Ion channels and channelopathies in glomeruli.

An essential step in renal function entails the formation of an ultrafiltrate that is delivered to the renal tubules for subsequent processing. This process, known as glomerular filtration, is controlled by intrinsic regulatory systems and by paracrine, neuronal, and endocrine signals that converge onto glomerular cells. In addition, the characteristics of glomerular fluid flow, such as the glomerular filtration rate and the glomerular filtration fraction, play an important role in determining blood flow to the rest of the kidney. Consequently, disease processes that initially affect glomeruli are the most likely to lead to end-stage kidney failure. The cells that comprise the glomerular filter, especially podocytes and mesangial cells, express many different types of ion channels that regulate intrinsic aspects of cell function and cellular responses to the local environment, such as changes in glomerular capillary pressure. Dysregulation of glomerular ion channels, such as changes in TRPC6, can lead to devastating glomerular diseases, and a number of channels, including TRPC6, TRPC5, and various ionotropic receptors, are promising targets for drug development. This review discusses glomerular structure and glomerular disease processes. It also describes the types of plasma membrane ion channels that have been identified in glomerular cells, the physiological and pathophysiological contexts in which they operate, and the pathways by which they are regulated and dysregulated. The contributions of these channels to glomerular disease processes, such as focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy, as well as the development of drugs that target these channels are also discussed.

Intersectin deficiency impairs cortico-striatal neurotransmission and causes obsessive-compulsive behaviors in mice.

The generation of appropriate behavioral responses involves dedicated neuronal circuits. The cortico-striatal-thalamo-cortical loop is especially important for the expression of motor routines and habits. Defects in this circuitry are closely linked to obsessive stereotypic behaviors, hallmarks of neuropsychiatric diseases including autism spectrum disorders (ASDs) and obsessive-compulsive disorders (OCDs). However, our knowledge of the essential synaptic machinery required to maintain balanced neurotransmission and plasticity within the cortico-striatal circuitry remains fragmentary. Mutations in the large synaptic scaffold protein intersectin1 (ITSN1) have been identified in patients presenting with ASD symptoms including stereotypic behaviors, although a causal relationship between stereotypic behavior and intersectin function has not been established. We report here that deletion of the two closely related proteins ITSN1 and ITSN2 leads to severe ASD/OCD-like behavioral alterations and defective cortico-striatal neurotransmission in knockout (KO) mice. Cortico-striatal function was compromised at multiple levels in ITSN1/2-depleted animals. Morphological analyses showed that the striatum of intersectin KO mice is decreased in size. Striatal neurons exhibit reduced complexity and an underdeveloped dendritic spine architecture. These morphological abnormalities correlate with defects in cortico-striatal neurotransmission and plasticity as well as reduced N-methyl-D-aspartate (NMDA) receptor currents as a consequence of postsynaptic NMDA receptor depletion. Our findings unravel a physiological role of intersectin in cortico-striatal neurotransmission to counteract ASD/OCD. Moreover, we delineate a molecular pathomechanism for the neuropsychiatric symptoms of patients carrying intersectin mutations that correlates with the observation that NMDA receptor dysfunction is a recurrent feature in the development of ASD/OCD-like symptoms.

Astrocyte ß-Adrenergic Receptor Activity Regulates NMDA Receptor Signaling of Medial Prefrontal Cortex Pyramidal Neurons.

Glutamate spillover from the synapse is tightly regulated by astrocytes, limiting the activation of extrasynaptically located NMDA receptors (NMDAR). The processes of astrocytes are dynamic and can modulate synaptic physiology. Though norepinephrine (NE) and ß-adrenergic receptor (ß-AR) activity can modify astrocyte volume, this has yet to be confirmed outside of sensory cortical areas, nor has the effect of noradrenergic signaling on glutamate spillover and neuronal NMDAR activity been explored. We monitored changes to astrocyte process volume in response to noradrenergic agonists in the medial prefrontal cortex of male and female mice. Both NE and the ß-AR agonist isoproterenol (ISO) increased process volume by ∼20%, significantly higher than changes seen when astrocytes had G-protein signaling blocked by GDPßS. We measured the effect of ß-AR signaling on evoked NMDAR currents. While ISO did not affect single stimulus excitatory currents of Layer 5 pyramidal neurons, ISO reduced NMDAR currents evoked by 10 stimuli at 50 Hz, which elicits glutamate spillover, by 18%. After isolating extrasynaptic NMDARs by blocking synaptic NMDARs with the activity-dependent NMDAR blocker MK-801, ISO similarly reduced extrasynaptic NMDAR currents in response to 10 stimuli by 18%. Finally, blocking ß-AR signaling in the astrocyte network by loading them with GDPßS reversed the ISO effect on 10 stimuli-evoked NMDAR currents. These results demonstrate that astrocyte ß-AR activity reduces extrasynaptic NMDAR recruitment, suggesting that glutamate spillover is reduced.

