Hisanao Izumi

Investigation of mechanisms of the flare and wheal reactions in human skin by band method

Following intradermal injection of histamine, substance P or neurotensin into the normal human forearm skin at the site 0.5 cm proximal to the band, the flare response developed as quickly on the distal side as on the proximal side of the band, whereas the wheal reaction was localized on the proximal side. Pretreatment of skin with capsaicin dramatically inhibited the histamine-induced flare response but had no effect on nicotine-induced axon reflex sweating. The usefulness of the band method is discussed.

Pharmacological properties of SAK3, a novel T-type voltage-gated Ca2+ channel enhancer

&NA; T‐type voltage‐gated Ca2+ channels (T‐VGCCs) function in the pathophysiology of epilepsy, pain and sleep. However, their role in cognitive function remains unclear. We previously reported that the cognitive enhancer ST101, which stimulates T‐VGCCs in rat cortical slices, was a potential Alzheimers disease therapeutic. Here, we introduce a more potent T‐VGCC enhancer, SAK3 (ethyl 8′‐methyl‐2′,4‐dioxo‐2‐(piperidin‐1‐yl)‐2′H‐spiro[cyclopentane‐1,3′‐imidazo [1,2‐a]pyridin]‐2‐ene‐3‐carboxylate), and characterize its pharmacological properties in brain. Based on whole cell patch‐clamp analysis, SAK3 (0.01–10 nM) significantly enhanced Cav3.1 currents in neuro2A cells ectopically expressing Cav3.1. SAK3 (0.1–10 nM nM) also enhanced Cav3.3 but not Cav3.2 currents in the transfected cells. Notably, Cav3.1 and Cav3.3 T‐VGCCs were localized in cholinergic neurve systems in hippocampus and in the medial septum. Indeed, acute oral administration of SAK3 (0.5 mg/kg, p.o.), but not ST101 (0.5 mg/kg, p.o.) significantly enhanced acetylcholine (ACh) release in the hippocampal CA1 region of naïve mice. Moreover, acute SAK3 (0.5 mg/kg, p.o.) administration significantly enhanced hippocampal ACh levels in olfactory‐bulbectomized (OBX) mice, rescuing impaired memory‐related behaviors. Treatment of OBX mice with the T‐VGCC‐specific blocker NNC 55‐0396 (12.5 mg/kg, i.p.) antagonized both enhanced ACh release and memory improvements elicited by SAK3 administration. We also observed that SAK3‐induced ACh releases were significantly blocked in the hippocampus from Cav3.1 knockout (KO) mice. These findings suggest overall that T‐VGCCs play a key role in cognition by enhancing hippocampal ACh release and that the cognitive enhancer SAK3 could be a candidate therapeutic in Alzheimers disease. HighlightsSAK3 enhances Cav3.1 and Cav3.3 T‐type Ca2+ channel currents.SAK3 promotes ACh release in the hippocampus via enhancing T‐type Ca2+ channel.Acute SAK3 administration improves memory deficits in olfactory‐bulbectomized mice.

Reduced CaM Kinase II and CaM Kinase IV Activities Underlie Cognitive Deficits in NCKX2 Heterozygous Mice

Among five members of the K+-dependent Na+/Ca2+ exchanger (NCKX) family (NCKX1–5), only NCKX2 is highly expressed in mouse brain. NCKX2 in plasma membranes mediates cytosolic calcium excretion through electrogenic exchange of 4 Na+ for 1 Ca2+ and 1 K+. Here, we observed significantly decreased levels of NCKX2 protein and mRNA in the CA1 region of APP23 mice, a model of Alzheimer’s disease. We also found that, like APP23 mice, heterozygous NCKX2-mutant mice exhibit mildly impaired hippocampal LTP and memory acquisition, the latter based on novel object recognition and passive avoidance tasks. When we addressed underlying mechanisms, we found that both CaMKII autophosphorylation and CaMKIV phosphorylation significantly decreased in CA1 regions of NCKX2+/− relative to control mice. Likewise, phosphorylation of GluA1 (Ser-831) and CREB (Ser-133), respective downstream targets of CaMKII and CaMKIV, also significantly decreased in the CA1 region. BDNF protein and mRNA levels significantly decreased in CA1 of NCKX2+/− relative to control mice. Finally, CaN activity increased in CA1 of NCKX2+/− mice. Our findings suggest that like APP23 mice, NCKX2+/− mice may exhibit impaired learning and hippocampal LTP due to decreased CaM kinase II and CaM kinase IV activities.

