Hiroyuki Mizoguchi

RAGE-mediated signaling contributes to intraneuronal transport of amyloid-β and neuronal dysfunction

Intracellular amyloid-β peptide (Aβ) has been implicated in neuronal death associated with Alzheimers disease. Although Aβ is predominantly secreted into the extracellular space, mechanisms of Aβ transport at the level of the neuronal cell membrane remain to be fully elucidated. We demonstrate that receptor for advanced glycation end products (RAGE) contributes to transport of Aβ from the cell surface to the intracellular space. Mouse cortical neurons exposed to extracellular human Aβ subsequently showed detectable peptide intracellularly in the cytosol and mitochondria by confocal microscope and immunogold electron microscopy. Pretreatment of cultured neurons from wild-type mice with neutralizing antibody to RAGE, and neurons from RAGE knockout mice displayed decreased uptake of Aβ and protection from Aβ-mediated mitochondrial dysfunction. Aβ activated p38 MAPK, but not SAPK/JNK, and then stimulated intracellular uptake of Aβ-RAGE complex. Similar intraneuronal co-localization of Aβ and RAGE was observed in the hippocampus of transgenic mice overexpressing mutant amyloid precursor protein. These findings indicate that RAGE contributes to mechanisms involved in the translocation of Aβ from the extracellular to the intracellular space, thereby enhancing Aβ cytotoxicity.

A natural scavenger of peroxynitrites, rosmarinic acid, protects against impairment of memory induced by Aβ25- 35

Peroxynitrite (ONOO(-))-mediated damage is regarded to be responsible for the cognitive dysfunction induced by amyloid beta protein (Abeta) in Alzheimers disease (AD). In the present study, we examined the protective effects of rosmarinic acid (RA), a natural scavenger of ONOO(-), on the memory impairment in a mouse model induced by acute i.c.v. injection of Abeta(25-35). Mice daily received i.p. several doses of RA after the injection of Abeta(25-35). RA prevented the memory impairments induced by Abeta(25-35) in the Y maze test and novel object recognition task. RA, at the effective lowest dose (0.25mg/kg), prevented Abeta(25-35)-induced nitration of proteins, an indirect indicator of ONOO(-) damage, in the hippocampus. At this dose, RA also prevented nitration of proteins and impairment of recognition memory induced by ONOO(-)-i.c.v.-injection. Co-injection of the non-memory-impairing dose of ONOO(-) with Abeta(25-35) blocked the protective effects of RA (0.25mg/kg). These results demonstrated that the memory protective effects of RA in the neurotoxicity of Abeta(25-35) is due to its scavenging of ONOO(-), and that daily consumption of RA may protect against memory impairments observed in AD.

Social isolation rearing-induced impairment of the hippocampal neurogenesis is associated with deficits in spatial memory and emotion-related behaviors in juvenile mice.

Experiences during brain development may influence the pathogenesis of developmental disorders. Thus, social isolation (SI) rearing after weaning is a useful animal model for studying the pathological mechanisms of such psychiatric diseases. In this study, we examined the effect of SI on neurogenesis in the hippocampal dentate gyrus (DG) relating to memory and emotion‐related behaviors. When newly divided cells were labeled with 5‐bromo‐2′‐deoxyuridine (BrdU) before SI, the number of BrdU‐positive cells and the rate of differentiation into neurons were significantly decreased after 4‐week SI compared with those in group‐housed mice. Repeated treatment of fluoxetine prevented the SI‐induced impairment of survival of newly divided cells and ameliorated spatial memory impairment and part of aggression in SI mice. Furthermore, we investigated the changes in gene expression in the DG of SI mice by using DNA microarray and real‐time PCR. We finally found that SI reduced the expression of development‐related genes Nurr1 and Npas4. These findings suggest that communication in juvenile is important in the survival and differentiation of newly divided cells, which may be associated with memory and aggression, and raise the possibility that the reduced expression of Nurr1 and/or Npas4 may contribute to the impairment of neurogenesis and memory and aggression induced by SI.

Behavioral abnormality and pharmacologic response in social isolation-reared mice

Social isolation (SI) rearing in rodents causes a variety of behavioral changes, including hyperlocomotion, anxiety, impulsivity, aggression, and learning and memory deficits. These behavioral abnormalities in rodents may be related to the symptoms in patients with neuropsychiatric disorders, such as attention-deficit hyperactivity disorder, obsessive-compulsive disorder, autism, schizophrenia and depression. In this study, we examined the effect of long-term SI rearing after weaning on emotional behaviors and cognitive function in mice. Furthermore, the effects of methylphenidate (MPH), clozapine (CLZ) and fluoxetine (FLX) on SI-induced behavioral changes were examined to measure the predictive validity of SI-reared mice as an animal model for these neuropsychiatric disorders. MPH improved SI-induced anxiety-like behavior in the elevated-plus maze test, but had no effect on aggressive behavior. In contrast, CLZ ameliorated aggressive behavior, but not anxiety-like behavior in SI-reared mice. Repeated FLX treatment prevented SI-induced aggressive behavior and social interaction deficits. These findings suggest that SI-induced behavioral abnormality is a psychobehavioral complex relevant to various clinical symptoms observed in neuropsychiatric disorders and that SI-reared mice are a useful animal model to study the pathophysiology/pathogenesis of these diseases.