Characterization of Mice Carrying a Neurodevelopmental Disease-Associated GluN2B(L825V) Variant.

N-Methyl-d-aspartate receptors (NMDARs), encoded by GRIN genes, are ionotropic glutamate receptors playing a critical role in synaptic transmission, plasticity, and synapse development. Genome sequence analyses have identified variants in GRIN genes in patients with neurodevelopmental disorders, but the underlying disease mechanisms are not well understood. Here, we have created and evaluated a transgenic mouse line carrying a missense variant Grin2bL825V , corresponding to a de novo GRIN2B variant encoding GluN2B(L825V) found in a patient with intellectual disability (ID) and autism spectrum disorder (ASD). We used HEK293T cells expressing recombinant receptors and primary hippocampal neurons prepared from heterozygous Grin2bL825V/+ (L825V/+) and wild-type (WT) Grin2b+/+ (+/+) male and female mice to assess the functional impact of the variant. Whole-cell NMDAR currents were reduced in neurons from L825V/+ compared with +/+ mice. The peak amplitude of NMDAR-mediated evoked excitatory postsynaptic currents (NMDAR-eEPSCs) was unchanged, but NMDAR-eEPSCs in L825V/+ neurons had faster deactivation compared with +/+ neurons and were less sensitive to a GluN2B-selective antagonist ifenprodil. Together, these results suggest a decreased functional contribution of GluN2B subunits to synaptic NMDAR currents in hippocampal neurons from L825V/+ mice. The analysis of the GluN2B(L825V) subunit surface expression and synaptic localization revealed no differences compared with WT GluN2B. Behavioral testing of mice of both sexes demonstrated hypoactivity, anxiety, and impaired sensorimotor gating in the L825V/+ strain, particularly affecting males, as well as cognitive symptoms. The heterozygous L825V/+ mouse offers a clinically relevant model of GRIN2B-related ID/ASD, and our results suggest synaptic-level functional changes that may contribute to neurodevelopmental pathology.

d-Serine Inhibits Non-ionotropic NMDA Receptor Signaling.

NMDA-type glutamate receptors (NMDARs) are widely recognized as master regulators of synaptic plasticity, most notably for driving long-term changes in synapse size and strength that support learning. NMDARs are unique among neurotransmitter receptors in that they require binding of both neurotransmitter (glutamate) and co-agonist (e.g., d-serine) to open the receptor channel, which leads to the influx of calcium ions that drive synaptic plasticity. Over the past decade, evidence has accumulated that NMDARs also support synaptic plasticity via ion flux-independent (non-ionotropic) signaling upon the binding of glutamate in the absence of co-agonist, although conflicting results have led to significant controversy. Here, we hypothesized that a major source of contradictory results might be attributed to variable occupancy of the co-agonist binding site under different experimental conditions. To test this hypothesis, we manipulated co-agonist availability in acute hippocampal slices from mice of both sexes. We found that enzymatic scavenging of endogenous co-agonists enhanced the magnitude of long-term depression (LTD) induced by non-ionotropic NMDAR signaling in the presence of the NMDAR pore blocker MK801. Conversely, a saturating concentration of d-serine completely inhibited LTD and spine shrinkage induced by glutamate binding in the presence of MK801 or Mg2+ Using a Förster resonance energy transfer (FRET)-based assay in cultured neurons, we further found that d-serine completely blocked NMDA-induced conformational movements of the GluN1 cytoplasmic domains in the presence of MK801. Our results support a model in which d-serine availability serves to modulate NMDAR signaling and synaptic plasticity even when the NMDAR is blocked by magnesium.

Hypothalamic Corticotropin-Releasing Hormone Contributes to Hypertension in Spontaneously Hypertensive Rats.

