D‐Serine metabolism in C6 glioma cells: Involvement of alanine‐serine‐cysteine transporter (ASCT2) and serine racemase (SRR) but not D‐amino acid oxidase (DAO)

D‐serine is an endogenous N‐methyl‐D‐aspartate (NMDA) receptor coagonist. It is synthesized from L‐serine by serine racemase (SRR), but many aspects of its metabolism remain unclear, especially in the forebrain, which lacks active D‐amino acid oxidase (DAO), the major D‐serine degradative enzyme. Candidate mechanisms include SRR operating in α,β‐eliminase mode (converting D‐serine to pyruvate) and regulation by serine transport, in which the alanine‐serine‐cysteine transporter ASCT2 is implicated. Here we report studies in C6 glioma cells, which “simulate” the forebrain, in that the cells express SRR and ASCT2 but lack DAO activity. We measured D‐serine, ASCT2, SRR, and DAO expression and DAO activity in two situations: after incubation of cells for 48 hr with serine isomers and after increased or decreased SRR expression by transfection and RNA interference, respectively. Incubation with serine enantiomers decreased [³H]D‐serine uptake and ASCT2 mRNA and increased SRR immunoreactivity but did not alter DAO immunoreactivity, and DAO activity remained undetectable. SRR overexpression increased D‐serine and pyruvate and decreased [³H]D‐serine uptake and ASCT2 mRNA but did not affect DAO. SRR knockdown did not alter any of the parameters. Our data suggest that D‐serine transport mediated by ASCT2 contributes prominently to D‐serine homeostasis when DAO activity is absent. The factors regulating D‐serine are important for understanding normal NMDA receptor function and because D‐serine, along with DAO and SRR, is implicated in the pathogenesis and treatment of schizophrenia. © 2010 Wiley‐Liss, Inc.     

Functional and immunocytochemical characterization of D‐serine transporters in cortical neuron and astrocyte cultures

D‐serine is an endogenous coagonist of N‐methyl‐D‐aspartate (NMDA) receptors that plays an important role in synaptic function, neuronal development, and excitotoxicity. Mechanisms of D‐serine transport are important in regulation of extracellular D‐serine concentration and therefore of these critical processes. D‐serine can be transported with low affinity through the Na⁺‐dependent amino acid transporter termed ASCT2, whereas high‐affinity D‐serine uptake has been reported through the Na⁺‐independent transporter termed asc‐1. We investigated immunoreactivity for ASCT2 and asc‐1 and D‐serine transport kinetics in cultured cortical neurons and astrocytes to gain insight into how D‐serine transporters regulate CNS D‐serine levels. Both neurons and astrocytes exhibited low‐affinity Na⁺‐dependent D‐serine uptake (K_T > 1 mM) with broad substrate selectivity that was consistent with uptake through ASCT2. Both neurons and astrocytes also stained positively for ASCT2 in immunocytochemistry studies. Neurons but not astrocytes stained positively for the high‐affinity D‐serine transporter asc‐1, but no evidence of functional asc‐1 could be detected in neurons with conditions that produced such activity in cortical synaptosomes. These data support ASCT2 function in both neuron and astrocyte cultures and identify a discrepancy between observed asc‐1 immunoreactivity and lack of functional asc‐1 activity in neuron cultures. Together these findings further our knowledge of the processes that govern D‐serine regulation. © 2009 Wiley‐Liss, Inc.     

