Yasushi Yabuki

Nobiletin treatment improves motor and cognitive deficits seen in MPTP-induced Parkinson model mice

Nobiletin, a polymethoxylated flavonoid found in citrus fruit peel, reportedly improves memory impairment in rodent models. Here we report its effect on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced motor and cognitive deficits. Nobiletin administration (50mg/kg i.p.) for 2 consecutive weeks improved motor deficits seen in MPTP-induced Parkinson model mice by 2weeks, an effect that continued until 2weeks after drug withdrawal. Drug treatment promoted similar rescue of MPTP-induced cognitive impairment at equivalent time points. Nonetheless, nobiletin treatment did not block loss of dopaminergic neurons seen in the MPTP-treated mouse midbrain, nor did it rescue decreased tyrosine hydroxylase (TH) protein levels seen in the striatum or hippocampal CA1 region of these mice. Interestingly, nobiletin administration (50mg/kg i.p.) rescued reduced levels of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation and phosphorylation at Thr-34 of dopamine- and cAMP-regulated phosphoprotein-32 (DARPP-32) in striatum and hippocampal CA1 to levels seen in sham-operated mice. Likewise, CaMKII- and cAMP kinase-dependent TH phosphorylation was significantly restored by nobiletin treatment. MPTP-induced reduction of dopamine contents in the striatum and hippocampal CA1 region was improved by nobiletin administration (50mg/kg i.p.). Acute intraperitoneal administration of nobiletin also enhanced dopamine release in striatum and hippocampal CA1, an effect partially inhibited by treatment with nifedipine (a L-type Ca(2+) channel inhibitor) or NNC 55-0396 (a T-type Ca(2+) channel inhibitor) and completely abolished by combined treatment with both. Overall, our study describes a novel nobiletin activity in brain and suggests that nobiletin rescues motor and cognitive dysfunction in MPTP-induced Parkinson model mice, in part by enhancing dopamine release.

Reduced calcium/calmodulin‐dependent protein kinase II activity in the hippocampus is associated with impaired cognitive function in MPTP‐treated mice

J. Neurochem. (2012) 120, 541–551.

Oral administration of glutathione improves memory deficits following transient brain ischemia by reducing brain oxidative stress

Oxidative stress aggravates brain injury following ischemia. The glutathione (GSH) system plays a pivotal role in combating oxidative stress in various cell types. To determine whether oral GSH administration elicits anti-oxidative effects, we assessed its potential neuroprotective effects in transient bilateral common carotid artery occlusion (BCCAO) mice. In naïve mice, acute oral administration of GSH significantly increased GSH levels by 1h in the cortex and hippocampus. Eleven days after BCCAO, untreated mice showed significantly decreased GSH levels and an inverse elevation of glutathione-disulfide (GSSG) levels in both the cortex and hippocampus. Oral administration of GSH (100 and 500 mg/kg p.o.) for 10 consecutive days after ischemia restored reduced GSH levels and inhibited GSSG elevation. Notably, post-administration of GSH (100 and 500 mg/kg p.o.) significantly prevented neuronal cell death in the hippocampal CA1 region in BCCAO mice, an effect closely correlated with decreased levels of oxidative markers such as 4-hydroxy-2-nonenal (4-HNE), 8-hydroxy-2-deoxyguanosine (8-OHdG) and nitrotyrosine in that region. Finally, GSH administration for 10 days improved memory deficits observed in BCCAO mice. Taken together, our findings indicate that the anti-oxidative effect of oral GSH administration ameliorates post-ischemia neuronal cell death and, in turn, may improve memory.

Melatonin reverses the decreases in hippocampal protein serine/threonine kinases observed in an animal model of autism.