Corticotropin-releasing hormone (CRH) is a neuropeptide regulating neuroendocrine and autonomic function. CRH mRNA and protein levels in the hypothalamic paraventricular nucleus (PVN) are increased in primary hypertension. However, the role of CRH in elevated sympathetic outflow in primary hypertension remains unclear. CRHR1 proteins were distributed in retrogradely labeled PVN presympathetic neurons with an increased level in the PVN tissue in adult spontaneously hypertensive rats (SHRs) compared with age-matched male Wistar-Kyoto (WKY) rats. CRH induced a more significant increase in the firing rate of PVN-rostral ventrolateral medulla (RVLM) neurons and sympathoexcitatory response in SHRs than in WKY rats, an effect that was blocked by preapplication of NMDA receptors (NMDARs) antagonist AP5 and PSD-95 inhibitor, Tat-N-dimer. Blocking CRHRs with astressin or CRHR1 with NBI35965 significantly decreased the firing rate of PVN-RVLM output neurons and reduced arterial blood pressure (ABP) and renal sympathetic nerve activity (RSNA) in SHRs but not in WKY, whereas blocking CRHR2 with antisauvagine-30 did not. Furthermore, Immunocytochemistry staining revealed that CRHR1 colocalized with NMDARs in PVN presympathetic neurons. Blocking CRHRs significantly decreased the NMDA currents in labeled PVN neurons. PSD-95-bound CRHR1 and PSD-95-bound GluN2A in the PVN were increased in SHRs. These data suggested that the upregulation of CRHR1 in the PVN is critically involved in the hyperactivity of PVN presympathetic neurons and elevated sympathetic outflow in primary hypertension.SIGNIFICANCE STATEMENT Our study found that corticotropin-releasing hormone receptor (CRHR)1 protein levels were increased in the paraventricular nucleus (PVN), and CRHR1 interacts with NMDA receptors (NMDARs) through postsynaptic density protein (PSD)-95 in the PVN neurons in primary hypertension. The increased CRHR1 and CRHR1-NMDAR-PSD-95 complex in the PVN contribute to the hyperactivity of the PVN presympathetic neurons and elevated sympathetic vasomotor tone in hypertension in SHRs. Thus, the antagonism of CRHR1 decreases sympathetic outflow and blood pressure in hypertension. These findings determine a novel role of CRHR1 in elevated sympathetic vasomotor tone in hypertension, which is useful for developing novel therapeutics targeting CRHR1 to treat elevated sympathetic outflow in primary hypertension. The CRHR1 receptor antagonists, which are used to treat health consequences resulting from chronic stress, are candidates to treat primary hypertension.

Linking activation of synaptic NMDA receptors-induced CREB signaling to brief exposure of cortical neurons to oligomeric amyloid-beta peptide.

Amyloid-beta peptide oligomers (AßO) have been considered "primum movens" for a cascade of events that ultimately cause selective neuronal death in Alzheimer's disease (AD). However, initial events triggered by AßO have not been clearly defined. Synaptic (Syn) N-methyl-d-aspartate receptors (NMDAR) are known to activate cAMP response element-binding protein (CREB), a transcriptional factor involved in gene expression related to cell survival, memory formation and synaptic plasticity, whereas activation of extrasynaptic (ESyn) NMDARs was linked to excitotoxic events. In AD brain, CREB phosphorylation/activation was shown to be altered, along with dyshomeostasis of intracellular Ca2+ (Ca2+ i). Thus, in this work, we analyze acute/early and long-term AßO-mediated changes in CREB activation involving Syn or ESyn NMDARs in mature rat cortical neurons. Our findings show that acute AßO exposure produce early increase in phosphorylated CREB, reflecting CREB activity, in a process occurring through Syn NMDAR-mediated Ca2+ influx. Data also demonstrate that AßO long-term (24 h) exposure compromises synaptic function related to Ca2+-dependent CREB phosphorylation/activation and nuclear CREB levels and related target genes, namely Bdnf, Gadd45γ, and Btg2. Data suggest a dual effect of AßO following early or prolonged exposure in mature cortical neurons through the activation of the CREB signaling pathway, linked to the activation of Syn NMDARs.

Characterization of tritiated JNJ-GluN2B-5 (3-[3H] 1-(azetidin-1-yl)-2-(6-(4-fluoro-3-methyl-phenyl)pyrrolo[3,2-b]pyridin-1-yl)ethanone), a high affinity GluN2B radioligand with selectivity over sigma receptors.

