Shiro Takei

DHA-PC and PSD-95 decrease after loss of synaptophysin and before neuronal loss in patients with Alzheimer's disease

Alzheimers disease (AD) is a progressive neurodegenerative disease that is characterized by senile plaques, neurofibrillary tangles, synaptic disruption, and neuronal loss. Several studies have demonstrated decreases of docosahexaenoic acid-containing phosphatidylcholines (DHA-PCs) in the AD brain. In this study, we used matrix-assisted laser desorption/ionization imaging mass spectrometry in postmortem AD brain to show that PC molecular species containing stearate and DHA, namely PC(18:0/22:6), was selectively depleted in the gray matter of patients with AD. Moreover, in the brain regions with marked amyloid β (Aβ) deposition, the magnitude of the PC(18:0/22:6) reduction significantly correlated with disease duration. Furthermore, at the molecular level, this depletion was associated with reduced levels of the postsynaptic protein PSD-95 but not the presynaptic protein synaptophysin. Interestingly, this reduction in PC(18:0/22:6) levels did not correlate with the degrees of Aβ deposition and neuronal loss in AD. The analysis of the correlations of key factors and disease duration showed that their effects on the disease time course were arranged in order as Aβ deposition, presynaptic disruption, postsynaptic disruption coupled with PC(18:0/22:6) reduction, and neuronal loss.

The Senescence-accelerated Mouse (SAM): A Higher Oxidative Stress and Age-dependent Degenerative Diseases Model

The SAM strain of mice is actually a group of related inbred strains consisting of a series of SAMP (accelerated senescence-prone) and SAMR (accelerated senescence-resistant) strains. Compared with the SAMR strains, the SAMP strains show a more accelerated senescence process, a shorter lifespan, and an earlier onset and more rapid progress of age-associated pathological phenotypes similar to human geriatric disorders. The higher oxidative stress status observed in SAMP mice is partly caused by mitochondrial dysfunction, and may be a cause of this senescence acceleration and age-dependent alterations in cell structure and function. Based on our recent observations, we discuss a possible mechanism for mitochondrial dysfunction resulting in the excessive production of reactive oxygen species, and a role for the hyperoxidative stress status in neurodegeneration in SAMP mice. These SAM strains can serve as a useful tool to understand the cellular mechanisms of age-dependent degeneration, and to develop clinical interventions.

Immunohistochemical localization of aggresomal proteins in glial cytoplasmic inclusions in multiple system atrophy

Y. Chiba, S. Takei, N. Kawamura, Y. Kawaguchi, K. Sasaki, S. Hasegawa‐Ishii, A. Furukawa, M. Hosokawa and A. Shimada (2012) Neuropathology and Applied Neurobiology38, 559–571

Characterization of a multidomain adaptor protein, p140Cap, as part of a pre-synaptic complex

p140Cap (Cas‐associated protein) is an adaptor protein considered to play pivotal roles in cell adhesion, growth and Src tyrosine kinase‐related signaling in non‐neuronal cells. It is also reported to interact with a pre‐synaptic membrane protein, synaptosome‐associated protein of 25 kDa, and may participate in neuronal secretion. However, properties and precise functions of p140Cap in neuronal cells are almost unknown. Here we show, using biochemical analyses, that p140Cap is expressed in rat brain in a developmental stage‐dependent manner, and is relatively abundant in the synaptic plasma membrane fraction in adults. Immunohistochemistry showed localization of p140Cap in the neuropil in rat brain and immunofluorescent analyses detected p140Cap at synapses of primary cultured rat hippocampal neurons. Electron microscopy further revealed localization at pre‐ and post‐synapses. Screening of p140Cap‐binding proteins identified a multidomain adaptor protein, vinexin, whose third Src‐homology 3 domain interacts with the C‐terminal Pro‐rich motif of p140Cap. Immunocomplexes between the two proteins were confirmed in COS7 and rat brain. We also clarified that a pre‐synaptic protein, synaptophysin, interacts with p140Cap. These results suggest that p140Cap is involved in neurotransmitter release, synapse formation/maintenance, and signaling.

Morphological impairments in microglia precede age-related neuronal degeneration in senescence-accelerated mice

