Kenichi Mishima

Osteopetrosis and thalamic hypomyelinosis with synaptic degeneration in DAP12-deficient mice

Deletions in the DAP12 gene in humans result in Nasu-Hakola disease, characterized by a combination of bone fractures and psychotic symptoms similar to schizophrenia, rapidly progressing to presenile dementia. However, it is not known why these disorders develop upon deficiency in DAP12, an immunoreceptor signal activator protein initially identified in the immune system. Here we show that DAP12-deficient (DAP12(-/-)) mice develop an increased bone mass (osteopetrosis) and a reduction of myelin (hypomyelinosis) accentuated in the thalamus. In vitro osteoclast induction from DAP12(-/-) bone marrow cells yielded immature cells with attenuated bone resorption activity. Moreover, immature oligodendrocytes were arrested in the vicinity of the thalamus, suggesting that the primary defects in DAP12(-/-) mice are the developmental arrest of osteoclasts and oligodendrocytes. In addition, the mutant mice also showed synaptic degeneration, impaired prepulse inhibition, which is commonly observed in several neuropsychiatric diseases in humans including schizophrenia, and aberrant electrophysiological profiles in the thalami. These results provide a molecular basis for a unique combination of skeletal and psychotic characteristics of Nasu-Hakola disease as well as for schizophrenia and presenile dementia.

Tau filament formation and associative memory deficit in aged mice expressing mutant (R406W) human tau

The R406W tau mutation found in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) causes a hereditary tauopathy clinically resembling Alzheimers disease. Expression of modest levels of the longest human tau isoform with this mutation under the control of the α-calcium–calmodulin-dependent kinase-II promoter in transgenic (Tg) mice resulted in the development of congophilic hyperphosphorylated tau inclusions in forebrain neurons. These inclusions appeared as early as 18 months of age. As with human cases, tau inclusions were composed of both mutant and endogenous wild-type tau, and were associated with microtubule disruption and flame-shaped transformations of the affected neurons. Straight tau filaments were recovered from Sarkosyl-insoluble fractions from only the aged Tg brains. Behaviorally, aged Tg mice had associative memory impairment without obvious sensorimotor deficits. Therefore, these mice that exhibit a phenotype mimicking R406W FTDP-17 provide an animal model for investigating the adverse properties associated with this mutation, which might potentially recapitulate some etiological events in Alzheimers disease.

Cannabidiol Prevents Cerebral Infarction Via a Serotonergic 5-Hydroxytryptamine1A Receptor–Dependent Mechanism

Background and Purpose— Cannabidiol has been reported to be a neuroprotectant, but the neuroprotective mechanism of cannabidiol remains unclear. We studied the neuroprotective mechanism of cannabidiol in 4-hour middle cerebral artery (MCA) occlusion mice. Methods— Male MCA occluded mice were treated with cannabidiol, abnormal cannabidiol, anandamide, methanandamide, cannabidiol plus capsazepine, and cannabidiol plus WAY100135 before and 3 hours after MCA occlusion. The infarct size was determined after 24 hours (2,3,5-triphenyltetrazolium chloride staining). Cerebral blood flow (CBF) was measured at, before and 1, 2, 3, and 4 hours after MCA occlusion. Results— Cannabidiol significantly reduced the infarct volume induced by MCA occlusion in a bell-shaped curve. Similarly, abnormal cannabidiol but not anandamide or methanandamide reduced the infarct volume. Moreover, the neuroprotective effect of cannabidiol was inhibited by WAY100135, a serotonin 5-hydroxytriptamine1A (5-HT1A) receptor antagonist but not capsazepine a vanilloid receptor antagonist. Cannabidiol increased CBF to the cortex, and the CBF was partly inhibited by WAY100135 in mice subjected to MCA occlusion. Conclusions— Cannabidiol and abnormal cannabidiol reduced the infarct volume. Furthermore, the neuroprotective effect of cannabidiol was inhibited by WAY100135 but not capsazepine, and the CBF increased by cannabidiol was partially reversed by WAY100135. These results suggested that the neuroprotective effect of cannabidiol may be related to the increase in CBF through the serotonergic 5-HT1A receptor.

