Norifumi Shioda

Aberrant Calcium/Calmodulin-Dependent Protein Kinase II (CaMKII) Activity Is Associated with Abnormal Dendritic Spine Morphology in the ATRX Mutant Mouse Brain

In humans, mutations in the gene encoding ATRX, a chromatin remodeling protein of the sucrose-nonfermenting 2 family, cause several mental retardation disorders, including α-thalassemia X-linked mental retardation syndrome. We generated ATRX mutant mice lacking exon 2 (ATRXΔE2 mice), a mutation that mimics exon 2 mutations seen in human patients and associated with milder forms of retardation. ATRXΔE2 mice exhibited abnormal dendritic spine formation in the medial prefrontal cortex (mPFC). Consistent with other mouse models of mental retardation, ATRXΔE2 mice exhibited longer and thinner dendritic spines compared with wild-type mice without changes in spine number. Interestingly, aberrant increased calcium/calmodulin-dependent protein kinase II (CaMKII) activity was observed in the mPFC of ATRXΔE2 mice. Increased CaMKII autophosphorylation and activity were associated with increased phosphorylation of the Rac1-guanine nucleotide exchange factors (GEFs) T-cell lymphoma invasion and metastasis 1 (Tiam1) and kalirin-7, known substrates of CaMKII. We confirmed increased phosphorylation of p21-activated kinases (PAKs) in mPFC extracts. Furthermore, reduced protein expression and activity of protein phosphatase 1 (PP1) was evident in the mPFC of ATRXΔE2 mice. In cultured cortical neurons, PP1 inhibition by okadaic acid increased CaMKII-dependent Tiam1 and kalirin-7 phosphorylation. Together, our data strongly suggest that aberrant CaMKII activation likely mediates abnormal spine formation in the mPFC. Such morphological changes plus elevated Rac1-GEF/PAK signaling seen in ATRXΔE2 mice may contribute to mental retardation syndromes seen in human patients.

The T-type voltage-gated calcium channel as a molecular target of the novel cognitive enhancer ST101: enhancement of long-term potentiation and CaMKII autophosphorylation in rat cortical slices

J. Neurochem. (2012) 121, 44–53.

Methyl pyruvate rescues mitochondrial damage caused by SIGMAR1 mutation related to amyotrophic lateral sclerosis.

BACKGROUND Amyotrophic lateral sclerosis (ALS) is a disease caused by motor neuron degeneration. Recently, a novel SIGMAR1 gene variant (p.E102Q) was discovered in some familial ALS patients. METHODS We address mechanisms underlying neurodegeneration caused by the mutation using Neuro2A cells overexpressing σ1R(E102Q), a protein of a SIGMAR1 gene variant (p.E102Q) and evaluate potential amelioration by ATP production via methyl pyruvate (MP) treatment. RESULTS σ1R(E102Q) overexpression promoted dissociation of the protein from the endoplasmic reticulum (ER) membrane and cytoplasmic aggregation, which in turn impaired mitochondrial ATP production and proteasome activity. Under ER stress conditions, overexpression of wild-type σ1R suppressed ER stress-induced mitochondrial injury, whereas σ1R(E102Q) overexpression aggravated mitochondrial damage and induced autophagic cell death. Moreover, σ1R(E102Q)-overexpressing cells showed aberrant extra-nuclear localization of the TAR DNA-binding protein (TDP-43), a condition exacerbated by ER stress. Treatment of cells with the mitochondrial Ca(2+) transporter inhibitor Ru360 mimicked the effects of σ1R(E102Q) overexpression, indicating that aberrant σ1R-mediated mitochondrial Ca(2+) transport likely underlies TDP-43 extra-nuclear localization, segregation in inclusion bodies, and ubiquitination. Finally, enhanced ATP production promoted by methyl pyruvate (MP) treatment rescued proteasome impairment and TDP-43 extra-nuclear localization caused by σ1R(E102Q) overexpression. CONCLUSIONS Our observations suggest that neurodegeneration seen in some forms of ALS are due in part to aberrant mitochondrial ATP production and proteasome activity as well as TDP-43 mislocalization resulting from the SIGMAR1 mutation. GENERAL SIGNIFICANCE ATP supplementation by MP represents a potential therapeutic strategy to treat ALS caused by SIGMAR1 mutation.