Oral vaccination with a viral vector containing Aβ cDNA attenuates age-related Aβ accumulation and memory deficits without causing inflammation in a mouse Alzheimer model

Immunotherapy with Aβ is expected to bring great improvement for Alzheimer disease (AD). However, clinical trials have been suspended because of meningoencephalitics, which accompanied lympho‐cytic infiltration. We have developed an oral vaccine for AD with a recombinant adeno‐associated viral vector carrying Aβ cDNA (AAV/Aβ). The vaccine reduces the amount of Aβ deposited without lymphocytic infiltration in APP transgenic (Tg2576) mice. In the present study, Tg2576 mice showed progressive cognitive impairments in the novel object recognition test, Y‐maze test, water maze test, and contextual conditioned fear learning test. A single oral administration of AAV/Aβ to Tg2576 mice at the age of 10 months alleviated progressive cognitive impairment with decreased Aβ deposition, insoluble Aβ, soluble Aβ oligomer (Aβ*56), microglial attraction, and synaptic degeneration induced in the brain regions at the age of 13 months. A histological analysis with hematoxylin and eosin and an immunohistochemical analysis with antibodies against CD3, CD4, CD8, and CD19 suggested there was no lymphocytic infiltration or microhemorrhage in the brain of AAV/Aβ‐vaccinated Tg2576 mice at 13 months of age. Taken together, these results suggest that immunotherapy with AAV/Aβ is a safe and effective treatment for AD.–Mouri, A., Noda, Y., Hara, H., Mizoguchi, H., Tabira, T., Nabeshima, T. Oral vaccination with a viral vector containing Aβ cDNA attenuates age‐related Aβ accumulation and memory deficits without causing inflammation in a mouse Alzheimer model. FASEB J. 21, 2135–2148 (2007)

Combined effect of neonatal immune activation and mutant DISC1 on phenotypic changes in adulthood

Gene-environment interaction may play a role in the etiology of schizophrenia. Transgenic mice expressing dominant-negative DISC1 (DN-DISC1 mice) show some histological and behavioral endophenotypes relevant to schizophrenia. Viral infection during neurodevelopment provides a major environmental risk for schizophrenia. Neonatal injection of polyriboinosinic-polyribocytidylic acid (polyI:C), which mimics innate immune responses elicited by viral infection, leads to schizophrenia-like behavioral alteration in mice after puberty. To study how gene-environmental interaction during neurodevelopment results in phenotypic changes in adulthood, we treated DN-DISC1 mice or wild-type littermates with injection of polyI:C during the neonatal stage, according to the published method, respectively, and the behavioral and histological phenotypes were examined in adulthood. We demonstrated that neonatal polyI:C treatment in DN-DISC1 mice resulted in the deficits of short-term, object recognition, and hippocampus-dependent fear memories after puberty, although polyI:C treatment by itself had smaller influences on wild-type mice. Furthermore, polyI:C-treated DN-DISC1 mice exhibited signs of impairment of social recognition and interaction, and augmented susceptibility to MK-801-induced hyperactivity as compared with vehicle-treated wild-type mice. Of most importance, additive effects of polyI:C and DN-DISC1 were observed by a marked decrease in parvalbumin-positive interneurons in the medial prefrontal cortex. These results suggest that combined effect of neonatal polyI:C treatment and DN-DISC1 affects some behavioral and histological phenotypes in adulthood.

Neonatal polyI:C treatment in mice results in schizophrenia-like behavioral and neurochemical abnormalities in adulthood

It has been reported that viral infection in the first and second trimesters of pregnancy in humans increases the risk of subsequently developing schizophrenia. To develop a mouse model of immune activation during the early postnatal period, neonatal ICR mice were repeatedly injected with polyriboinosinic-polyribocytidilic acid (polyI:C; an inducer of strong innate immune responses) for 5 days (postnatal day 2-6) which may correspond, in terms of brain development, to the early second trimester in human. Cognitive and emotional behavior as well as the extracellular level of glutamate in the hippocampus were analyzed at the age of 10-12 weeks old. PolyI:C-treated mice showed anxiety-like behavior, impairment of object recognition memory and social behavior, and sensorimotor gating deficits, as compared to the saline-treated control group. Depolarization-evoked glutamate release in the hippocampus was impaired in polyI:C-treated mice compared to saline-treated control mice. Furthermore, to investigate the effect of neonatal immune activation on the expression levels of schizophrenia-related genes, we analyzed mRNA levels in the hippocampus 2 and 24h after polyI:C treatment. No significant differences or only transient and marginal changes were observed between polyI:C-treated and saline-treated control mice in the expression levels of schizophrenia-related genes in the hippocampus.