Here, we describe the characterization of a radioligand selective for GluN2B-containing NMDA receptors, 3-[3H] 1-(azetidin-1-yl)-2-(6-(4-fluoro-3-methyl-phenyl)pyrrolo[3,2-b]pyridin-1-yl)ethanone ([3H]-JNJ- GluN2B-5). In rat cortical membranes, the compound bound to a single site, and the following kinetic parameters were measured; association rate constant Kon = 0.0066 ± 0.0006 min-1 nM-1, dissociation rate constant Koff = 0.0210 ± 0.0001 min-1 indicating calculated KD = Koff/Kon = 3.3 ± 0.4 nM, (mean ± SEM, n = 3). The equilibrium dissociation constant determined from saturation binding experiments in rat cortex was KD of 2.6 ± 0.3 nM (mean ± SEM, n = 3). In contrast to the widely used GluN2B radioligand [3H]-Ro 25-6981, whose affinity Ki for sigma 1 and sigma 2 receptors are 2 and 189 nM, respectively, [3H]-JNJ-GluN2B-5 exhibits no measurable affinity for sigma 1 and sigma 2 receptors (Ki > 10 µM for both) providing distinct selectivity advantages. Anatomical distribution of [3H]-JNJ-GluN2B-5 binding sites in rat, mouse, dog, monkey, and human brain tissue was studied using in vitro autoradiography, which showed high specific binding in the hippocampus and cortex and negligible binding in the cerebellum. Enhanced selectivity for GluN2B-containing receptors translated to a good signal-to-noise ratio in both in vitro radioligand binding and in vitro autoradiography assays. In conclusion, [3H]-JNJ-GluN2B-5 is a high-affinity GluN2B radioligand with excellent signal-to-noise ratio and unprecedented selectivity.

Tryptophan metabolism as a 'reflex' feature of neuroimmune communication: Sensor and effector functions for the indoleamine-2, 3-dioxygenase kynurenine pathway.

Although the central nervous system (CNS) and immune system were regarded as independent entities, it is now clear that immune system cells can influence the CNS, and neuroglial activity influences the immune system. Despite the many clinical implications for this 'neuroimmune interface', its detailed operation at the molecular level remains unclear. This narrative review focuses on the metabolism of tryptophan along the kynurenine pathway, since its products have critical actions in both the nervous and immune systems, placing it in a unique position to influence neuroimmune communication. In particular, since the kynurenine pathway is activated by pro-inflammatory mediators, it is proposed that physical and psychological stressors are the stimuli of an organismal protective reflex, with kynurenine metabolites as the effector arm co-ordinating protective neural and immune system responses. After a brief review of the neuroimmune interface, the general perception of tryptophan metabolism along the kynurenine pathway is expanded to emphasize this environmentally driven perspective. The initial enzymes in the kynurenine pathway include indoleamine-2,3-dioxygenase (IDO1), which is induced by tissue damage, inflammatory mediators or microbial products, and tryptophan-2,3-dioxygenase (TDO), which is induced by stress-induced glucocorticoids. In the immune system, kynurenic acid modulates leucocyte differentiation, inflammatory balance and immune tolerance by activating aryl hydrocarbon receptors and modulates pain via the GPR35 protein. In the CNS, quinolinic acid activates N-methyl-D-aspartate (NMDA)-sensitive glutamate receptors, whereas kynurenic acid is an antagonist: the balance between glutamate, quinolinic acid and kynurenic acid is a significant regulator of CNS function and plasticity. The concept of kynurenine and its metabolites as mediators of a reflex coordinated protection against stress helps to understand the variety and breadth of their activity. It should also help to understand the pathological origin of some psychiatric and neurodegenerative diseases involving the immune system and CNS, facilitating the development of new pharmacological strategies for treatment.

Mechanisms of synapse-to-nucleus calcium signalling in striatal neurons and impairments in Huntington's disease.

