Hisashi Kitagawa

Mechanism of action of aripiprazole predicts clinical efficacy and a favourable side-effect profile

The antipsychotic efficacy of aripiprazole is not generally associated with extrapyramidal symptoms, cardiovascular effects, sedation or elevations in serum prolactin that characterize typical or atypical antipsychotics. The aim of this study was to clarify the mechanism of action of aripiprazole that underlies its favourable clinical profiles. The preclinical efficacy and side-effect profiles of aripiprazole were evaluated usingseveral pharmaco-behavioural test systems in mice and rats, both in vivo and ex vivo, and compared with those of other conventional and atypical antipsychotics. Each of the antipsychotics induced catalepsy and inhibited apomorphine-induced stereotypy. The catalepsy liability ratios for these drugs were 6.5 for aripiprazole, 4.7 for both olanzapine and risperidone. The ptosis liability ratios for aripiprazole, olanzapine and risperidone were 14, 7.2 and 3.3, respectively. Aripiprazole slightly increased DOPA accumulation in the forebrain of reserpinised mice, reduced 5-HTP accumulation at the highest dose and exhibited a weaker inhibition of 5-methoxy-N,N-dimethyl-tryptamine-induced head twitches. Aripiprazole did not inhibit physostigmine- or norepinephrine-induced lethality in rats. In conclusion, aripiprazole shows a favourable preclinical efficacy and side-effect profile compared to a typical antipsychotics. This profile may result from its high affinity partialagonist activity at D2 and 5-HT1A receptors and its antagonism of 5-HT2A receptors.

Adenovirus-mediated gene transfer of glial cell line-derived neurotrophic factor prevents ischemic brain injury after transient middle cerebral artery occlusion in rats

To examine a possible protective effect of exogenous glial cell line-derived neurotrophic factor (GDNF) gene expression against ischemic brain injury, a replication-defective adenoviral vector containing GDNF gene (Ad-GDNF) was directly injected into the cerebral cortex at 1 day before 90 minutes of transient middle cerebral artery occlusion (MCAO) in rats. 2,3,5-Triphenyltetrazolium chloride staining showed that infarct volume of the Ad-GDNF-injected group at 24 hours after the transient MCAO was significantly smaller than that of vehicle- or Ad-LacZ-treated group. Enzyme-linked immunosorbent assay (ELISA) for immunoreactive GDNF demonstrated that GDNF gene products in the Ad-GDNF-injected group were higher than those of vehicle-treated group at 24 hours after transient MCAO. Immunoreactive GDNF staining was obviously detected in the cortex around the needle track just before or 24 hours after MCAO in the Ad-GDNF group, whereas no or slight GDNF staining was detected in the vehicle group. The numbers of TUNEL, immunoreactive caspase-3, and cytochrome c-positive neurons induced in the ipsilateral cerebral cortex at 24 hours after transient MCAO were markedly reduced by the Ad-GDNF group. These results suggest that the successful exogenous GDNF gene transfer ameliorates ischemic brain injury after transient MCAO in association with the reduction of apoptotic signals.

Immunoreactive Akt, PI3-K and ERK protein kinase expression in ischemic rat brain.

In order to clarify the role of protein kinases in ischemic brain injury, the spatiotemporal expression of immunoreactive serine-threonine kinase Akt, phosphatidylinositol 3-kinase (PI3-K) and extracellular signal-regulated kinase (ERK) were examined at 3, 8, or 24 h after permanent middle cerebral artery occlusion (MCAO) in rats. Weak staining for these protein kinases was found in both cortical and caudate neurons in sham controls. The staining for Akt-1 and PI3-K was increased at 3-8 h in the ischemic penumbral region and declined at 24 h. A slight induction of these kinases was observed in the ischemic core region. Robust expression of ERK was noted at 3-8 h in most neurons in the area of ischemia. At 24 h, ERK continued to be expressed in the ischemic penumbra, but decreased in the ischemic core. These findings suggest that the signaling for Akt and PI3-K are different from the ERK dependent signal transduction during ischemic brain injury.

