-CA8WT viral particles via sciatic nerve injection demonstrates retrograde transport to dorsal root ganglia (DRG) producing prolonged V5-CA8WT expression, pITPR1 and pTrkA inhibition, and profound analgesia and anti-hyperalgesia in male C57BL/6J mice. AAV8-V5-CA8WT mediated overexpression prevented and treated allodynia and hyperalgesia associated with chronic neuropathic pain produced by the spinal nerve ligation (SNL) model. These AAV8-V5-CA8 data provide a proof-of-concept for precision medicine through targeted gene therapy of NGF-responsive somatosensory neurons as a long-acting local analgesic able to prevent and treat chronic neuropathic pain through regulating TrkA signaling, ITPR1 activation, and intracellular free calcium release by ITPR1.

Ultra-Slow Single-Vessel BOLD and CBV-Based fMRI Spatiotemporal Dynamics and Their Correlation with Neuronal Intracellular Calcium Signals

Functional MRI has been used to map brain activity and functional connectivity based on the strength and temporal coherence of neurovascular-coupled hemodynamic signals. Here, single-vessel fMRI reveals vessel-specific correlation patterns in both rodents and humans. In anesthetized rats, fluctuations in the vessel-specific fMRI signal are correlated with the intracellular calcium signal measured in neighboring neurons. Further, the blood-oxygen-level-dependent (BOLD) signal from individual venules and the cerebral-blood-volume signal from individual arterioles show correlations at ultra-slow (<0.1Hz), anesthetic-modulated rhythms. These data support a model that links neuronal activity to intrinsic oscillations in the cerebral vasculature, with a spatial correlation length of ∼2mm for arterioles. In complementary data from awake human subjects, the BOLD signal is spatially correlated among sulcus veins and specified intracortical veins of the visual cortex at similar ultra-slow rhythms. These data support the use of fMRI to resolve functional connectivity at the level of single vessels.

Novel botanical drug DA-9803 prevents deficits in Alzheimer\u2019s mouse models

BackgroundAlzheimer’s disease (AD) is a neurodegenerative disorder characterized by deposition of amyloid plaques and disruption of neural circuitry, leading to cognitive decline. Animal models of AD deposit senile plaques and exhibit structural and functional deficits in neurons and neural networks. An effective treatment would prevent or restore these deficits, including calcium dyshomeostasis observed with in-vivo imaging.MethodsWe examined the effects of DA-9803, a multimodal botanical drug, in 5XFAD and APP/PS1 transgenic mice which underwent daily oral treatment with 30 or 100 mg/kg DA-9803 or vehicle alone. Behavioral testing and longitudinal imaging of amyloid deposits and intracellular calcium in neurons with multiphoton microscopy was performed.ResultsChronic administration of DA-9803 restored behavioral deficits in 5XFAD mice and reduced amyloid-β levels. DA-9803 also prevented progressive amyloid plaque deposition in APP/PS1 mice. Elevated calcium, detected in a subset of neurons before the treatment, was restored and served as a functional indicator of treatment efficacy in addition to the behavioral readout. In contrast, mice treated with vehicle alone continued to progressively accumulate amyloid plaques and calcium overload.ConclusionsIn summary, treatment with DA-9803 prevented structural and functional outcome measures in mouse models of AD. Thus, DA-9803 shows promise as a novel therapeutic approach for Alzheimer’s disease.

Positive N-Methyl-D-Aspartate Receptor Modulation by Rapastinel Promotes Rapid and Sustained Antidepressant-Like Effects.

BackgroundModulation of glutamatergic synaptic transmission by N-methyl-D-aspartate receptors can produce rapid and sustained antidepressant effects. Rapastinel (GLYX-13), initially described as a N-methyl-D-aspartate receptor partial glycine site agonist, exhibits rapid antidepressant effect in rodents without the accompanying dissociative effects of N-methyl-D-aspartate receptor antagonists.MethodsThe relationship between rapastinel’s in vitro N-methyl-D-aspartate receptor pharmacology and antidepressant efficacy was determined by brain microdialysis and subsequent pharmacological characterization of therapeutic rapastinel concentrations in N-methyl-D-aspartate receptor-specific radioligand displacement, calcium mobilization, and medial prefrontal cortex electrophysiology assays.ResultsBrain rapastinel concentrations of 30 to 100 nM were associated with its antidepressant-like efficacy and enhancement of N-methyl-D-aspartate receptor-dependent neuronal intracellular calcium mobilization. Modulation of N-methyl-D-aspartate receptors by rapastinel was independent of D-serine concentrations, and glycine site antagonists did not block rapastinel’s effect. In rat medial prefrontal cortex slices, 100 nM rapastinel increased N-methyl-D-aspartate receptor-mediated excitatory postsynaptic currents and enhanced the magnitude of long-term potentiation without any effect on miniature EPSCs or paired-pulse facilitation responses, indicating postsynaptic action of rapastinel. A critical amino acid within the NR2 subunit was identified as necessary for rapastinel’s modulatory effect.ConclusionRapastinel brain concentrations associated with antidepressant-like activity directly enhance medial prefrontal cortex N-methyl-D-aspartate receptor activity and N-methyl-D-aspartate receptor-mediated synaptic plasticity in vitro. At therapeutic concentrations, rapastinel directly enhances N-methyl-D-aspartate receptor activity through a novel site independent of the glycine coagonist site. While both rapastinel and ketamine physically target N-methyl-D-aspartate receptors, the 2 molecules have opposing actions on N-methyl-D-aspartate receptors. Modest positive modulation of N-methyl-D-aspartate receptors by rapastinel represents a novel pharmacological approach to promote well-tolerated, rapid, and sustained improvements in mood disorders.