Reduced expression of the ATRX gene, a chromatin-remodeling factor, causes hippocampal dysfunction in mice

Mutations of the ATRX gene, which encodes an ATP‐dependent chromatin‐remodeling factor, were identified in patients with α‐thalassemia X‐linked mental retardation (ATR‐X) syndrome. There is a milder variant of ATR‐X syndrome caused by mutations in the Exon 2 of the gene. To examine the impact of the Exon 2 mutation on neuronal development, we generated ATRX mutant (ATRXΔE2) mice. Truncated ATRX protein was produced from the ATRXΔE2 mutant allele with reduced expression level. The ATRXΔE2 mice survived and reproduced normally. There was no significant difference in Morris water maze test between wild‐type and ATRXΔE2 mice. In a contextual fear conditioning test, however, total freezing time was decreased in ATRXΔE2 mice compared to wild‐type mice, suggesting that ATRXΔE2 mice have impaired contextual fear memory. ATRXΔE2 mice showed significantly reduced long‐term potentiation in the hippocampal CA1 region evoked by high‐frequency stimulation. Moreover, autophosphorylation of calcium‐calmodulin‐dependent kinase II (αCaMKII) and phosphorylation of glutamate receptor, ionotropic, AMPA 1 (GluR1) were decreased in the hippocampi of the ATRXΔE2 mice compared to wild‐type mice. These findings suggest that ATRXΔE2 mice may have fear‐associated learning impairment with the dysfunction of αCaMKII and GluR1. The ATRXΔE2 mice would be useful tools to investigate the role of the chromatin‐remodeling factor in the pathogenesis of abnormal behaviors and learning impairment.

CaMKIIδB Mediates Aberrant NCX1 Expression and the Imbalance of NCX1/SERCA in Transverse Aortic Constriction-Induced Failing Heart

Ca2+/calmodulin-dependent protein kinase II δB (CaMKIIδB) is one of the predominant isoforms of CaMKII in the heart. The precise role of CaMKIIδB in the transcriptional cross-talk of Ca2+-handling proteins during heart failure remains unclear. In this work, we aim to determine the mechanism of CaMKIIδB in modulating the expression of sarcolemmal Na+–Ca2+ exchange (NCX1). We also aim to address the potential effects of calmodulin antagonism on the imbalance of NCX1 and sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) during heart failure. Eight weeks after transverse aortic constriction (TAC)-induced heart failure in mice, we found that the heart weight/tibia length (HW/TL) ratio and the lung weight/body weight (LW/BW) ratio increased by 59% and 133%, respectively. We further found that the left ventricle-shortening fraction decreased by 40% compared with the sham-operated controls. Immunoblotting revealed that the phosphorylation of CaMKIIδB significantly increased 8 weeks after TAC-induced heart failure. NCX1 protein levels were also elevated, whereas SERCA2 protein levels decreased in the same animal model. Moreover, transfection of active CaMKIIδB significantly increased NCX1 protein levels in adult mouse cardiomyocytes via class IIa histone deacetylase (HDAC)/myocyte enhancer factor-2 (MEF2)-dependent signaling. In addition, pharmacological inhibition of calmodulin/CaMKIIδB activity improved cardiac function in TAC mice, which partially normalized the imbalance between NCX1 and SERCA2. These data identify NCX1 as a cellular target for CaMKIIδB. We also suggest that the CaMKIIδB-induced imbalance between NCX1 and SERCA2 is partially responsible for the disturbance of intracellular Ca2+ homeostasis and the pathological process of heart failure.

Distinct cardioprotective effects of 17β-estradiol and dehydroepiandrosterone on pressure overload-induced hypertrophy in ovariectomized female rats.

