Inhibition of miR-96-5p in the mouse brain increases glutathione levels by altering NOVA1 expression.

Glutathione (GSH) is an important antioxidant that plays a critical role in neuroprotection. GSH depletion in neurons induces oxidative stress and thereby promotes neuronal damage, which in turn is regarded as a hallmark of the early stage of neurodegenerative diseases. The neuronal GSH level is mainly regulated by cysteine transporter EAAC1 and its inhibitor, GTRAP3-18. In this study, we found that the GTRAP3-18 level was increased by up-regulation of the microRNA miR-96-5p, which was found to decrease EAAC1 levels in our previous study. Since the 3'-UTR region of GTRAP3-18 lacks the consensus sequence for miR-96-5p, an unidentified protein should be responsible for the intermediate regulation of GTRAP3-18 expression by miR-96-5p. Here, we discovered that RNA-binding protein NOVA1 functions as an intermediate protein for GTRAP3-18 expression via miR-96-5p. Moreover, we show that intra-arterial injection of a miR-96-5p-inhibiting nucleic acid to living mice by a drug delivery system using microbubbles and ultrasound decreased the level of GTRAP3-18 via NOVA1 and increased the levels of EAAC1 and GSH in the dentate gyrus of the hippocampus. These findings suggest that the delivery of a miR-96-5p inhibitor to the brain would efficiently increase the neuroprotective activity by increasing GSH levels via EAAC1, GTRAP3-18 and NOVA1.

Rab1a rescues the toxicity of PRAF3.

The PRA1-superfamily member PRAF3 plays pivotal roles in membrane traffic as a GDI displacement factor via physical interaction with a variety of Rab proteins, as well as in the modulation of antioxidant glutathione through its interaction with EAAC1 (SLC1A1). Overproduction of PRAF3 is known to be toxic to the host cells, although the factors capable of cancelling the toxicity remained unknown. We here show that Rab1a can rescue the cytotoxicity caused by PRAF3 possibly by "positively" regulating ER-Golgi trafficking, cancelling the "negative" modulation by PRAF3. Our results illuminate the close physiological relationship between PRAF3 and Rab proteins.

Rhythmic oscillations of the microRNA miR-96-5p play a neuroprotective role by indirectly regulating glutathione levels.

Glutathione (GSH) is a key antioxidant that plays an important neuroprotective role in the brain. Decreased GSH levels are associated with neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. Here we show that a diurnal fluctuation of GSH levels is correlated with neuroprotective activity against oxidative stress in dopaminergic cells. In addition, we found that the cysteine transporter excitatory amino acid carrier 1 (EAAC1), which is involved in neuronal GSH synthesis, is negatively regulated by the microRNA miR-96-5p, which exhibits a diurnal rhythm. Blocking miR-96-5p by intracerebroventricular administration of an inhibitor increased the level of EAAC1 as well as that of GSH and had a neuroprotective effect against oxidative stress in the mouse substantia nigra. Our results suggest that the diurnal rhythm of miR-96-5p may play a role in neuroprotection by regulating neuronal GSH levels via EAAC1.

Alpha(1A)-adrenergic control of piloerection and palpebral fissure width in rats.

We determined the receptor subtypes of α1-adrenoceptor, which is involved in autonomic functions induced by methamphetamine (METH) in rats. An intraperitoneal injection of METH provoked the autonomic responses piloerection, eyelid retraction, and ejaculation. Pretreatment with prazosin, a nonselective α1-adrenoceptor antagonist, completely abolished the above METH-induced responses. Prazosin also provoked eyelid ptosis in saline controls. The effects of prazosin were mimicked only by a selective α1A-adrenoceptor antagonist, silodosin, not by selective α1B or α1D antagonists. These results suggest that α1A-adrenoceptor participates in the regulation of piloerection, palpebral fissure width, and ejaculation in rats.

Involvement of the α(1D)-adrenergic receptor in methamphetamine-induced hyperthermia and neurotoxicity in rats.