Effect of Noopept on Dynamics of Intracellular Calcium in Neurons of Cultured Rat Hippocampal Slices.

A neuroprotective and nootropic drug Noopept increased the frequency of spontaneous calcium transients in neurons of CA1 radial layer in cultured rat hippocampal slices. In contrast, the drug exerted no significant effect on intracellular calcium concentration and its dynamics in neurons of hippocampal CA1 pyramidal layer.

Validation of electrical stimulation models: intracellular calcium measurement in three-dimensional scaffolds.

Peripheral nerve injury can be disabling. Regeneration is limited by the rate of axonal extension, and proximal injury to peripheral nerves can take over a year to reach target organs. Electrical stimulation (ES) has been shown to increase the rate of neurite growth, though the mechanism is not yet well understood. In our prior manuscript, we developed a computational model that demonstrates how ES can functionally elevate intracellular calcium concentration ([Ca2+]i) based on ES intensity and duration. In this article, we validate the computation model for the [Ca2+]i changes in neuron soma. Embryonic chicken dorsal root ganglion cells were suspended in 3-dimensional collagen scaffolds. Fura-2 was used to measure [Ca2+]i in response to biphasic ES pulses ranging from 70 to 60,000 V/m in intensity and from 10 µs to 100 ms in duration. The computational model most closely matched the experimental data of the neurons with the highest [Ca2+]i elevation for ES pulses 100 µs or greater in duration. Nickel (200 µM) and cadmium (200 µM) blocked 98-99% of the [Ca2+]i rise, indicating that the rise in [Ca2+]i in response to ES is via voltage-dependent calcium channels. The average [Ca2+]i rise in response to ES was about one-tenth of the peak rise. Therefore, the computational model is validated for elevating [Ca2+]i of neurons and can be used as a tool for designing efficacious ES protocols for improving neuronal regeneration.NEW & NOTEWORTHY Electrical stimulation is used to enhance neuron growth, and the role of neuronal intracellular calcium concentration ([Ca2+]i) is an area of research interest. Widely varying stimulation parameters in the literature make it difficult to compare stimulation protocols. The results in this manuscript are the first to show neuronal [Ca2+]i in response to a broad and defined range of electrical pulse durations and intensities. These results validate our previously published novel computational model of [Ca2+]i.

Altered Ca2+ homeostasis induces Calpain-Cathepsin axis activation in sporadic Creutzfeldt-Jakob disease

Sporadic Creutzfeldt-Jakob disease (sCJD) is the most prevalent form of human prion disease and it is characterized by the presence of neuronal loss, spongiform degeneration, chronic inflammation and the accumulation of misfolded and pathogenic prion protein (PrPSc). The molecular mechanisms underlying these alterations are largely unknown, but the presence of intracellular neuronal calcium (Ca2+) overload, a general feature in models of prion diseases, is suggested to play a key role in prion pathogenesis.Here we describe the presence of massive regulation of Ca2+ responsive genes in sCJD brain tissue, accompanied by two Ca2+-dependent processes: endoplasmic reticulum stress and the activation of the cysteine proteases Calpains 1/2. Pathogenic Calpain proteins activation in sCJD is linked to the cleavage of their cellular substrates, impaired autophagy and lysosomal damage, which is partially reversed by Calpain inhibition in a cellular prion model. Additionally, Calpain 1 treatment enhances seeding activity of PrPSc in a prion conversion assay. Neuronal lysosomal impairment caused by Calpain over activation leads to the release of the lysosomal protease Cathepsin S that in sCJD mainly localises in axons, although massive Cathepsin S overexpression is detected in microglial cells. Alterations in Ca2+ homeostasis and activation of Calpain-Cathepsin axis already occur at pre-clinical stages of the disease as detected in a humanized sCJD mouse model.Altogether our work indicates that unbalanced Calpain-Cathepsin activation is a relevant contributor to the pathogenesis of sCJD at multiple molecular levels and a potential target for therapeutic intervention.

Characterisation of neuroprotective efficacy of modified poly-arginine-9 (R9) peptides using a neuronal glutamic acid excitotoxicity model.

In a recent study, we highlighted the importance of cationic charge and arginine residues for the neuroprotective properties of poly-arginine and arginine-rich peptides. In this study, using cortical neuronal cultures and an in vitro glutamic acid excitotoxicity model, we examined the neuroprotective efficacy of different modifications to the poly-arginine-9 peptide (R9). We compared an unmodified R9 peptide with R9 peptides containing the following modifications: (i) C-terminal amidation (R9-NH2); (ii) N-terminal acetylation (Ac-R9); (iii) C-terminal amidation with N-terminal acetylation (Ac-R9-NH2); and (iv) C-terminal amidation with D-amino acids (R9D-NH2). The three C-terminal amidated peptides (R9-NH2, Ac-R9-NH2, and R9D-NH2) displayed neuroprotective effects greater than the unmodified R9 peptide, while the N-terminal acetylated peptide (Ac-R9) had reduced efficacy. Using the R9-NH2 peptide, neuroprotection could be induced with a 10min peptide pre-treatment, 1-6h before glutamic acid insult, or when added to neuronal cultures up to 45min post-insult. In addition, all peptides were capable of reducing glutamic acid-mediated neuronal intracellular calcium influx, in a manner that reflected their neuroprotective efficacy. This study further highlights the neuroprotective properties of poly-arginine peptides and provides insight into peptide modifications that affect efficacy.