The ageing brain is characterized by degenerative changes in both neurons and glia. Although neurons are known to lose dendritic complexity with ageing, age‐related changes in the morphology of microglia have not been well documented. We investigated potential age‐related changes in microglial morphology using mouse models. Senescence‐accelerated mouse prone 10 (SAMP10) in which neuronal degeneration begins to appear around 8 months of age and becomes progressively remarkable with advancing age was used as a model of brain ageing. Senescence‐accelerated mouse resistant 1 (SAMR1) in which age‐related neuronal changes are inconspicuous was used as usual‐ageing controls. Hippocampal sections prepared from 3‐, 8‐ and 14‐month‐old SAMP10 and 3‐, 8‐, 14‐ and 24‐month‐old SAMR1 mice were stained immunohistochemically with anti‐Iba‐1 antibody to highlight microglia. Stick figures of individual microglia reflecting the length and complexity of cytoplasmic processes were made by camera lucida drawing. Parameters representing morphological features of microglia were quantified using an image analyzer: area of convex closure, cell body area, number of primary processes, maximal branch order, combined projection length, number of segments and number of tips. Pathological changes of processes such as beading and clusters of fragmented twigs were counted. In microglia of 3‐ and 8‐month‐old SAMP10 mice, combined projection length was shorter and numbers of segments and tips were smaller than those in age‐matched SAMR1 mice. Similar changes were detected in SAMR1 mice at age 14 months and older. Microglia of SAMP10 mice at all ages were characterized by having frequent pathological changes in processes, which were not remarkable in SAMR1 mice at any age. These morphological abnormalities in microglia of SAMP10 mice preceded the onset of neuronal degeneration and may lead to making brain tissue less protective to neurons. We propose that preceding abnormalities in microglia may contribute to the vulnerability to age‐related neuronal degeneration in SAMP10 mice.

Preferential localization of prostamide/prostaglandin F synthase in myelin sheaths of the central nervous system

Prostaglandin (PG) F(₂α) is a product of cyclooxygenase (COX)-catalyzed metabolism of arachidonic acid and exerts biological functions in various tissues. Prostaglandin ethanolamide (prostamide) F(₂α) is a COX-2-catalyzed metabolite of arachidonoyl ethanolamide (anandamide) that induces pharmacological actions in ocular tissues. Although PGF(₂α) is one of the most abundant prostaglandins in the brain, function of PGF(₂α) in the central nervous system (CNS) has not been extensively investigated. Recently identified prostamide/PGF synthase catalyzes the reductions of prostamide H₂ to prostamide F(₂α) and PGH₂ to PGF(₂α), chiefly in the CNS. We examined tissue distribution of the enzyme in the CNS by immunohistochemistry, double immunofluorescence, and immuno-electron microscopy. We confirmed histological findings by immunofluorescence analyses of brain cell cultures. Prostamide/PGF synthase was expressed preferentially in the white matter bundles of the entire CNS of adult mice with less marked expression in neuronal cell bodies. The enzyme was colocalized with myelin basic protein (MBP) in myelin sheaths but not in axons. At the ultrastructural level, the enzyme was localized to myelin sheaths. Expression of the enzyme increased between P9 and P14 during the postnatal development, presumably in accordance with myelinogenesis. Cultured oligodendrocytes at 7 days in vitro expressed the enzyme in cytoplasmic processes where the enzyme was colocalized with MBP. Immunoreactivity for COX-2 was detected in white matter and cultured oligodendrocytes. Relatively selective localization of prostamide/PGF synthase suggests that myelin sheaths of the CNS may serve as the sites for producing prostamide F(₂α) and/or PGF(₂α), which may contribute to the formation and maintenance of central myelin.

Exome sequencing of senescence-accelerated mice (SAM) reveals deleterious mutations in degenerative disease-causing genes

BackgroundSenescence-accelerated mice (SAM) are a series of mouse strains originally derived from unexpected crosses between AKR/J and unknown mice, from which phenotypically distinct senescence-prone (SAMP) and -resistant (SAMR) inbred strains were subsequently established. Although SAMP strains have been widely used for aging research focusing on their short life spans and various age-related phenotypes, such as immune dysfunction, osteoporosis, and brain atrophy, the responsible gene mutations have not yet been fully elucidated.ResultsTo identify mutations specific to SAMP strains, we performed whole exome sequencing of 6 SAMP and 3 SAMR strains. This analysis revealed 32,019 to 38,925 single-nucleotide variants in the coding region of each SAM strain. We detected Ogg1 p.R304W and Mbd4 p.D129N deleterious mutations in all 6 of the SAMP strains but not in the SAMR or AKR/J strains. Moreover, we extracted 31 SAMP-specific novel deleterious mutations. In all SAMP strains except SAMP8, we detected a p.R473W missense mutation in the Ldb3 gene, which has been associated with myofibrillar myopathy. In 3 SAMP strains (SAMP3, SAMP10, and SAMP11), we identified a p.R167C missense mutation in the Prx gene, in which mutations causing hereditary motor and sensory neuropathy (Dejerine-Sottas syndrome) have been identified. In SAMP6 we detected a p.S540fs frame-shift mutation in the Il4ra gene, a mutation potentially causative of ulcerative colitis and osteoporosis.ConclusionsOur data indicate that different combinations of mutations in disease-causing genes may be responsible for the various phenotypes of SAMP strains.