Microglia Activated with the Toll-Like Receptor 9 Ligand CpG Attenuate Oligomeric Amyloid β Neurotoxicity in in Vitro and in Vivo Models of Alzheimer’s Disease

Soluble oligomeric amyloid beta (oAbeta) 1-42 causes synaptic dysfunction and neuronal injury in Alzheimers disease (AD). Although accumulation of microglia around senile plaques is a hallmark of AD pathology, the role of microglia in oAbeta1-42 neurotoxicity is not fully understood. Here, we showed that oAbeta but not fibrillar Abeta was neurotoxic, and microglia activated with unmethylated DNA CpG motif (CpG), a ligand for Toll-like receptor 9, attenuated oAbeta1-42 neurotoxicity in primary neuron-microglia co-cultures. CpG enhanced microglial clearance of oAbeta1-42 and induced higher levels of the antioxidant enzyme heme oxygenase-1 in microglia without producing neurotoxic molecules such as nitric oxide and glutamate. Among subclasses of CpGs, class B and class C activated microglia to promote neuroprotection. Moreover, intracerebroventricular administration of CpG ameliorated both the cognitive impairments induced by oAbeta1-42 and the impairment of associative learning in Tg2576 mouse model of AD. We propose that CpG may be an effective therapeutic strategy for limiting oAbeta1-42 neurotoxicity in AD.

Blockade of Gap Junction Hemichannel Suppresses Disease Progression in Mouse Models of Amyotrophic Lateral Sclerosis and Alzheimer's Disease

BACKGROUND Glutamate released by activated microglia induces excitotoxic neuronal death, which likely contributes to non-cell autonomous neuronal death in neurodegenerative diseases, including amyotrophic lateral sclerosis and Alzheimers disease. Although both blockade of glutamate receptors and inhibition of microglial activation are the therapeutic candidates for these neurodegenerative diseases, glutamate receptor blockers also perturbed physiological and essential glutamate signals, and inhibitors of microglial activation suppressed both neurotoxic/neuroprotective roles of microglia and hardly affected disease progression. We previously demonstrated that activated microglia release a large amount of glutamate specifically through gap junction hemichannel. Hence, blockade of gap junction hemichannel may be potentially beneficial in treatment of neurodegenerative diseases. METHODS AND FINDINGS In this study, we generated a novel blood-brain barrier permeable gap junction hemichannel blocker based on glycyrrhetinic acid. We found that pharmacologic blockade of gap junction hemichannel inhibited excessive glutamate release from activated microglia in vitro and in vivo without producing notable toxicity. Blocking gap junction hemichannel significantly suppressed neuronal loss of the spinal cord and extended survival in transgenic mice carrying human superoxide dismutase 1 with G93A or G37R mutation as an amyotrophic lateral sclerosis mouse model. Moreover, blockade of gap junction hemichannel also significantly improved memory impairments without altering amyloid β deposition in double transgenic mice expressing human amyloid precursor protein with K595N and M596L mutations and presenilin 1 with A264E mutation as an Alzheimers disease mouse model. CONCLUSIONS Our results suggest that gap junction hemichannel blockers may represent a new therapeutic strategy to target neurotoxic microglia specifically and prevent microglia-mediated neuronal death in various neurodegenerative diseases.

The allosteric potentiation of nicotinic acetylcholine receptors by galantamine ameliorates the cognitive dysfunction in beta amyloid25-35 i.c.v.-injected mice: involvement of dopaminergic systems.

Galantamine, a drug for Alzheimer’s disease, is a novel cholinergic agent with a dual mode of action, which inhibits acetylcholinesterase and allosterically modulates nicotinic acetylcholine receptors (nAChRs), as a result stimulates catecholamine neurotransmission. In the present study, we investigated whether galantamine exerts cognitive improving effects through the allosteric modulation of nAChR in the intracerebroventricular beta amyloid (Aβ)25−35-injected animal model of Alzheimer’s disease. Galantamine (3 mg/kg p.o.) significantly increased the extracellular dopamine release in the hippocampus of saline- and Aβ25−35-injected mice. The effects of nicotine on the extracellular dopamine release were potentiated by galantamine, but antagonized by mecamylamine, a nAChR antagonist. Aβ25−35-injected mice, compared with saline-injected mice, could not discriminate between new and familiar objects in the novel object recognition test and exhibited less freezing response in the fear-conditioning tasks, suggesting Aβ25−35 induced cognitive impairment. Galantamine improved the Aβ25−35-induced cognitive impairment in the novel object recognition and fear-conditioning tasks. These improving effects of galantamine were blocked by the treatment with mecamylamine, SCH-23390, a dopamine-D1 receptor antagonist, and sulpiride, a dopamine-D2 receptor antagonist, but not by scopolamine, a muscarinic acetylcholine receptor antagonist. This study provides the first in vivo evidence that galantamine augments dopaminergic neurotransmission within the hippocampus through the allosteric potentiation of nAChRs. The improving-effects of galantamine on the Aβ25−35-induced cognitive impairment may be mediated through the activation of, at least in part, dopaminergic systems, and the enhancement of dopamine release may be one of multiple mechanisms underlying the therapeutic benefit of galantamine.