Reduced expression of Na + /Ca 2+ exchangers is associated with cognitive deficits seen in Alzheimer's disease model mice

ABSTRACT Na+/Ca2+ exchangers (NCXs) are expressed primarily in the plasma membrane of most cell types, where they mediate electrogenic exchange of one Ca2+ for three Na+ ions, depending on Ca2+ and Na+ electrochemical gradients across the membrane. Three mammalian NCX isoforms (NCX1, NCX2, and NCX3) are each encoded by a distinct gene. Here, we report that NCX2 and NCX3 protein and mRNA levels are relatively reduced in hippocampal CA1 of APP23 and APP‐KI mice. Likewise, NCX2+/− or NCX3+/− mice exhibited impaired hippocampal LTP and memory‐related behaviors. Moreover, relative to controls, calcium/calmodulin‐dependent protein kinase II (CaMKII) autophosphorylation significantly decreased in NCX2+/− mouse hippocampus but increased in hippocampus of NCX3+/− mice. NCX2 or NCX3 heterozygotes displayed impaired maintenance of hippocampal LTP, a phenotype that in NCX2+/− mice was correlated with elevated calcineurin activity and rescued by treatment with the calcineurin (CaN) inhibitor FK506. Likewise, FK506 treatment significantly restored impaired hippocampal LTP in APP‐KI mice. Moreover, Ca2+ clearance after depolarization following high frequency stimulation was slightly delayed in hippocampal CA1 regions of NCX2+/− mice. Electron microscopy revealed relatively decreased synaptic density in CA1 of NCX2+/− mice, while the number of spines with perforated synapses in CA1 significantly increased in NCX3+/− mice. We conclude that memory impairment seen in NCX2+/− and NCX3+/− mice reflect dysregulated hippocampal CaMKII activity, which alters dendritic spine morphology, findings with implications for memory deficits seen in Alzheimers disease model mice. HighlightsThe mRNA and protein levels of NCX2 and NCX3 in CA1 significantly reduced in APP23 and APP‐KI mouse brain.NCX2+/− or NCX3+/− mice exhibited impaired memory‐related behaviors.NCX2+/− and NCX3+/− mice dysregulated hippocampal CaMKII activity.

Neuroprotective effects of protein tyrosine phosphatase 1B inhibitor on cerebral ischemia/reperfusion in mice

Akt (Protein kinase B, PKB), a serine/threonine kinase, plays a critical role in cell development, growth, and survival. Akt phosphorylation mediates a neuroprotective effect against ischemic injury. Recently, a protein-tyrosine phosphatase-1B (PTP1B) inhibitor (KY-226) was developed to elicit anti-diabetic and anti-obesity effects via enhancement of insulin signaling. Previously, we reported that the nonselective PTP1B inhibitor, sodium orthovanadate, rescued neurons from delayed neuronal death during brain ischemia. In this study, we confirmed the ameliorative effects of KY-226 on ischemia/reperfusion (I/R) injury using a murine model of middle cerebral artery occlusion (MCAO). ICR mice were subjected to MCAO for 2 h followed by reperfusion. Although KY-226 permeability was poor through the blood-brain barrier (BBB) of normal mice, it could penetrate through the BBB of mice after I/R insult. Intraperitoneal KY-226 administration elicited dose-dependent reductions in infarcted brain areas and improved neurological deficits. The neuroprotective effects of KY-266 were obtained when administered within 0.5 h after reperfusion. KY-226 (10 mg/kg) also restored reduced Akt phosphorylation and eNOS phosphorylation (Ser-1177) levels following I/R insult. Moreover, 10 mg/kg of KY-226 improved I/R-induced decreased extracellular signal-regulated kinase (ERK) phosphorylation. Furthermore, KY-226 attenuated the generation of reactive oxygen species (ROS) in mouse cortex. These results suggest that KY-226 may act as a novel therapeutic candidate for ischemic stroke. Activation of Akt and ERK possibly underlie the neuroprotective mechanism of KY-226.

SAK3, AN T-TYPE CA2+ CHANNEL STIMULATOR INHIBITS AMYLOID BETA ACCUMULATION

Background:We recently developed a novel cognitive enhancer of an Alzheimer disease (AD) therapeutic candidate ST101 (Phase II in USA) which stimulates T-type voltage-gated Ca channels (T-VGCC) (Moriguchi et al., J Neurochem 2012;121:44-53). In Phase II trial, ST101 by combination is donepezil is significantly improved cognition in AD patients. Methods:We here introduced another spiroimidazopyridine derivatives (SAK compounds) (PCT/JP2013/051388), which are much stronger than ST101 when assessed by Ca conductance of T-VGCC with neuronal cells transfected Cav3.1 gene. We evaluated its cognitive improvement and its inhibition of amyloid beta (1-42) accumulation in AD model (APP23) mice. Results: SAK3 treatment significantly enhanced mouse hippocampal LTP. Likewise, SAK3 administration in vivo enhanced the hippocampal glutamate release in mouse hippocampus. The SAK3-induced glutamate release was inhibited by treatment with T-VGCC inhibitor. Consistent with these observations, SAK3 improved cognition in novel objective recognition task in APP23 mice. We also confirmed that the cognitive impairment in AD model mice is improved by 2-3 month treatment with SAK3 (05.mg/kg). The prolonged administration of SAK3 also suppressed the amyloid beta (1-42) accumulation in ADmodel mice. Conclusions: Taken together, the novel T-VGCC stimulator SAK3 elicits the enhancement of glutamate release in the hippocampus, thereby likely improving cognitive impairment in AD mice. We are looking for a partner company to conduct the preclinical studies to develop SAK3 as novel AD therapeutics.