Simultaneous efflux of endogenous D‐ser and L‐glu from single acute hippocampus slices during oxygen glucose deprivation

D‐serine and L‐glutamate play crucial roles in excitotoxicity through N‐methyl‐D‐aspartate receptor coactivation, but little is known about the temporal profile of efflux during cerebral ischemia. We utilized a newly designed brain slice microperfusion device coupled offline to capillary electrophoresis laser‐induced fluorescence to monitor dynamic efflux of endogenous D‐ser and L‐glu in response to oxygen glucose deprivation (OGD) in single acute hippocampus slices. Efflux profiles with 2‐min temporal resolution in response to 24‐min OGD show that efflux of D‐ser slightly precedes efflux of L‐glu by one 2‐min sampling interval. Thus both coagonists are available to activate NMDA receptors by the time when glu is released. The magnitude of D‐ser efflux relative to baseline values is, however, less than that for L‐glu. Peak efflux during OGD, expressed as pre‐OGD baseline values, was as follows: D‐ser 254% ± 24%, L‐glu 1,675% ± 259%, L‐asp 519% ± 128%, and L‐thr 313% ± 33%. L‐glutamine efflux was shown to decrease significantly in response to OGD. The microperfusion/CE‐LIF approach shows several promising attributes for studying endogenous chemical efflux from single, acute brain slices. © 2009 Wiley‐Liss, Inc.     

Searching for cognitive enhancement in the Morris water maze: better and worse performance in D‐amino acid oxidase knockout (Dao−/−) mice

A common strategy when searching for cognitive‐enhancing drugs has been to target the N‐methyl‐d ‐aspartate receptor (NMDAR), given its putative role in synaptic plasticity and learning. Evidence in favour of this approach has come primarily from studies with rodents using behavioural assays like the Morris water maze. D‐amino acid oxidase (DAO) degrades neutral D‐amino acids such as D‐serine, the primary endogenous co‐agonist acting at the glycine site of the synaptic NMDAR. Inhibiting DAO could therefore provide an effective and viable means of enhancing cognition, particularly in disorders like schizophrenia, in which NMDAR hypofunction is implicated. Indirect support for this notion comes from the enhanced hippocampal long‐term potentiation and facilitated water maze acquisition of ddY/Dao⁻ mice, which lack DAO activity due to a point mutation in the gene. Here, in Dao knockout (Dao^−/−) mice, we report both better and worse water maze performance, depending on the radial distance of the hidden platform from the side wall of the pool. Dao^−/− mice displayed an increased innate preference for swimming in the periphery of the maze (possibly due to heightened anxiety), which facilitated the discovery of a peripherally located platform, but delayed the discovery of a centrally located platform. By contrast, Dao^−/− mice exhibited normal performance in two alternative assays of long‐term spatial memory: the appetitive and aversive Y‐maze reference memory tasks. Taken together, these results question the proposed relationship between DAO inactivation and enhanced long‐term associative spatial memory. They also have generic implications for how Morris water maze studies are performed and interpreted.     

Potential roles of D‐serine and serine racemase in experimental temporal lobe epilepsy

To confirm the roles of D‐serinergic gliotransmission in epilepsy, we investigated the relationship between spatiotemporally specific glial responses and the D‐serine/serine racemase system in mesial temporal structures following status epilepticus (SE). In control animals, D‐serine and serine racemase immunoreactivities were detected mainly in astrocytes. After SE, D‐serine and serine racemase immunoreactivities were increased in astrocytes. Double‐immunofluorescence study revealed that up‐regulation of serine racemase immunoreactivity was relevant not to D‐serine immunoreactivity but to nestin or vimentin immunoreactivity. Neither D‐serine nor serine racemase was found in naïve or reactive microglia. In addition, phosphorylated N‐methyl‐D‐aspartate (NMDA) receptor subunit 1 (pNR1‐Ser896) immunoreactivity in the hippocampus was increased compared with controls. Increased D‐serine immunoreactivity showed direct correlation with the phosphorylation of Ser896 of NR1. Given the findings of our previous study, these findings suggest that D ‐serine and serine racemase in astrocytes may play roles in neuronal hyperexcitability via a cooperative activation of NMDA receptors. Furthermore, serine racemase may be involved in migration and differentiation of immature astrocytes, which is relevant to reactive astrogliosis. © 2010 Wiley‐Liss, Inc.     