Huntington's disease (HD) is a monogenic disorder with autosomal dominant inheritance. In HD patients, neurons in the striatum and cortex degenerate, leading to motor, psychiatric and cognitive disorders. Dysregulated synaptic function and calcium handling are common in many neurodegenerative diseases, including HD. N-methyl-D-aspartate (NMDA) receptor function is enhanced in HD at extrasynaptic sites, altering the balance of calcium-dependent neuronal survival versus death signalling pathways. Endoplasmic reticulum (ER) calcium handling is also abnormal in HD. The ER, which is continuous with the nuclear envelope, is purportedly involved in nuclear calcium signalling; based on this, we hypothesised that nuclear calcium signalling is altered in HD. We explored this hypothesis with calcium imaging techniques, including simultaneous epifluorescent imaging of cytosolic and nuclear calcium using jRCaMP1b and GCaMP3 sensors, respectively, in striatal spiny projection neurons in cortical-striatal co-cultures from the YAC128 mouse model of HD. Our data show contributions from a variety of calcium channels to nuclear calcium signalling. NMDA receptors (NMDARs) play an essential role in initiating action potential-dependent calcium signalling to the nucleus, and ryanodine receptors (RyR) contribute to both cytosolic and nuclear calcium signals. Unlike previous reports in glutamatergic hippocampal and cortical neurons, we found that in GABAergic striatal neurons, L-type voltage-gated calcium channels (CaV) contribute to cytosolic, but not nuclear calcium signalling. Calcium imaging also suggests impairments in nuclear calcium signalling in HD striatal neurons, where spontaneous action potential-dependent calcium transients in the nucleus were smaller in YAC128 striatal neurons compared to those of wild-type (WT). Our results elucidate mechanisms involved in action potential-dependent nuclear calcium signalling in GABAergic striatal neurons, and have revealed a clear deficit in this signalling in HD.

Long term effects of peripubertal stress on the thalamic reticular nucleus of female and male mice.

Adverse experiences during infancy and adolescence have an important and enduring effect on the brain and are predisposing factors for mental disorders, particularly major depression. This impact is particularly notable in regions with protracted development, such as the prefrontal cortex. The inhibitory neurons of this cortical region are altered by peripubertal stress (PPS), particularly in female mice. In this study we have explored whether the inhibitory circuits of the thalamus are impacted by PPS in male and female mice. This diencephalic structure, as the prefrontal cortex, also completes its development during postnatal life and is affected by adverse experiences. The long-term changes induced by PPS were exclusively found in adult female mice. We have found that PPS increases depressive-like behavior and induces changes in parvalbumin-expressing (PV+) cells of the thalamic reticular nucleus (TRN). We observed reductions in the volume of the TRN, together with those of parameters related to structures/molecules that regulate the plasticity and connectivity of PV+ cells: perineuronal nets, matricellular structures surrounding PV+ neurons, and the polysialylated form of the neural cell adhesion molecule (PSA-NCAM). The expression of the GluN1, but not of GluN2C, NMDA receptor subunit was augmented in the TRN after PPS. An increase in the fluorescence intensity of PV+ puncta was also observed in the synaptic output of TRN neurons in the lateral posterior thalamic nucleus. These results demonstrate that the inhibitory circuits of the thalamus, as those of the prefrontal cortex, are vulnerable to the effects of aversive experiences during early life, particularly in females. This vulnerability is probably related to the protracted development of the TRN and might contribute to the development of psychiatric disorders.

Novel Therapeutic Approach for Excitatory/Inhibitory Imbalance in Neurodevelopmental and Neurodegenerative Diseases.

Excitatory/inhibitory (E/I) balance, defined as the balance between excitation and inhibition of synaptic activity in a neuronal network, accounts in part for the normal functioning of the brain, controlling, for example, normal spike rate. In many pathological conditions, this fine balance is perturbed, leading to excessive or diminished excitation relative to inhibition, termed E/I imbalance, reflected in network dysfunction. E/I imbalance has emerged as a contributor to neurological disorders that occur particularly at the extremes of life, including autism spectrum disorder and Alzheimer's disease, pointing to the vulnerability of neuronal networks at these critical life stages. Hence, it is important to develop approaches to rebalance neural networks. In this review, we describe emerging therapies that can normalize the E/I ratio or the underlying abnormality that contributes to the imbalance in electrical activity, thus improving neurological function in these maladies.

Putative neural mechanisms underlying release-mode-specific abnormalities in dopamine neural activity in a schizophrenia-like model: The distinct roles of glutamate and serotonin in the impaired regulation of dopamine neurons.