Lower global cognitive function scores are a common symptom of autism spectrum disorders (ASDs). This study investigates the effects of melatonin on hippocampal serine/threonine kinase signaling in an experimental ASD model. We found that chronic melatonin (1.0 or 5.0 mg/kg/day, 28 days) treatment significantly rescued valproic acid (VPA, 600 mg/kg)‐induced decreases in CaMKII (Thr286), NMDAR1 (Ser896), and PKA (Thr197) phosphorylation in the hippocampus without affecting total protein levels. Compared with control rats, the immunostaining of pyramidal neurons in the hippocampus revealed a decrease in immunolabeling intensity for phospho‐CaMKII (Thr286) in the hippocampus of VPA‐treated rats, which was ameliorated by chronic melatonin treatment. Consistent with the elevation of CaMKII/PKA/PKC phosphorylation observed in melatonin‐treated rat, long‐term potentiation (LTP) was enhanced after chronic melatonin (5.0 mg/kg) treatment, as reflected by extracellular field potential slopes that increased from 56 to 60 min (133.4 ± 3.9% of the baseline, P < 0.01 versus VPA‐treated rats) following high‐frequency stimulation (HFS) in hippocampal slices. Accordingly, melatonin treatment also significantly improved social behavioral deficits at postnatal day 50 in VPA‐treated rats. Taken together, the increased phosphorylation of CaMKII/PKA/PKC signaling might contribute to the beneficial effects of melatonin on autism symptoms.

FABP3 Protein Promotes α-Synuclein Oligomerization Associated with 1-Methyl-1,2,3,6-tetrahydropiridine-induced Neurotoxicity

Background: αSyn toxicity is triggered by oligomerization of αSyn, and its formation is partly regulated by PUFAs. Results: MPTP-induced neurotoxicity and αSyn oligomerization are attenuated in Fabp3−/− mice. Conclusion: FABP3 is implicated in arachidonic acid-induced αSyn oligomerization and promotes dopaminergic cell death. Significance: FABP3 aggravates MPTP-induced neuronal toxicity and αSyn accumulation. α-Synuclein (αSyn) accumulation in dopaminergic (DA) neurons is partly regulated by long-chain polyunsaturated fatty acids. We found that fatty acid-binding protein 3 (FABP3, H-FABP), a factor critical for arachidonic acid (AA) transport and metabolism in brain, is highly expressed in DA neurons. Fabp3 knock-out (Fabp3−/−) mice were resistant to 1-methyl-1,2,3,6-tetrahydropiridine-induced DA neurodegeneration in the substantia nigra pars compacta and showed improved motor function. Interestingly, FABP3 interacted with αSyn in the substantia nigra pars compacta, and αSyn accumulation following 1-methyl-1,2,3,6-tetrahydropiridine treatment was attenuated in Fabp3−/− compared with wild-type mice. We confirmed that FABP3 overexpression aggravates AA-induced αSyn oligomerization and promotes cell death in PC12 cells, whereas overexpression of a mutant form of FABP3 lacking fatty-acid binding capacity did not. Taken together, αSyn oligomerization in DA neurons is likely aggravated by AA through FABP3 in Parkinson disease pathology.

Aberrant CaMKII activity in the medial prefrontal cortex is associated with cognitive dysfunction in ADHD model rats.

Attention-deficit/hyperactivity disorder (ADHD) is a heterogeneous neurobehavioral disorder accompanied by cognitive and learning deficits, which is prevalent among boys. Juvenile male stroke-prone spontaneously hypertensive rats (SHRSP) exhibit ADHD-like behaviors including cognitive deficits and represent one animal model of ADHD. Here, we define a mechanism underlying cognitive dysfunction observed in SHRSP. Acute methylphenidate (MPH: 1mg/kg, p.o.) administration to SHRSP significantly improved not only inattention in a Y-maze task but also cognitive dysfunction in a novel object recognition test. Interestingly, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity, which is essential for memory and learning acquisition, was excessively elevated in the medial prefrontal cortex (mPFC) but not in the hippocampal CA1 region of SHRSP compared with Wistar-Kyoto (WKY) rats. We also confirmed that elevated CaMKII autophosphorylation in the mPFC causes increased phosphorylation of the CaMKII substrate α-amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid-type glutamate receptor subunit 1 (GluR1) (Ser-831). Ca(2+)-dependent phosphorylation levels of factors such as extracellular signal-regulated kinase (ERK) and protein kinase C (PKC) were unchanged in the SHRSP mPFC. Also, protein levels of the dopamine D2 receptor (D2R) but not the dopamine D1 receptor (D1R) were increased in the SHRSP mPFC. Acute MPH (1mg/kg, p.o.) administration attenuated aberrant CaMKII activity and increased GluR1 phosphorylation observed in SHRSP. Taken together, we propose that cognitive impairment in SHRSP is associated with aberrant CaMKII activity in the mPFC.

Decreased CaMKII and PKC activities in specific brain regions are associated with cognitive impairment in neonatal ventral hippocampus-lesioned rats.