The glycine transporter GlyT1 controls N‐methyl‐D‐aspartic acid receptor coagonist occupancy in the mouse retina

We examined the role of GlyT1, the high‐affinity glycine transporter, in the mouse retina with an emphasis on the role of glycine as a coagonist of N‐methyl‐D‐aspartic acid (NMDA) receptors. We pursued this objective by studying heterozygote mice deficient in the GlyT1 transporter (GlyT1^?/+) and compared those results with wild‐type (WT) littermate controls (GlyT1^+/+). Capillary electrophoresis was used to separate and quantitatively measure glycine release from isolated retina preparations; pharmacologically blocking GlyT1 with N‐[3‐([1,1‐biphenyl]‐4‐yloxy)‐3‐(4‐fluorophenyl)propyl]‐N‐methylglycine in the WT retina generated a significantly larger accumulation of glycine into the bathing environment when compared with the GlyT1^?/+ retinas. The relative occupancy state of the NMDA receptor coagonist sites was tested using whole‐cell recordings from ganglion cells while bath applying D‐serine or D‐serine + NMDA. The interpretation of these studies was simplified by blocking post‐synaptic inhibition with picrotoxinin and strychnine. NMDA receptor coagonist sites were more saturated and less enhanced by D‐serine in the GlyT1^?/+ mice compared with the WT controls. Immunoblots of NMDA receptor subunits (NR1, NR2A and NR2B) in WT and GlyT1^?/+ animals showed that the NR1 subunits were identical. These observations are discussed in view of contemporary issues about NMDA receptor coagonist function in the vertebrate retina and the role of glycine vs. D‐serine as the endogenous coagonist.     

Effects of (R)‐(−)‐5‐methyl‐1‐nicotinoyl‐2‐pyrazoline on glutamate transporter 1 and cysteine/glutamate exchanger as well as ethanol drinking behavior in male,alcohol‐preferring rats

Alcohol consumption is largely associated with alterations in the extracellular glutamate concentrations in several brain reward regions. We recently showed that glutamate transporter 1 (GLT‐1) is downregulated following chronic exposure to ethanol for 5 weeks in alcohol‐preferring (P) rats and that upregulation of the GLT‐1 levels in nucleus accumbens and prefrontal cortex results, in part, in attenuating ethanol consumption. Cystine glutamate antiporter (xCT) is also downregulated after chronic ethanol exposure in P rats, and its upregulation could be valuable in attenuating ethanol drinking. This study examines the effect of a synthetic compound, (R)‐(?)‐5‐methyl‐1‐nicotinoyl‐2‐pyrazoline (MS‐153), on ethanol drinking and expressions of GLT‐1 and xCT in the amygdala and the hippocampus of P rats. P rats were exposed to continuous free‐choice access to water, 15% and 30% ethanol, and food for 5 weeks, after which they received treatments of MS‐153 or vehicle for 5 days. The results show that MS‐153 treatment significantly reduces ethanol consumption. It was revealed that GLT‐1 and xCT expressions were downregulated in both the amygdala and the hippocampus of ethanol–vehicle‐treated rats (ethanol–vehicle group) compared with water‐control animals. MS‐153 treatment upregulated GLT‐1 and xCT expressions in these brain regions. These findings demonstrate an important role for MS‐153 in these glutamate transporters for the attenuation of ethanol‐drinking behavior. © 2015 Wiley Periodicals, Inc.     

Effects of asenapine on prefrontal N‐methyl‐D‐aspartate receptor‐mediated transmission: Involvement of dopamine D1 receptors