Methamphetamine (METH) is a psychostimulant that damages nigrostriatal dopaminergic terminals, primarily by enhancing dopamine and glutamate release. α1-adrenergic receptor (AR) subtype involved in METH-induced neurotoxicity in rats was investigated using selective α1-AR antagonists. METH neurotoxicity was evaluated by (1) measuring body temperature; (2) determining tyrosine hydroxylase (TH) immunoreactivity levels; (3) examining levels of dopamine and its metabolites; and (4) assessing glial fibrillary acidic protein (GFAP) and microglial immunoreactivity in the striatum. METH caused a decrease in dopamine and TH levels and induced hyperthermia which is an exacerbating factor of METH neurotoxicity. Concurrently, METH increased GFAP expression and the number of activated microglia. Pretreatment with prazosin, a nonselective α1-AR antagonist, completely abolished METH-induced decrease in both dopamine and TH and caused a partial reduction in hyperthermia. Prazosin also prevented METH-induced increase in both GFAP expression and the number of activated microglia. In vivo microdialysis analysis revealed that prazosin, however, does not alter the METH-induced dopamine release in the striatum. The neuroprotective effects of prazosin could be mimicked by a selective α(1D) antagonist, BMY 7378, but not by selective α(1A) or α(1B) antagonists. These results suggest that the α(1D)-AR is involved in METH-induced hyperthermia and neurotoxicity in rats.

Anticonvulsant action of indazole.

Here we report that indazole is characterized as a potential anticonvulsant, inhibiting pentylenetetrazole-, electroshock- and strychnine-induced convulsions in mice (ED50's: 39.9, 43.2 and 82.4 mg/kg, respectively) but not bicuculline- and picrotoxin-induced convulsions. The median toxic dose (TD(50)) of indazole was 52.3 mg/kg by the minimal motor impairment test. Therefore, nontoxic doses produced anticonvulsant activity against pentylenetetrazole- and electroshock-induced seizures. Indazole (50 mg/kg) had no effect on spontaneous activity but induced hypothermia. It also inhibited the metabolism of dopamine and 5-hydroxytryptamine in the brain in vivo and the activities of monoamine oxidase A and B in vitro, with IC(50) values of 20.6 µM and 16.3 µM, respectively. However, these inhibitory effects do not account for the anticonvulsant activity because treatment with typical monoamine oxidase inhibitors such as pargyline or tranylcypromine did not completely reproduce the anticonvulsant activity of indazole. In the animal seizure models tested, the anticonvulsant profile of indazole most resembled that of gabapentin and somewhat resembled those of the AMPA/kainate antagonist NBQX and the sodium channel inhibitor phenytoin, but differed from that of benzodiazepine. The isobolographic analyses showed that the interactive mode of indazole with gabapentin, NBQX or phenytoin is additive. These results suggest that indazole has anticonvulsant activity and multiple mechanisms.

Increased neuronal glutathione and neuroprotection in GTRAP3-18-deficient mice.

Glutathione (GSH) is an important neuroprotective molecule in the brain. The strategy to increase neuronal GSH level is a promising approach to the treatment of neurodegenerative diseases. However, the regulatory mechanism by which neuron-specific GSH synthesis is facilitated remains elusive. Glutamate transporter-associated protein 3-18 (GTRAP3-18) is an endoplasmic reticulum protein interacting with excitatory amino acid carrier 1 (EAAC1), which is a neuronal glutamate/cysteine transporter. To investigate the potential regulatory mechanism to increase neuronal GSH level in vivo, we generated GTRAP3-18-deficient (GTRAP3-18(-/-)) mice using a gene-targeting approach. Disruption of the GTRAP3-18 gene resulted in increased EAAC1 expression in the plasma membrane, increased neuronal GSH content and neuroprotection against oxidative stress. In addition, GTRAP3-18(-/-) mice performed better in motor/spatial learning and memory tests than wild-type mice. Therefore, the suppression of GTRAP3-18 increases neuronal resistance to oxidative stress by increasing GSH content and also facilitates cognitive function. The present results may provide a molecular basis for the development of treatments for neurodegenerative diseases.

Chronic treatment with a selective ligand for the sigma-1 receptor chaperone, SA4503, up-regulates BDNF protein levels in the rat hippocampus.

Numerous studies have shown the sigma-1 receptor chaperone (previously designated as the sigma-1 receptor) to play an important role in various neuronal functions, and selective ligands for sigma-1 receptor chaperone have been found to have therapeutic potential in certain psychiatric disorders and some forms of neuronal damage. Brain-derived neurotrophic factor (BDNF) is postulated to be related to the pharmacological action of sigma ligands, though the functional interaction between sigma-1 receptor chaperone ligands and BDNF remains to be clarified. This study was undertaken to investigate whether or not administration of SA4503, a selective ligand for sigma-1 receptor chaperone, affects BDNF levels in several regions of the rat brain. Rats were injected with SA4503 (0.3, 1 and 3 mg/(kg day), i.p.) once or repeatedly for 2 or 4 weeks. BDNF protein levels were estimated by Western blot analysis in the striatum, midbrain, frontal cortex, hippocampus and thalamus. A single injection of SA4503 did not change BDNF protein levels, while chronic injection for 2 or 4 weeks tended to increase BDNF levels in the hippocampus. In particular, 1 mg/kg of SA4503 daily for 2 weeks led to a statistically significant, i.e. twofold, increase in the BDNF protein level in the hippocampus. On the other hand, the TrkB receptor, a primary receptor for BDNF, exists in truncated and full-length isoforms, hippocampal levels of which were unaffected by SA4503 treatments. Our findings indicate that chronic administration of SA4503 may regulate region-specific BDNF functions in the rat brain.