Abnormalities in dopamine function might be related to psychiatric disorders such as schizophrenia. Even at the same concentration, dopamine exerts opposite effects on information processing in the prefrontal cortex depending on independent dopamine release modes known as tonic and phasic releases. This duality of dopamine prevents a blanket interpretation of the implications of dopamine abnormalities for diseases on the basis of absolute dopamine levels. Moreover, the mechanisms underlying the mode-specific dopamine abnormalities are not clearly understood. Here, I show that the two modes of dopamine release in the prefrontal cortex of a schizophrenia-like model are disrupted by different mechanisms. In the schizophrenia-like model established by perinatal exposure to inflammatory cytokine, epidermal growth factor, tonic release was enhanced and phasic release was decreased in the prefrontal cortex. I examined the activity of dopamine neurons in the ventral tegmental area (VTA), which sends dopamine projections to the prefrontal cortex, under anaesthesia. The activation of VTA dopamine neurons during excitatory stimulation (local application of glutamate or N-methyl-d-aspartic acid [NMDA]), which is associated with phasic activity, was blunt in this model. Dopaminergic neuronal activity in the resting state related to tonic release was increased by disinhibition of the dopamine neurons due to the impairment of 5HT2 (5HT2A) receptor-regulated GABAergic inputs. Moreover, chronic administration of risperidone ameliorated this disinhibition of dopaminergic neurons. These results provide an idea about the mechanism of dopamine disturbance in schizophrenia and may be informative in explaining the effects of atypical antipsychotics as distinct from those of typical drugs.

Implications of high homocysteine levels in migraine pain: An experimental study of the excitability of peripheral meningeal afferents in rats with hyperhomocysteinemia.

OBJECTIVES: Investigation of chronic homocysteine action on the excitability and N-methyl-D-aspartate (NMDA) sensitivity of the peripheral trigeminovascular system of rats. BACKGROUND: Migraine is a neurological disease that affects 15%-20% of the general population. Epidemiological observations show that an increase of the sulfur-containing amino acid homocysteine in plasma-called hyperhomocysteinemia-is associated with a high risk of migraine, especially migraine with aura. In animal studies, rats with hyperhomocysteinemia demonstrated mechanical allodynia, photophobia, and anxiety, and higher sensitivity to cortical spreading depression. In addition, rats with hyperhomocysteinemia were more sensitive in a model of chronic migraine induced by nitroglycerin which indicated the involvement of peripheral nociceptive mechanisms. The present work aimed to analyze the excitability of meningeal afferents and neurons isolated from the trigeminal ganglion of rats with prenatal hyperhomocysteinemia. METHODS: Experiments were performed on male rats born from females fed with a methionine-rich diet before and during pregnancy. The activity of meningeal afferents was recorded extracellularly in hemiskull preparations ex vivo and action potentials were characterized using cluster analysis. The excitability of trigeminal ganglion neurons was assessed using whole-cell patch clamp recording techniques and calcium imaging studies. Meningeal mast cells were stained using toluidine blue. RESULTS: The baseline extracellular recorded electrical activity of the trigeminal nerve was higher in the hyperhomocysteinemia group with larger amplitude action potentials. Lower concentrations of KCl caused an increase in the frequency of action potentials of trigeminal afferents recorded in rat hemiskull ex vivo preparations. In trigeminal ganglion neurons of rats with hyperhomocysteinemia, the current required to elicit at least one action potential (rheobase) was lower, and more action potentials were induced in response to stimulus of 2 × rheobase. In controls, short-term application of homocysteine and its derivatives increased the frequency of action potentials of the trigeminal nerve and induced Ca2+ transients in neurons, which are associated with the activation of NMDA receptors. At the same time, in rats with hyperhomocysteinemia, we did not observe an increased response of the trigeminal nerve to NMDA. Similarly, the parameters of Ca2+ transients induced by NMDA, homocysteine, and its derivatives were not changed in rats with hyperhomocysteinemia. Acute incubation of the meninges in homocysteine and homocysteinic acid did not change the state of the mast cells, whereas in the model of hyperhomocysteinemia, an increased degranulation of mast cells in the meninges was observed. CONCLUSIONS: Our results demonstrated higher excitability of the trigeminal system of rats with hyperhomocysteinemia. Together with our previous finding about the lower threshold of generation of cortical spreading depression in rats with hyperhomocysteinemia, the present data provide evidence of homocysteine as a factor that increases the sensitivity of the peripheral migraine mechanisms, and the control of homocysteine level may be an important strategy for reducing the risk and/or severity of migraine headache attacks.

Adapting the endoplasmic reticulum proteostasis rescues epilepsy-associated NMDA receptor variants.