Asenapine is a novel psychopharmacologic agent being developed for schizophrenia and bipolar disorder. Like clozapine, asenapine facilitates cortical dopaminergic and N‐methyl‐D ‐aspartate (NMDA) receptor‐mediated transmission in rats. The facilitation of NMDA‐induced currents in cortical pyramidal cells by clozapine is dependent on dopamine and D₁ receptor activation. Moreover, previous results show that clozapine prevents and reverses the blockade of NMDA‐induced currents and firing activity in the pyramidal cells by the noncompetitive NMDA receptor antagonist phencyclidine (PCP). Here, we investigated the effects of asenapine in these regards using intracellular electrophysiological recording in vitro. Asenapine (5 nM) significantly facilitated NMDA‐induced currents (162 ± 15% of control) in pyramidal cells of the medial prefrontal cortex (mPFC). The asenapine‐induced facilitation was blocked by the D₁ receptor antagonist SCH23390 (1 μM). Furthermore, the PCP‐induced blockade of cortical NMDA‐induced currents was effectively reversed by 5 nM asenapine. Our results demonstrate a clozapine‐like facilitation of cortical NMDA‐induced currents by asenapine that involves prefrontal dopamine and activation of D₁ receptors. Asenapine and clozapine also share the ability to reverse functional PCP‐induced hypoactivity of cortical NMDA receptors. The ability of asenapine to increase both cortical dopaminergic and NMDA receptor‐mediated glutamatergic transmission suggests that this drug may have an advantageous effect not only on positive symptoms in patients with schizophrenia, but also on negative and cognitive symptoms. Synapse 64:870–874, 2010. © 2010 Wiley‐Liss, Inc.     

Regulatory volume increase in astrocytes exposed to hypertonic medium requires β1‐adrenergic Na+/K+‐ATPase stimulation and glycogenolysis

The cotransporter of Na⁺, K⁺, 2Cl^–, and water, NKKC1, is activated under two conditions in the brain, exposure to highly elevated extracellular K⁺ concentrations, causing astrocytic swelling, and regulatory volume increase in cells shrunk in response to exposure to hypertonic medium. NKCC1‐mediated transport occurs as secondary active transport driven by Na⁺/K⁺‐ATPase activity, which establishes a favorable ratio for NKCC1 operation between extracellular and intracellular products of the concentrations of Na⁺, K⁺, and Cl^– × Cl^–. In the adult brain, astrocytes are the main target for NKCC1 stimulation, and their Na⁺/K⁺‐ATPase activity is stimulated by elevated K⁺ or the β‐adrenergic agonist isoproterenol. Extracellular K⁺ concentration is normal during regulatory volume increase, so this study investigated whether the volume increase occurred faster in the presence of isoproterenol. Measurement of cell volume via live cell microscopic imaging fluorescence to record fluorescence intensity of calcein showed that this was the case at isoproterenol concentrations of ≥1 µM in well‐differentiated mouse astrocyte cultures incubated in isotonic medium with 100 mM sucrose added. This stimulation was abolished by the β₁‐adrenergic antagonist betaxolol, but not by ICI118551, a β₂‐adrenergic antagonist. A large part of the β₁‐adrenergic signaling pathway in astrocytes is known. Inhibitors of this pathway as well as the glycogenolysis inhibitor 1,4‐dideoxy‐1,4‐imino‐D‐arabinitol hydrochloride and the NKCC1 inhibitors bumetanide and furosemide abolished stimulation by isoproterenol, and it was weakened by the Na⁺/K⁺‐ATPase inhibitor ouabain. These observations are of physiological relevance because extracellular hypertonicity occurs during intense neuronal activity. This might trigger a regulatory volume increase, associated with the post‐excitatory undershoot. © 2014 Wiley Periodicals, Inc.     

Wfs1‐deficient animals have brain‐region‐specific changes of Na+, K+‐ATPase activity and mRNA expression of α1 and β1 subunits