Protective effect against Parkinson's disease-related insults through the activation of XBP1.

The accumulation of misfolded and unfolded proteins in endoplasmic reticulum (ER) induces ER stress, activating the unfolded protein response (UPR). Recent evidence has suggested the relationship between UPR and dopaminergic neuronal cell death in Parkinsons disease (PD); however, it remains unclear whether it makes sense to modulate UPR, to mitigate the progression of PD. In this study, we investigated a role of the IRE1 alpha-XBP1 pathway in the survival of dopaminergic cells, under stress induced by PD-related insults. The exogenous expression of the active-form XBP1 (XBP1s) protein had protective effects against cell death induced by 1-methyl-4-phenylpyridinium (MPP+) and proteasome inhibitors. Moreover, adenoviral XBP1s expression significantly suppressed the degeneration of dopaminergic neurons in the mouse model of PD, as induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). These results demonstrate that the enhancement of XBP1 could be a novel PD therapeutic strategy.

1H MRS identifies lactate rise in the striatum of MPTP-treated C57BL/6 mice

Mitochondrial dysfunction has been implicated in the death of nigrostriatal dopaminergic neurons in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐treated experimental models of Parkinsons disease (PD). Here we utilized proton magnetic resonance spectroscopy (1H MRS) to identify changes in energy metabolism in the striatum of MPTP‐treated C57BL/6 mice. Remarkable increases in lactate/creatine (Lac/Cr) ratio were observed at 2 h and then quickly returned to about the basal level by 7 h after injection of MPTP. Neurochemical and Western blot analyses revealed that dopamine contents and protein levels of tyrosine hydroxylase and dopamine transporter in the striatum were profoundly decreased at 3 days after MPTP treatment. Pretreatment with deprenyl, a monoamine oxidase B inhibitor, or GBR‐12909, a dopamine uptake inhibitor, almost completely attenuated both the increases in striatal Lac/Cr ratio and the subsequent loss of dopaminergic nerve terminals in MPTP‐treated mice. The present study indicates that 1H MRS is a sensitive measure of biochemical alterations of the brain in a mouse model of PD, and further shows that the increases in striatal Lac/Cr ratio induced by MPTP may be associated with mitochondrial energy crisis, followed by dopaminergic neurotoxicity.

Intracerebral adenosine infusion improves neurological outcome after transient focal ischemia in rats

Abstract In order to elucidate the role of adenosine in brain ischemia, the possible protective effects of adenosine on ischemic brain injury were investigated in a rat model of brain ischemia both in vitro and in vivo. Exogenous adenosine dose-dependently rescued cortical neuronal cells from injury after glucose deprivation in vitro. Adenosine (1 mM) also significantly reduced hypoglycemia/hypoxia-induced glutamate release from the hippocampal slice. In a rat model of transient middle cerebral artery occlusion (MCAO), extracellular adenosine concentration was increased immediately after occlusion, and then returned to the baseline by 30 min after reperfusion. Adenosine infusion through a microdialysis probe into the ipsilateral striatum (1 mM adenosine, 2 µl min-1, total 4.5 h from the occlusion to 3 h after reperfusion) showed a significant improvement in the neurological outcome, and about 25% reduction of infarct volume, although the effect did not reach statistical significance, compared with the vehicle-treated group at 20 h after 90 min of MCAO. These results demonstrated the neuroprotective effect of adenosine against ischemic brain injury both in vitro and in vivo, suggesting the possible therapeutic application of adenosine regulating agents, which inhibit adenosine uptake or metabolism to enhance or maintain extracellular endogenous adenosine levels, for stroke treatment. [Neurol Res 2002; 24: 317-323]

Ameliorative effect of glial cell line-derived neurotrophic factor on brain edema formation after permanent middle cerebral artery occlusion in rats

Glial cell line-derived neurotrophic factor (GDNF) was applied topically on the brain surface immediately after permanent middle cerebral artery (MCA) occlusion in rats. In contrast to the cases treated with vehicle, a formation of brain edema was significantly reduced at one day by the treatment with GDNF. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) staining was markedly reduced in the cases with GDNF treatment at 12 h after MCA occlusion. However, the induction of immunoreactive 70-kd heat shock protein (HSP70) was slightly ameliorated by the GDNF treatment. The present results suggest that the treatment with GDNF has a significant effect on ameliorating brain edema formation after continuous brain ischemia, and the effect is greatly associated with the reduction of apoptotic changes, but slightly with that of stress response of cells.