The GRIN genes encoding N-methyl-D-aspartate receptor (NMDAR) subunits are remarkably intolerant to variation. Many pathogenic NMDAR variants result in their protein misfolding, inefficient assembly, reduced surface expression, and impaired function on neuronal membrane, causing neurological disorders including epilepsy and intellectual disability. Here, we investigated the proteostasis maintenance of NMDARs containing epilepsy-associated variations in the GluN2A subunit, including M705V and A727T. In the transfected HEK293T cells, we showed that the two variants were targeted to the proteasome for degradation and had reduced functional surface expression. We demonstrated that the application of BIX, a known small molecule activator of an HSP70 family chaperone BiP (binding immunoglobulin protein) in the endoplasmic reticulum (ER), dose-dependently enhanced the functional surface expression of the M705V and A727T variants in HEK293T cells. Moreover, BIX (10 µM) increased the surface protein levels of the M705V variant in human iPSC-derived neurons. We revealed that BIX promoted folding, inhibited degradation, and enhanced anterograde trafficking of the M705V variant by modest activation of the IRE1 pathway of the unfolded protein response. Our results suggest that adapting the ER proteostasis network restores the folding, trafficking, and function of pathogenic NMDAR variants, representing a potential treatment for neurological disorders resulting from NMDAR dysfunction.

STIM2 regulates NMDA receptor endocytosis that is induced by short-term NMDA receptor overactivation in cortical neurons.

Recent findings suggest an important role for the dysregulation of stromal interaction molecule (STIM) proteins, activators of store-operated Ca2+ channels, and the prolonged activation of N-methyl-D-aspartate receptors (NMDARs) in the development of neurodegenerative diseases. We previously demonstrated that STIM silencing increases Ca2+ influx through NMDAR and STIM-NMDAR2 complexes are present in neurons. However, the interplay between NMDAR subunits (GluN1, GluN2A, and GluN2B) and STIM1/STIM2 with regard to intracellular trafficking remains unknown. Here, we found that the activation of NMDAR endocytosis led to an increase in STIM2-GluN2A and STIM2-GluN2B interactions in primary cortical neurons. STIM1 appeared to migrate from synaptic to extrasynaptic sites. STIM2 silencing inhibited post-activation NMDAR translocation from the plasma membrane and synaptic spines and increased NMDAR currents. Our findings reveal a novel molecular mechanism by which STIM2 regulates NMDAR synaptic trafficking by promoting NMDAR endocytosis after receptor overactivation, which may suggest protection against excessive uncontrolled Ca2+ influx through NMDARs.

Astrocytic NMDA Receptors.

Astrocytic NMDA receptors (NMDARs) are heterotetramers, whose expression and properties are largely determined by their subunit composition. Astrocytic NMDARs are characterized by a low sensitivity to magnesium ions and low calcium conductivity. Their activation plays an important role in the regulation of various intracellular processes, such as gene expression and mitochondrial function. Astrocytic NMDARs are involved in calcium signaling in astrocytes and can act through the ionotropic and metabotropic pathways. Astrocytic NMDARs participate in the interactions of the neuroglia, thus affecting synaptic plasticity. They are also engaged in the astrocyte-vascular interactions and contribute to the regulation of vascular tone. Astrocytic NMDARs are involved in various pathologies, such as ischemia and hyperammonemia, and their blockade prevents negative changes in astrocytes during these diseases.

NMDA Receptors and Indices of Energy Metabolism in Erythrocytes: Missing Link to the Assessment of Efficiency of Oxygen Transport in Hepatic Encephalopathy.