Mutations in the WFS1 gene, which encodes the endoplasmic reticulum (ER) glycoprotein, cause Wolfram syndrome, a disease characterized by juvenile‐onset diabetes mellitus, optic atrophy, deafness, and different psychiatric abnormalities. Loss of neuronal cells and pancreatic β‐cells in Wolfram syndrome patients is probably related to the dysfunction of ER stress regulation, which leads to cell apoptosis. The present study shows that Wfs1‐deficient mice have brain‐region‐specific changes in Na⁺,K⁺‐ATPase activity and in the expression of the α₁ and β₁ subunits. We found a significant (1.6‐fold) increase of Na‐pump activity and β₁ subunit mRNA expression in mice lacking the Wfs1 gene in the temporal lobe compared with their wild‐type littermates. By contrast, exposure of mice to the elevated plus maze (EPM) model of anxiety decreased Na‐pump activity 1.3‐fold in the midbrain and dorsal striatum and 2.0‐fold in the ventral striatum of homozygous animals compared with the nonexposed group. Na‐pump α₁‐subunit mRNA was significantly decreased in the dorsal striatum and midbrain of Wfs1‐deficient homozygous animals compared with wild‐type littermates. In the temporal lobe, an increase in the activity of the Na‐pump is probably related to increased anxiety established in Wfs1‐deficient mice, whereas the blunted dopamine function in the forebrain of Wfs1‐deficient mice may be associated with a decrease of Na‐pump activity in the dorsal and ventral striatum and in the midbrain after exposure to the EPM. © 2014 Wiley Periodicals, Inc.     

Differential contributions of Ca2+‐activated K+ channels and Na+/K+‐ATPases to the generation of the slow afterhyperpolarization in CA1 pyramidal cells

In many types of CNS neurons, repetitive spiking produces a slow afterhyperpolarization (sAHP), providing sustained, intrinsically generated negative feedback to neuronal excitation. Changes in the sAHP have been implicated in learning behaviors, in cognitive decline in aging, and in epileptogenesis. Despite its importance in brain function, the mechanisms generating the sAHP are still controversial. Here we have addressed the roles of M‐type K⁺ current (I_M), Ca²⁺‐gated K⁺ currents (I_Ca(K)'s) and Na⁺/K⁺‐ATPases (NKAs) current to sAHP generation in adult rat CA1 pyramidal cells maintained at near‐physiological temperature (35 °C). No evidence for I_M contribution to the sAHP was found in these neurons. Both I_Ca(K)'s and NKA current contributed to sAHP generation, the latter being the predominant generator of the sAHP, particularly when evoked with short trains of spikes. Of the different NKA isoenzymes, α₁‐NKA played the key role, endowing the sAHP a steep voltage‐dependence. Thus normal and pathological changes in α₁‐NKA expression or function may affect cognitive processes by modulating the inhibitory efficacy of the sAHP.     

1‐[2‐(4‐Benzyloxyphenoxy)Ethyl]Imidazole inhibits monoamine oxidase B and protects against neuronal loss and behavioral impairment in rodent models of Parkinson's disease

Monoamine oxidase B (MAO‐B) is well known as a therapeutic target for Parkinson's disease (PD). MAO‐B inhibitors retain antiparkinsonism abilities to improve motor function and prevent neuronal loss by decreasing dopamine metabolism and oxidative stress in the brain. From the study to find novel antiparkinsonism drugs that can inhibit MAO‐B activity, neuronal loss, and behavioral deficits in the mouse model of PD, we identified that 1‐[2‐(4‐benzyloxyphenoxy)ethyl]imidazole (BPEI) or safinamide strongly and selectively inhibited MAO‐B activities in a dose‐dependent manner (IC₅₀ of BPEI and safinamide for MAO‐B were 0.016 and 0.0021 µM and for MAO‐A were 70.0 and 370 µM, respectively). In ex vivo studies after an administration (30 mg/kg, i.p.) of BPEI or safinamide to normal mice, the MAO‐B activity in the brain was reduced by up to 90.6% or 82.4% at 1.0 hr. BPEI (20 mg/kg, i.p.) or safinamide (20 mg/kg, i.p.) significantly reversed the behavioral impairments, dopamine levels in the striatum, and neuronal loss in the substantia nigra of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐treated mice compared with the MPTP‐alone‐treated group. In the 6‐hydroxydopamine‐induced PD rat model, behavioral improvement by levodopa sparing activity was observed in the BPEI‐ or safinamide‐treated (20 mg/kg, i.p.) rats. Moreover, BPEI revealed additional curative activities for nonmotor symptoms of PD such as pain, anxiety, epilepsy, and depression in rodent disease models. Therefore, BPEI has broad therapeutic potential for treating motor symptoms via strong and selective inhibitory effects on MAO‐B, with additional benefits for comorbid symptoms in PD. © 2015 Wiley Periodicals, Inc.     