Induction of DNA fragmentation and HSP72 immunoreactivity by adenovirus‐mediated gene transfer in normal gerbil hippocampus and ventricle

Foreign genes have been successfully transferred and expressed in experimental animal brains using adenoviral vectors. However, it is not fully understood whether adenovirus‐mediated gene transfer causes stressful or cytotoxic injury in brain.

In vitro functional characteristics of dopamine D2 receptor partial agonists in second and third messenger-based assays of cloned human dopamine D2Long receptor signalling

Aripiprazole, (+)terguride, OPC-4392 and (—)3-PPP have been classified as dopamine D2 receptor partial agonists based largely on their activity in second messenger-based assays of dopamine D2 receptor signalling. Nevertheless, signal transduction amplification might result in these compounds behaving as dopamine D2 receptor full agonists at a more downstream level of signalling. We compared the intrinsic activity (Emax, expressed as a percentage of the maximal effect of dopamine) of aripiprazole, (+)terguride, OPC-4392 and (—)3-PPP using second (calcium (Ca2+) mobilization) and third (extracellular signal-regulated kinase 2 (ERK2) phosphoprotein expression) messenger readouts of cloned human dopamine D2long (hD2L) receptor signalling in CHO cells. These compounds were all less potent and displayed lower intrinsic activity in the Ca2+ assay (aripiprazole = 24.3%, (+)terguride = 56.9%, OPC-4392 = 58.6% and (—)3-PPP = 75.1%), and aripiprazole (Emax = 54.5%) displayed a substantially lower intrinsic activity than (+)terguride (Emax = 92.3%), OPC-4392 (Emax = 93.1%) and (—)3-PPP (Emax = 101.1%) in the more downstream-based ERK2 phosphoprotein expression assay. These drug effects on Ca2+ mobilization and ERK2 phosphoprotein expression were mediated through dopamine hD2L receptors, as they all were blocked by (—)raclopride, whereas (—)raclopride and other dopamine D2 receptor antagonists (haloperidol, risperidone, ziprasidone, olanzapine, clozapine and quetiapine) were inactive on their own in both assays. These data are consistent with clinical evidence that only dopamine D2 receptor partial agonists with a sufficiently low enough intrinsic activity will prove effective against the positive symptoms of schizophrenia, and also highlight the importance of using downstream-based assays in the discovery of novel D2 receptor partial agonist therapeutics.

Altered emotional behaviors in the diabetes mellitus OLETF type 1 congenic rat.

GPR10 is a G-protein-coupled receptor expressed in thalamic and hypothalamic brain regions, including the reticular thalamic nucleus (RTN) and periventricular nucleus (Pev), and the endogenous ligand for this receptor, prolactin-releasing peptide (PrRP), has demonstrated regulatory effects on the stress response. We produced a congenic rat by introducing the Dmo1 allele from the OLETF rat which encodes the amino acid sequences of GPR10 with a truncated NH2-terminus, into the Brown-Norway background. Using receptor autoradiography, we determined a lack of specific [125I]PrRP binding in the RTN and Pev of these mutant rats compared to the control rats. Furthermore, intracerebroventricular injection of PrRP did not induce a significant increase of c-fos-like immunoreactivity in the paraventricular nucleus of the mutant rats compared to the control rats. The mutant rats also displayed a less anxious-like phenotype in three behavioral-based models of anxiety-like behavior (open field, elevated plus maze and defensive withdrawal test). These data show the mutant congenic rat, of which GPR10 neither binds nor responds to PrRP, expresses less anxious-like phenotypes. On the basis of these observations, the GPR10 might be a novel target for the developing new drugs against anxiety and/or other stress-related diseases.