Kou Takahashi

Small-molecule activator of glutamate transporter EAAT2 translation provides neuroprotection

Glial glutamate transporter EAAT2 plays a major role in glutamate clearance in synaptic clefts. Several lines of evidence indicate that strategies designed to increase EAAT2 expression have potential for preventing excitotoxicity, which contributes to neuronal injury and death in neurodegenerative diseases. We previously discovered several classes of compounds that can increase EAAT2 expression through translational activation. Here, we present efficacy studies of the compound LDN/OSU-0212320, which is a pyridazine derivative from one of our lead series. In a murine model, LDN/OSU-0212320 had good potency, adequate pharmacokinetic properties, no observed toxicity at the doses examined, and low side effect/toxicity potential. Additionally, LDN/OSU-0212320 protected cultured neurons from glutamate-mediated excitotoxic injury and death via EAAT2 activation. Importantly, LDN/OSU-0212320 markedly delayed motor function decline and extended lifespan in an animal model of amyotrophic lateral sclerosis (ALS). We also found that LDN/OSU-0212320 substantially reduced mortality, neuronal death, and spontaneous recurrent seizures in a pilocarpine-induced temporal lobe epilepsy model. Moreover, our study demonstrated that LDN/OSU-0212320 treatment results in activation of PKC and subsequent Y-box-binding protein 1 (YB-1) activation, which regulates activation of EAAT2 translation. Our data indicate that the use of small molecules to enhance EAAT2 translation may be a therapeutic strategy for the treatment of neurodegenerative diseases.

Glutamate transporter EAAT2: regulation, function, and potential as a therapeutic target for neurological and psychiatric disease

Glutamate is the predominant excitatory neurotransmitter in the central nervous system. Excitatory amino acid transporter 2 (EAAT2) is primarily responsible for clearance of extracellular glutamate to prevent neuronal excitotoxicity and hyperexcitability. EAAT2 plays a critical role in regulation of synaptic activity and plasticity. In addition, EAAT2 has been implicated in the pathogenesis of many central nervous system disorders. In this review, we summarize current understanding of EAAT2, including structure, pharmacology, physiology, and functions, as well as disease relevancy, such as in stroke, Parkinson’s disease, epilepsy, amyotrophic lateral sclerosis, Alzheimer’s disease, major depressive disorder, and addiction. A large number of studies have demonstrated that up-regulation of EAAT2 protein provides significant beneficial effects in many disease models suggesting EAAT2 activation is a promising therapeutic approach. Several EAAT2 activators have been identified. Further understanding of EAAT2 regulatory mechanisms could improve development of drug-like compounds that spatiotemporally regulate EAAT2.

Increased glial glutamate transporter EAAT2 expression reduces epileptogenic processes following pilocarpine-induced status epilepticus.

Several lines of evidence indicate that glutamate plays a crucial role in the initiation of seizures and their propagation; abnormal glutamate release causes synchronous firing of large populations of neurons, leading to seizures. In the present study, we investigated whether enhanced glutamate uptake by increased glial glutamate transporter EAAT2, the major glutamate transporter, could prevent seizure activity and reduce epileptogenic processes. EAAT2 transgenic mice, which have a 1.5-2 fold increase in EAAT2 protein levels as compared to their non-transgenic counterparts, were tested in a pilocarpine-induced status epilepticus (SE) model. Several striking phenomena were observed in EAAT2 transgenic mice compared with their non-transgenic littermates. First, the post-SE mortality rate and chronic seizure frequency were significantly decreased. Second, neuronal degeneration in hippocampal subfields after SE were significantly reduced. Third, the SE-induced neurogenesis and mossy fiber sprouting were significantly decreased. The severity of cell loss in epileptic mice was positively correlated with that of mossy fiber sprouting and chronic seizure frequency. Our results suggest that increased EAAT2 expression can protect mice against SE-induced death, neuropathological changes, and chronic seizure development. This study suggests that enhancing EAAT2 protein expression is a potential therapeutic approach.

Restored glial glutamate transporter EAAT2 function as a potential therapeutic approach for Alzheimer’s disease

Takahashi et al. demonstrate that restoring glial glutamate transporter EAAT2 function improves cognitive functions and synaptic integrity while reducing amyloid plaques in a sustained fashion after treatment cessation.