ObjectiveWe recently reported decreased &sgr;1 receptor expression in the heart after abdominal aortic stenosis in bilateral ovariectomized rats. Here, we use ovariectomized female rats to investigate the distinct cardioprotective effects of 17&bgr;-estradiol (E2) and dehydroepiandrosterone (DHEA) in pressure overload (PO)–induced cardiac dysfunction. MethodsE2 (0.1 mg/kg) and DHEA (30 mg/kg) were administered to rats subcutaneously and orally, respectively, for 14 days starting 2 weeks after aortic banding. ResultsBoth E2 and DHEA treatments significantly inhibited PO-induced increases both in heart weight/body weight ratio and lung weight/body weight ratios. Both E2 and DHEA also ameliorated hypertrophy-induced impairment of left ventricular end-diastolic pressure, left ventricular–developed pressure, left ventricular contraction and relaxation (±dp/dt) rates, heart rate, and mean arterial blood pressure. Notably, DHEA but not E2 administration rescued decreased PO-induced &sgr;1 receptor reduction in the heart. Coadministration with N,N-Dipropyl-2-[4-methoxy-3-(2-phenylethoxy) phenyl]-ethylamine monohydrochloride, an &sgr;1 receptor antagonist, inhibited DHEA-induced amelioration of heart dysfunction without altering E2-induced cardioprotection. Mechanistically, both E2 and DHEA treatments significantly restored PO-induced decreases in protein kinase B (Akt) phosphorylation and Akt-mediated endothelial nitric oxide synthase (eNOS) phosphorylation (Ser1179). N,N-Dipropyl-2-[4-methoxy-3-(2-phenylethoxy) phenyl]-ethylamine monohydrochloride treatment totally abolished DHEA-induced Akt and eNOS phosphorylation without altering E2-induced Akt-eNOS activation. ConclusionsTaken together, these results from an ovariectomized rat model of PO-induced cardiac dysfunction show that DHEA but not E2 elicits a cardioprotective action through &sgr;1 receptor activation. DHEA-induced Akt-eNOS activation through &sgr;1 receptors is probably associated with its cardioprotective activity.

Fluvoxamine rescues mitochondrial Ca2 + transport and ATP production through σ1-receptor in hypertrophic cardiomyocytes

AIMS We previously reported that fluvoxamine, a selective serotonin reuptake inhibitor with high affinity for the σ1-receptor (σ1R), ameliorates cardiac hypertrophy and dysfunction via σ1R stimulation. Although σ1R on non-cardiomyocytes interacts with the IP3 receptor (IP3R) to promote mitochondrial Ca(2+) transport, little is known about its physiological and pathological relevance in cardiomyocytes. MAIN METHODS Here we performed Ca(2+) imaging and measured ATP production to define the role of σ1Rs in regulating sarcoplasmic reticulum (SR)-mitochondrial Ca(2+) transport in neonatal rat ventricular cardiomyocytes treated with angiotensin II to promote hypertrophy. KEY FINDING These cardiomyocytes exhibited imbalances in expression levels of σ1R and IP3R and impairments in both phenylephrine-induced mitochondrial Ca(2+) mobilization from the SR and ATP production. Interestingly, σ1R stimulation with fluvoxamine rescued impaired mitochondrial Ca(2+) mobilization and ATP production, an effect abolished by treatment of cells with the σ1R antagonist, NE-100. Under physiological conditions, fluvoxamine stimulation of σ1Rs suppressed intracellular Ca(2+) mobilization through IP3Rs and ryanodine receptors (RyRs). In vivo, chronic administration of fluvoxamine to TAC mice also rescued impaired ATP production. SIGNIFICANCE These results suggest that σ1R stimulation with fluvoxamine promotes SR-mitochondrial Ca(2+) transport and mitochondrial ATP production, whereas σ1R stimulation suppresses intracellular Ca(2+) overload through IP3Rs and RyRs. These mechanisms likely underlie in part the anti-hypertrophic and cardioprotective action of the σ1R agonists including fluvoxamine.

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.