Impaired social interaction and reduced anxiety-related behavior in vasopressin V1a receptor knockout mice

The arginine vasopressin (AVP) system plays an important role in social behavior. Autism, with its hallmark disturbances in social behavior, has been associated with the V1a receptor (V1aR) gene. Furthermore, impairments of social function are often observed in symptoms of schizophrenia. Subchronic phencyclidine (PCP) produces behaviors relating to certain aspects of schizophrenic symptoms such as impairing social interaction in animals and it reduces the density of V1aR binding sites in several brain regions. Here, we report that V1aR knockout (KO) mice exhibited impairment of social behavior in a social interaction test, and showed reduced anxiety-related behavior in elevated plus-maze and marble-burying behavior tests. Given the current findings, the V1aR may be involved in the regulation of social interaction, and V1aR KO mice could be used as an animal model of psychiatric disorders associated with social behavior deficits, such as autism and schizophrenia.

Magnetic resonance imaging shows delayed ischemic striatal neurodegeneration.

Brief focal ischemia leading to temporary neurological deficits induces delayed hyperintensity on T1‐weighted magnetic resonance imaging (MRI) in the striatum of humans and rats. The T1 hyperintensity may stem from biochemical alterations including manganese (Mn) accumulation after ischemia. To clarify the significance of this MRI modification, we investigated the changes in the dorsolateral striatum of rats from 4 hours through 16 weeks after a 15‐minute period of middle cerebral artery occlusion (MCAO), for MRI changes, Mn concentration, neuronal number, reactivities of astrocytes and microglia/macrophages, mitochondrial Mn‐superoxide dismutase (Mn‐SOD), glutamine synthetase (GS), and amyloid precursor protein. The cognitive and behavioral studies were performed in patients and rats and compared with striatal T1 hyperintensity to show whether alteration in brain function correlated with MRI and histological changes. The T1‐weighted MRI signal intensity of the dorsolateral striatum increased from 5 days to 4 weeks after 15‐minute MCAO, and subsequently decreased until 16 weeks. The Mn concentration of the dorsolateral striatum increased after ischemia in concert with induction of Mn‐SOD and GS in reactive astrocytes. The neuronal survival ratio in the dorsolateral striatum decreased significantly from 4 hours through 16 weeks, accompanied by extracellular amyloid precursor protein accumulation and chronic glial/inflammatory responses. The patients and rats with neuroradiological striatal degeneration had late‐onset cognitive and/or behavioral declines after brief focal ischemia. This study suggests that (1) the hyperintensity on T1‐weighted MRI after mild ischemia may involve tissue Mn accumulation accompanied by Mn‐SOD and GS induction in reactive astrocytes, (2) the MRI changes correspond to striatal neurodegeneration with a chronic inflammatory response and signs of oxidative stress, and (3) the subjects with these MRI changes are at risk for showing a late impairment of brain function even though the transient ischemia is followed by total neurological recovery. Ann Neurol 2003;54:732–747

Selective and long-term learning impairment following neonatal hypoxic-ischemic brain insult in rats

We examined four different learning and memory tasks in rats which had been subjected to left carotid artery ligation followed by 2 h hypoxia (8% oxygen) when they were 7 days old. The examination began on the 4th week after insult and continued to 18 weeks post-insult. Compared with the control group, the hypoxic-ischemic group showed significant learning impairments in choice reaction time tasks relating to the attention process, and in plus-maze tasks and water maze tasks which examine long-term reference memory. In eight-arm radial maze tasks representing both short-term working memory and long-term reference memory, inferiority of the hypoxic-ischemic group was transient. Results of the sensorimotor test were normal in the hypoxic-ischemic group although slight flexion and twisting in the right forelimb was observed in 30% of the hypoxic-ischemic group when suspended by the tail. These abnormalities did not affect the results of learning tests. Findings of the study indicate that left-side brain damage produced by hypoxia-ischemia at 7 days of age resulted in selective and long-lasting learning and memory impairment.

Delayed Treatment With Minocycline Ameliorates Neurologic Impairment Through Activated Microglia Expressing a High-Mobility Group Box1–Inhibiting Mechanism