Induction of cysteine string protein after chronic antidepressant treatment in rat frontal cortex

We have previously identified 204 partial cDNA fragments (ADRG1-204) as antidepressant related genes/expressed sequence tags. Then, we developed our original cDNA microarrays, on which the 194 clones out of ADRG1-204 were spotted. With this ADRG microarray, we found that the expression of a spot, ADRG55, which representing cysteine string protein (CSP), was significantly increased in rat brain after chronic treatment with a selective serotonin reuptake inhibitor, sertraline. In the present study, reverse transcription-polymerase chain reaction analysis confirmed the induction of CSP at mRNA levels in rat frontal cortex after chronic treatment with two different classes of antidepressants, imipramine or sertraline. Western blot analysis also revealed that CSP-immunoreactivity was increased after antidepressant treatment. In conclusion, our data suggest that CSP is one of the common functional molecules induced after chronic antidepressant treatment.

Riluzole rapidly attenuates hyperemotional responses in olfactory bulbectomized rats, an animal model of depression.

Growing evidence indicates that the glutamatergic neurotransmitter system is central to the neurobiology and treatment of depression. Riluzole, a drug currently used to slow the progression of amyotrophic lateral sclerosis (ALS), directly affects the glutamatergic system. In this study, we investigated the effects of riluzole in olfactory bulbectomy (OBX) rats, an animal model of depression. The olfactory bulbs in rats were removed by suction. The emotionality of rats was measured by scoring their responses to given stimuli, i.e., attack, startle, struggle, and fight responses. The OBX rats chronically treated with vehicle for 7 days at 14 days following surgery showed significant increases in emotionality responses. Single (1st day administration) and subchronic (7th day administration) riluzole treatment (1-10 mg/kg, po) significantly and dose-dependently reduced hyperemotional responses in OBX rats. Both single and subchronic riluzole treatment (10 mg/kg, po) had no significant effects on the emotional responses in sham operated rats. In addition, we demonstrated that single riluzole treatment (10 mg/kg, po) significantly decreased extracellular glutamate levels in medial prefrontal cortex of OBX rats by in vivo microdialysis. We provide the first experimental evidence that riluzole rapidly attenuated hyperemotional responses in OBX rats, an animal model of depression.

Antidepressant-like effects of the delta-opioid receptor agonist SNC80 ([(+)-4-[(alphaR)-alpha-[(2S,5R)-2,5-dimethyl-4-(2-propenyl)-1-piperazinyl]-(3-methoxyphenyl)methyl]-N,N-diethylbenzamide) in an olfactory bulbectomized rat model.

The responses of olfactory bulbectomized (OBX) rats to antidepressant treatment are similar to those of depressed patients since chronic administration of an antidepressant reverses OBX-induced behavioral and physiological changes. Previously, using several animal models, it was demonstrated that single treatment with delta-opioid receptor agonists produced an antidepressant-like effect. This study examined the antidepressant effects resulting from subchronic exposure for 8 days to the delta-opioid receptor agonist SNC80 in an OBX rat model of depression. The olfactory bulbs were removed by suction. The emotionality of rats was measured by scoring their responses to given stimuli, i.e., attack, startle, struggle, and fight responses. The OBX rats chronically treated with vehicle for 7 days at 14 days following surgery showed a significant increase in emotionality score and a decrease in the time spent and entries in the open arm of a plus-maze. In the case of OBX rats, these changes were dose- and time-dependently reversed by chronic SNC80 treatment (1-10 mg/kg, s.c.) for 7 days, as same as desipramine (10 mg/kg, i.p.). Moreover, the concentration of 5-HT and its metabolite 5-HIAA in the frontal cortex, hippocampus, and amygdala were decreased in OBX rats, and these changes were also normalized by SNC80 treatment, rather than desipramine treatment. In addition, SNC80 also significantly reversed the loss of TH-positive cells produced by OBX in the dorsal raphe. In conclusion, we demonstrated that subchronic SNC80 treatment could completely reverse OBX-induced behavioral abnormalities and defects in serotonergic function.