Neonatal ventral hippocampus (NVH)-lesioned rats represent a neurodevelopmental impairment model of schizophrenia. Previous observations indicate that postpubertal NVH-lesioned rats exhibit impairments in prepulse inhibition (PPI), spontaneous locomotion and social interaction behavior. Here, we document the neurochemical basis of those defects. PPI impairment but not cognitive impairment was improved by acute risperidone treatment (0.30mg/kgi.p.). Immunohistochemical analyses using anti-autophosphorylated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) antibody indicated significantly reduced CaMKII autophosphorylation, especially in the medial prefrontal cortex (mPFC), striatum and hippocampal CA1 region, of NVH-lesioned rats relative to control animals. We also confirmed that reduced CaMKII autophoshorylation in the mPFC, striatum and hippocampal CA1 region causes decreased phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid-type glutamate receptor subunit 1 (GluR1) (Ser 831), a CaMKII substrate. Like CaMKII, PKCα (Ser 657) autophosphorylation and NR1 (Ser 896) phosphorylation were decreased both in the mPFC and CA1 region. Interestingly, phosphorylation of DARPP-32 (Thr 34) was decreased in the mPFC but increased in the striatum and CA1 region of NVH-lesioned rats compared to controls. Risperidone treatment restored increased DARPP-32 phosphorylation in the striatum and CA1 regions of NVH-lesioned rats but did not rescue CaMKII and PKCα autophosphorylation. Taken together, we find that impaired cognition observed in NVH-lesioned rats is associated with decreased CaMKII and PKCα activities in memory-related brain regions, changes not rescued by risperidone treatment.

Oral l-Citrulline administration improves memory deficits following transient brain ischemia through cerebrovascular protection

L-citrulline (L-Cit) is known to increase nitric oxide (NO) production via the increase of L-arginine (L-Arg) concentration in the blood and improve endothelial dysfunction in cardiovascular diseases. However, little is known about the effects of L-Cit on cerebrovascular dysfunction. Here we showed that oral L-Cit administration prevents cerebrovascular injury following cerebral ischemia using a 20-min bilateral common carotid artery occlusion (BCCAO) mouse model. After BCCAO ischemia, mice were treated with L-Cit (50, 75, or 100 mg/kg p.o.) for 10 days once a day. L-Cit administration not only prevented neuronal cell death but also prevented capillary loss in the hippocampal region following brain ischemia. The cerebrovascular protective effect of L-Cit was associated with the restoration of endothelial nitric oxide synthase (eNOS) expression in the hippocampus. In addition, we devised a novel protocol to analyze NOx(-) (NO(2-) and NO(3-)) productions following L-Arg infusion using in vivo microdialysis and revealed that decreased L-Arg-induced NOx(-) levels were improved in the hippocampus of BCCAO mice following repeated L-Cit administration. Finally, memory deficits following brain ischemia were improved by oral administration of L-Cit. In summary, L-Cit is a potential therapeutic agent that protects cerebrovascular injury and in turn prevents neuronal cell death. Thereby, oral L-Cit administration improves cognitive deficits following brain ischemia.

Blockade of the KATP channel Kir6.2 by memantine represents a novel mechanism relevant to Alzheimer’s disease therapy

Here, we report a novel target of the drug memantine, ATP-sensitive K+ (KATP) channels, potentially relevant to memory improvement. We confirmed that memantine antagonizes memory impairment in Alzheimer’s model APP23 mice. Memantine increased CaMKII activity in the APP23 mouse hippocampus, and memantine-induced enhancement of hippocampal long-term potentiation (LTP) and CaMKII activity was totally abolished by treatment with pinacidil, a specific opener of KATP channels. Memantine also inhibited Kir6.1 and Kir6.2 KATP channels and elevated intracellular Ca2+ concentrations in neuro2A cells overexpressing Kir6.1 or Kir6.2. Kir6.2 was preferentially expressed at postsynaptic regions of hippocampal neurons, whereas Kir6.1 was predominant in dendrites and cell bodies of pyramidal neurons. Finally, we confirmed that Kir6.2 mutant mice exhibit severe memory deficits and impaired hippocampal LTP, impairments that cannot be rescued by memantine administration. Altogether, our studies show that memantine modulates Kir6.2 activity, and that the Kir6.2 channel is a novel target for therapeutics to improve memory impairment in Alzheimer disease patients.

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.