Background and Purpose— Minocycline, a semisynthetic tetracycline antibiotic, has been reported to ameliorate brain injury and inhibit microglial activation after focal cerebral ischemia. However, the cerebroprotective mechanism of minocycline remains unclear. In the present study, we investigated that mechanism of minocycline in a murine model of 4-hour middle cerebral artery (MCA) occlusion. Methods— One day after 4-hour MCA occlusion, minocycline was administered intraperitoneally for 14 days. Neurologic scores were measured 1, 7, and 14 days after cerebral ischemia. Motor coordination was evaluated at 14 days by the rota-rod test at 10 rpm. Activated microglia and high-mobility group box1 (HMGB1), a cytokine-like mediator, were also evaluated by immunostaining and Western blotting. In addition, terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling immunostaining was carried out 14 days after cerebral ischemia. Results— Repeated treatment with minocycline (1, 5, and 10 mg/kg) for 14 days improved neurologic score, motor coordination on the rota-rod test, and survival in a dose-dependent manner. Minocycline decreased the expression of Iba1, a marker of activated microglia, as assessed by both immunostaining and Western blotting. Moreover, minocycline decreased the activation of microglia expressing HMGB1 within the brain and also decreased both brain and plasma HMGB1 levels. Additionally, minocycline significantly decreased the number of terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling–positive cells and prevented ischemic brain atrophy 14 days after cerebral ischemia. Conclusions— Our results suggest that minocycline inhibits activated microglia expressing HMGB1 and decreases neurologic impairment induced by cerebral ischemia. Minocycline will have a palliative action and open new therapeutic possibilities for treatment of postischemic injury via an HMGB1-inhibiting mechanism.

ADAMTS13 gene deletion aggravates ischemic brain damage: a possible neuroprotective role of ADAMTS13 by ameliorating postischemic hypoperfusion.

Reperfusion after brain ischemia causes thrombus formation and microcirculatory disturbances, which are dependent on the platelet glycoprotein Ib-von Willebrand factor (VWF) axis. Because ADAMTS13 cleaves VWF and limits platelet-dependent thrombus growth, ADAMTS13 may ameliorate ischemic brain damage in acute stroke. We investigated the effects of ADAMTS13 on ischemia-reperfusion injury using a 30-minute middle cerebral artery occlusion model in Adamts13(-/-) and wild-type mice. After reperfusion for 0.5 hours, the regional cerebral blood flow in the ischemic cortex was decreased markedly in Adamts13(-/-) mice compared with wild-type mice (P < .05), which also resulted in a larger infarct volume after 24 hours for Adamts13(-/-) compared with wild-type mice (P < .01). Thus, Adamts13 gene deletion aggravated ischemic brain damage, suggesting that ADAMTS13 may protect the brain from ischemia by regulating VWF-platelet interactions after reperfusion. These results indicate that ADAMTS13 may be a useful therapeutic agent for stroke.

Inhibition of Reactive Astrocytes with Fluorocitrate Retards Neurovascular Remodeling and Recovery after Focal Cerebral Ischemia in Mice

Glial scarring is traditionally thought to be detrimental after stroke. But emerging studies now suggest that reactive astrocytes may also contribute to neurovascular remodeling. Here, we assessed the effects and mechanisms of metabolic inhibition of reactive astrocytes in a mouse model of stroke recovery. Five days after stroke onset, astrocytes were metabolically inhibited with fluorocitrate (FC, 1 nmol). Markers of reactive astrocytes (glial fibrillary acidic protein (GFAP), HMGB1), markers of neurovascular remodeling (CD31, synaptophysin, PSD95), and behavioral outcomes (neuroscore, rotarod latency) were quantified from 1 to 14 days. As expected, focal cerebral ischemia induced significant neurological deficits in mice. But over the course of 14 days after stroke onset, a steady improvement in neuroscore and rotarod latencies were observed as the mice spontaneously recovered. Reactive astrocytes coexpressing GFAP and HMGB1 increased in peri-infarct cortex from 1 to 14 days after cerebral ischemia in parallel with an increase in the neurovascular remodeling markers CD31, synaptophysin, and PSD95. Compared with stroke-only controls, FC-treated mice demonstrated a significant decrease in HMGB1-positive reactive astrocytes and neurovascular remodeling, as well as a corresponding worsening of behavioral recovery. Our results suggest that reactive astrocytes in peri-infarct cortex may promote neurovascular remodeling, and these glial responses may aid functional recovery after stroke.

Neuroprotective effect of γ-glutamylethylamide (theanine) on cerebral infarction in mice

In the present study, we examined the neuroprotective effect of γ-glutamylethylamide (theanine) on the ischemic brain damage in a middle cerebral artery occlusion model in mice. Theanine was injected i.p. 3 h after the occlusion or immediately before and 3 h after the occlusion. Theanine (1 mg/kg) significantly decreased the size of the cerebral infarcts 1 day after the occlusion. In contrast, theanine did not affect the cerebral blood flow, brain temperature and physiological variables (pH, pCO2, pO2 and hematocrit) in this model. These results suggest that theanine directly provides neuroprotection against focal cerebral ischemia and may be clinically useful for preventing cerebral infarction.