Dexamethasone indirectly induces Ndrg2 expression in rat astrocytes

N‐myc downstream‐regulated gene 2 (Ndrg2) has been associated with cell proliferation, differentiation, and apoptosis. Ndrg2 expression in the brain is induced by glucocorticoid treatment or chronic stress in vivo. It has been postulated that glucocorticoid‐induced Ndrg2 expression in astrocytes is regulated by the glucocorticoid response element half‐site (GRE1/2) upstream of the Ndrg2 transcription site. Here we examined the mechanisms of dexamethasone‐induced Ndrg2 expression in rat astrocytes. Ndrg2 mRNA expression in primary astrocytes was significantly increased after 24 hr of exposure to dexamethasone in a concentration‐dependent manner. Dexamethasone‐induced Ndrg2 mRNA and protein expression was blocked by pretreatment with RU486, a glucocorticoid receptor antagonist. Moreover, dexamethasone‐induced Ndrg2 mRNA expression was reduced by pretreatment with the protein synthesis inhibitor cycloheximide. The Ndrg2 reporter assay showed that deletion of a putative GRE1/2, located upstream of Ndrg2, did not affect induction by dexamethasone. A region between –755 and –701 bp from the transcription start site was shown to regulate induction by dexamethasone using promoter constructs progressively deleted from the 5′ to 3′ ends. This region contained the predicted transcription factor binding sites for early B‐cell factor 1 (Ebf1), nuclear factor‐κB (NFκB), and paired box gene 5 (Pax5). Mutation at the NFκB‐ or Pax5‐binding site, but not the Ebf1 binding site, abolished dexamethasone‐induced promoter activation. These results indicate that Ndrg2 expression was indirectly induced by dexamethasone at the DNA level, potentially by the binding of NFκB or Pax5 to the transcription factor binding sites, and GRE1/2 was not involved in this induction.

Ubiquitin ligase Kf-1 is involved in the endoplasmic reticulum-associated degradation pathway

Kf-1 was first identified as a gene showing enhanced expression in the cerebral cortex of a sporadic Alzheimers disease patient. To date, however, the functional properties of Kf-1 protein remain unknown. In this study, immunohistochemical analysis showed that Kf-1 immunoreactivity was detected in rat hippocampus and cerebral cortex neurons. Interestingly, it was colocalized with endoplasmic reticulum (ER) marker. To investigate the specific function of Kf-1 protein, we generated Myc tagged wild type Kf-1 (Myc-Kf-1WT) and RING finger domain deletion mutant of Kf-1 (Myc-Kf-1DeltaR), and then transfected in HEK293 cells. Myc-Kf-1WT displayed a reticular pattern typical of ER localization, with large perinuclear aggregates and colocalized with ER marker, calnexin. Myc-Kf-1WT facilitated ubiquitination of endogenous proteins, whereas Myc-Kf-1DeltaR did not show ubiquitin ligase activity. In addition, we found that Kf-1 interacted with components of the ER-associated degradation (ERAD) pathway, including Derlin-1 and VCP. Taken together, these properties suggest that Kf-1 is an ER ubiquitin ligase involved in the ERAD pathway.

Prg1 is regulated by the basic helix-loop-helix transcription factor Math2

Math2 (NEX‐1/NeuroD6) is a member of the basic helix‐loop‐helix transcription factor family and is involved in neuronal differentiation and maturation. In this study, we identified the genes targeted by Math2 using DNA microarrays and cultured rat cortical cells transfected with Math2. Of the genes regulated by Math2, we focused on plasticity‐related gene 1 (Prg1). Prg1 expression induced by Math2 was confirmed in cultured rat cortical cells and PC12 cells analyzed by real‐time quantitative PCR. In the promoter region of rat Prg1, we identified four E‐boxes [designated −E1 to −E4 (CANNTG)] recognized by the basic helix‐loop‐helix transcription factor. Using chromatin immunoprecipitation assays, we found that Math2 directly bound to at least one of these E‐boxes. The Prg1 reporter assay showed that −E1 was critical for the regulation of Math2‐mediated Prg1 expression. Investigation of the functional roles of Math2 and Prg1 in PC12 cells revealed that 72 h after transfection with either Math2 or Prg1, neurite length and number were significantly induced. Co‐transfection with Prg1‐siRNA completely inhibited Math2‐mediated morphological changes. Our results suggest that Math2 directly regulates Prg1 expression and that the Math2–Prg1 cascade plays an important role in neurite outgrowth in PC12 cells.