Activities of 7-nitroindazole and 1-(2-(trifluoromethylphenyl)-imidazole independent of neuronal nitric-oxide synthase inhibition.

7-Nitroindazole (NI) is a widely used inhibitor of neuronal nitricoxide synthase (nNOS) used to study the role of the neuronal NO pathway in the nervous system. 7-NI prevents convulsions, including 2-amino-4-methylphosphinobutyric acid (glufosinate)-induced convulsions, in experimental models. Herein, we examined nNOS involvement in glufosinate-induced convulsions and the specificity of 7-NI for nNOS. Another nNOS inhibitor, 1-[2-(trifluoromethyl)phenyl]imidazole (TRIM), inhibited NOS activity in vivo, and it prevented glufosinate-induced convulsions. In contrast, an endothelial NOS inhibitor, N(5)-(1-iminoethyl)-l-ornithine, inhibited NOS activity in vivo, but it did not prevent the convulsions. These results suggest the involvement of nNOS in glufosinate-induced convulsions. However, a nonspecific NOS inhibitor, N(omega)-nitro-l-arginine methyl ester, inhibited NOS activity in vivo, but it failed to prevent glufosinate-induced convulsions. 6-NI and indazole, which did not inhibit NOS activity in vivo, suppressed glufosinate-induced convulsions. Moreover, glufosinate elicited convulsions in nNOS-deficient mice. These results suggest the anticonvulsant effects of 7-NI and TRIM on glufosinate-induced convulsions do not involve nNOS inhibition, instead possibly being related to an undefined property of nitrogen-containing chemical structures.

Repeated immobilization stress increases uncoupling protein 1 expression and activity in Wistar rats.

Repeat immobilization-stressed rats are leaner and have improved cold tolerance due to enhancement of brown adipose tissue (BAT) thermogenesis. This process likely involves stress-induced sympathetic nervous system activation and adrenocortical hormone release, which dynamically enhances and suppresses uncoupling protein 1 (UCP1) function, respectively. To investigate whether repeated immobilization influences UCP1 thermogenic properties, we assessed UCP1 mRNA, protein expression, and activity (GDP binding) in BAT from immobilization-naive or repeatedly immobilized rats (3 h daily for 4 weeks) and sham operated or adrenalectomized (ADX) rats. UCP1 properties were assessed before (basal) and after exposure to 3 h of acute immobilization. Basal levels of GDP binding and UCP1 expression was significantly increased (140 and 140%) in the repeated immobilized group. Acute immobilization increased GDP binding in both naive (180%) and repeated immobilized groups (220%) without changing UCP1 expression. In ADX rats, basal GDP binding and UCP1 gene expression significantly increased (140 and 110%), and acute immobilization induced further increase. These data demonstrate that repeated immobilization resulted in enhanced UCP1 function, suggesting that enhanced BAT thermogenesis contributes to lower body weight gain through excess energy loss and an improved ability to maintain body temperature during cold exposure.

Enhanced gene expression of endothelial nitric oxide synthase in brown adipose tissue during cold exposure.

It has been shown that norepinephrine (NE) can mediate vasodilatation by stimulating the production of nitric oxide (NO) in brown adipose tissue (BAT), resulting in an increase in BAT blood flow. We speculated that constitutive NO synthase (NOS) is involved in this NO production. However, it is not known whether constitutive NOS is expressed in BAT. To answer this question, we assessed the expression of two types of constitutive NOS, endothelial (eNOS) and neuronal NOS (nNOS), in BAT of rats. eNOS was abundantly expressed in both BAT and isolated brown adipocytes, whereas nNOS was not. Cold exposure, which is known to stimulate NE release from sympathetic nerve terminals in BAT, led to a significant increase in eNOS mRNA in this tissue. In contrast, very low levels of inducible NOS (iNOS) mRNA were expressed, and cold stimulation failed to increase iNOS mRNA levels in BAT. These results suggest that eNOS is the primary isoform that is responsible for NO production in BAT and that its expression may be under sympathetic control.