The comparison of mouse full metallothionein‐1 versus α and β domains and metallothionein‐1‐to‐3 mutation following traumatic brain injury reveals different biological motifs

Traumatic injury to the brain is one of the leading causes of injury‐related death or disability, but current therapies are limited. Previously it has been shown that the antioxidant proteins metallothioneins (MTs) are potent neuroprotective factors in animal models of brain injury. The exogenous administration of MTs causes effects consistent with the roles proposed from studies in knock‐out mice. We herewith report the results comparing full mouse MT‐1 with the independent α and β domains, alone or together, in a cryoinjury model. The lesion of the cortex caused the mice to perform worse in the horizontal ladder beam and the rota‐rod tests; all the proteins showed a modest effect in the former test, while only full MT‐1 improved the performance of animals in the rota‐rod, and the α domain showed a rather detrimental effect. Gene expression analysis by RNA protection assay demonstrated that all proteins may alter the expression of host‐response genes such as GFAP, Mac1 and ICAM, in some cases being the β domain more effective than the α domain or even the full MT‐1. A MT‐1‐to‐MT‐3 mutation blunted some but not all the effects caused by the normal MT‐1, and in some cases increased its potency. Thus, splitting the two MT‐1 domains do not seem to eliminate all MT functions but certainly modifies them, and different motifs seem to be present in the protein underlying such functions. © 2010 Wiley‐Liss, Inc.     

Novel D3 dopamine receptor‐preferring agonist D‐264: Evidence of neuroprotective property in Parkinson's disease animal models induced by 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine and lactacystin

Parkinson's disease (PD), a progressive neurodegenerative movement disorder, is known to be caused by diverse pathological conditions resulting from dysfunction of the ubiquitin‐proteasome system (UPS), mitochondria, and oxidative stress leading to preferential nigral dopamine (DA) neuron degeneration in the substantia nigra. In the present study, we evaluated the novel D3 receptor‐preferring agonist D‐264 in a mouse model of PD to evaluate its neuroprotective properties against both the nigrostriatal dopaminergic toxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐ and the proteasome inhibitor lactacystin‐induced dopaminergic degeneration. C57BL/6 male mice either were given MPTP by intraperitoneal injection twice per day for 2 successive days at a dose 20 mg/kg or were microinjected with lactacystin bilaterally (1.25 μg/side) into the medial forebrain bundle (MFB). Pretreatment with D‐264 (1 mg/kg and 5 mg/kg, intraperitoneally, once per day), started 7 days before administration of MPTP or lactacystin. We found that D‐264 significantly improved behavioral performance, attenuated both MPTP‐ and lactacystin‐induced DA neuron loss, and blocked proteasomal inhibition and microglial activation in the substantia nigra (SN). Furthermore, D‐264 treatment was shown to increase the levels of brain‐derived neurotrophic factor (BDNF) and glial cell line‐derived factor (GDNF) in MPTP‐ and lactacystin‐treated mice, possibly indicating, at least in part, the mechanism of neuroprotection by D‐264. Furthermore, pretreatment with the D3 receptor antagonist U99194 significantly altered the effect of neuroprotection conferred by D‐264. Collectively, our study demonstrates that D‐264 can prevent neurodegeneration induced by the selective neurotoxin MPTP and the UPS inhibitor lactacystin. The results indicate that D‐264 could potentially serve as a symptomatic and neuroprotective treatment agent for PD. © 2010 Wiley‐Liss, Inc.