Defects in cytokine-mediated neuroprotective glial responses to excitotoxic hippocampal injury in senescence-accelerated mouse

Aging is a result of damage accumulation, and understanding of the mechanisms of aging requires exploration of the cellular and molecular systems functioning to control damage. Senescence-accelerated mouse prone 10 (SAMP10) has been established as an inbred strain exhibiting accelerated aging with an earlier onset of cognitive impairment due to neurodegeneration than the senescence-resistant control (SAMR1) strain. We hypothesized that tissue-protective responses of glial cells are impaired in SAMP10 mice. We injected kainic acid (KA) to induce hippocampal injury and studied how cytokines were upregulated on Day 3 using 3-month-old SAMP10 and SAMR1 mice. Following microarray-based screening for upregulated genes, we performed real-time RT-PCR and immunohistochemistry. Results indicated well-orchestrated cytokine-mediated glial interactions in the injured hippocampus of SAMR1 mice, in which microglia-derived interferon (IFN)-γ stimulated astrocytes via IFN-γ receptor and thereby induced expression of CXCL10 and macrophage inflammatory protein (MIP)-1α, and activated microglia produced granulocyte-macrophage colony-stimulating factor (GM-CSF) and osteopontin (OPN). OPN was the most strongly upregulated cytokine. CD44, an OPN receptor, was also strongly upregulated in the neuropil, especially on neurons and astrocytes. KA-induced hippocampal upregulation of these cytokines was strikingly reduced in SAMP10 mice compared to SAMR1 mice. On Day 30 after KA injection, SAMP10 but not SAMR1 mice exhibited hippocampal layer atrophy. Since the OPN-CD44 system is essential for neuroprotection and remodeling, these findings highlight the defects of SAMP10 mice in cytokine-mediated neuroprotective glia-neuron interactions, which may be associated with the mechanism underlying the vulnerability of SAMP10 mice to age-related neurodegeneration.

Niemann‐Pick disease type C1 predominantly involving the frontotemporal region, with cortical and brainstem Lewy bodies: An autopsy case

Niemann‐Pick disease type C (NPC) is an autosomal recessive neurovisceral lipid storage disorder. Two disease‐causing genes (NPC1 and NPC2) have been identified. NPC is characterized by neuronal and glial lipid storage and NFTs. Here, we report a man with juvenile‐onset progressive neurological deficits, including pyramidal signs, ataxia, bulbar palsy, vertical supranuclear ophthalmoplegia, and psychiatric symptoms; death occurred at age 37 before definitive clinical diagnosis. Post mortem gross examination revealed a unique distribution of brain atrophy, predominantly in the frontal and temporal lobes. Microscopically, lipid storage in neurons and widely distributed NFTs were observed. Lipid storage cells appeared in systemic organs and filipin staining indicated intracellular cholesterol accumulation in hepatic macrophages. Electron microscopy revealed accumulation of lipids and characteristic oligolamellar inclusions. These findings suggested an NPC diagnosis. Neuronal loss and gliosis were frequently accompanied by NFTs and occurred in the frontal and temporal cortices, hippocampus, amygdala, basal forebrain, basal ganglia, thalamus, substantia nigra and brain stem nuclei. Lewy bodies (LBs) were observed in most, but not all, regions where NFTs were evident. In contrast, neuronal lipid storage occurred in more widespread areas, including the parietal and occipital cortices where neurodegeneration with either NFTs or LBs was minimal. Molecular genetic analysis demonstrated that the patient had compound heterozygous mutations in the cysteine‐rich loop (A1017T and Y1088C) of the NPC1 gene. To our knowledge there has been no previous report of the A1017T mutation. The pathological features of this patient support the notion that NPC has an aspect of α‐synucleinopathy, and long‐term survivors of NPC may develop a frontotemporal‐predominant distribution of brain atrophy.

Immunohistochemical demonstration of increased prostaglandin F2α levels in the rat hippocampus following kainic acid-induced seizures

Prostaglandin (PG) F(2α) is one of the major prostanoids biosynthesized by cyclooxygenases (COXs) from arachidonic acid. Although it has been reported that there is a selective surge in PGF(2α) production in the hippocampus during kainic acid (KA)-induced seizure activity, the precise intra-hippocampal distribution of PGF(2α) has not been elucidated due to the paucity of effective histological techniques for detecting PGs in tissues. We investigated the tissue distribution of PGF(2α) in the rat hippocampus 30 min after KA injection by developing fixation and immunohistological-staining methods. To detect PGF(2α) directly on histological sections, we used systemic perfusion fixation with water-soluble carbodiimide fixative, followed by immersion of the brains in Zambonis fixative. We then performed immunofluorescence staining with anti-PGF(2α) antibody, with negative control experiments used to confirm the staining specificity. Definitive immunolabeling for PGF(2α) was evident most markedly in pyramidal cells of the hippocampal cornu Ammonis (CA) 3 sector and neurons of the hilus in KA-treated rats. Immunolabeling for PGF(2α) was also evident in granule cells of the dentate gyrus. Double immunfluorescence staining revealed that PGF(2α)-immunopositive neurons expressed cytosolic phospholipases A(2), COX-2, and FP receptor. These results suggest that the major source of PGF(2α) production immediately after KA injection was neurons of the hippocampal CA3 sector, hilus and dentate gyrus. These neurons exert PGF(2α)-mediated functions via FP receptors in an autocrine/paracrine manner and may play pathophysiological roles in the acute phase (30 min) of excitotoxicity.