Hepatic encephalopathy (HE) is a neuropsychiatric syndrome that develops in patients with severe liver dysfunction and/or portocaval shunting. Despite more than a century of research into the relationship between liver damage and development of encephalopathy, pathogenetic mechanisms of hepatic encephalopathy have not yet been fully elucidated. It is generally recognized, however, that the main trigger of neurologic complications in hepatic encephalopathy is the neurotoxin ammonia/ammonium, concentration of which in the blood increases to toxic levels (hyperammonemia), when detoxification function of the liver is impaired. Freely penetrating into brain cells and affecting NMDA-receptor-mediated signaling, ammonia triggers a pathological cascade leading to the sharp inhibition of aerobic glucose metabolism, oxidative stress, brain hypoperfusion, nerve cell damage, and formation of neurological deficits. Brain hypoperfusion, in turn, could be due to the impaired oxygen transport function of erythrocytes, because of the disturbed energy metabolism that occurs in the membranes and inside erythrocytes and controls affinity of hemoglobin for oxygen, which determines the degree of oxygenation of blood and tissues. In our recent study, this causal relationship was confirmed and novel ammonium-induced pro-oxidant effect mediated by excessive activation of NMDA receptors leading to impaired oxygen transport function of erythrocytes was revealed. For a more complete evaluation of "erythrocytic" factors that diminish brain oxygenation and lead to encephalopathy, in this study, activity of the enzymes and concentration of metabolites of glycolysis and Rapoport-Lubering shunt, as well as morphological characteristics of erythrocytes from the rats with acute hyperammoniemia were determined. To elucidate the role of NMDA receptors in the above processes, MK-801, a non-competitive receptor antagonist, was used. Based on the obtained results it can be concluded that it is necessary to consider ammonium-induced morphofunctional disorders of erythrocytes and hemoglobinemia which can occur as a result of alterations in highly integrated networks of metabolic pathways may act as an additional systemic "erythrocytic" pathogenetic factor to prevent the onset and progression of cerebral hypoperfusion in hepatic encephalopathy accompanied by hyperammonemia.

Selective disruption of synaptic NMDA receptors of the hippocampal trisynaptic circuit in Aß pathology.

Synaptic dysfunction is an early feature in Alzheimer's disease (AD) pathogenesis and a major morphological correlate of memory deficits. Given the main synaptic location of N-methyl-D-aspartate receptors (NMDARs), their dysregulation has been implicated in these pathological effects. Here, to detect possible alterations in the expression and synaptic localisation of the GluN1 subunit in the brain of amyloidogenic APP/PS1 mice, we employed histoblot and SDS-digested freeze-fracture replica labelling (SDS-FRL) techniques. Histoblots showed that GluN1 expression was significantly reduced in the hippocampus in a layer-dependent manner, in the cortex and the caudate putamen of APP/PS1 transgenic mice at 12 months of age but was unaltered at 1 and 6 months. Using quantitative SDS-FRL, we unravelled the molecular organisation of GluN1 in seven excitatory synapse populations at a high spatial resolution in the CA1 and CA3 fields and the DG of the hippocampus in 12-month-old APP/PS1 mice. In the CA1 field, the labelling density for GluN1 in the excitatory synapses established on spines and interneurons, was significantly reduced in APP/PS1 mice compared to age-matched wild-type mice in the stratum lacunosum-moleculare but unaltered in the stratum radiatum. In the CA3 field, synaptic GluN1 was reduced in mossy fibre-CA3 pyramidal cell synapses but unaltered in the A/C-CA3 pyramidal cell synapses. In the DG, the density of GluN1 in granule cell-perforant pathway synapses was reduced in APP/PS1 mice. Altogether, our findings provide evidence of specific alterations of synaptic GluN1 in the trisynaptic circuit of the hippocampus in Aß pathology. This differential vulnerability in the disruption of NMDARs may be involved in the mechanisms causing abnormal network activity of the hippocampal circuit and cognitive impairment characteristic of APP/PS1 mice.

Dopaminergic neuromodulation of prefrontal cortex activity requires the NMDA receptor coagonist d-serine.

Prefrontal control of cognitive functions critically depends upon glutamatergic transmission and N-methyl D-aspartate (NMDA) receptors, the activity of which is regulated by dopamine. Yet whether the NMDA receptor coagonist d-serine is implicated in the dopamine-glutamate dialogue in the prefrontal cortex (PFC) and other brain areas remains unexplored. Here, using electrophysiological recordings, we show that d-serine is required for the fine-tuning of glutamatergic neurotransmission, neuronal excitability, and synaptic plasticity in the PFC through the actions of dopamine at D1 and D3 receptors. Using in vivo microdialysis, we show that D1 and D3 receptors exert a respective facilitatory and inhibitory influence on extracellular levels and activity of d-serine in the PFC, with actions expressed primarily via the cAMP/protein kinase A (PKA) signaling cascade. Further, using functional magnetic resonance imaging (fMRI) and behavioral assessment, we show that d-serine is required for the potentiation of cognition by D3R blockade as revealed in a test of novel object recognition memory. Collectively, these results unveil a key role for d-serine in the dopaminergic neuromodulation of glutamatergic transmission and PFC activity, findings with clear relevance to the pathogenesis and treatment of diverse brain disorders involving alterations in dopamine-glutamate cross-talk.