Developmental defects and aberrant accumulation of endogenous psychosine in oligodendrocytes in a murine model of Krabbe disease.

Krabbe disease (KD), or globoid cell leukodystrophy, is an inherited lysosomal storage disease with leukodystrophy caused by a mutation in the galactosylceramidase (GALC) gene. The majority of patients show the early onset form of KD dominated by cerebral demyelination with apoptotic oligodendrocyte (OL) death. However, the initial pathophysiological changes in developing OLs remain poorly understood. Here, we show that OLs of twitcher mice, an authentic mouse model of KD, exhibited developmental defects and impaired myelin formation in vivo and in vitro. In twitcher mouse brain, abnormal myelination and reduced expression of myelin genes during the period of most active OL differentiation and myelination preceded subsequent progressive OL death and demyelination. Importantly, twitcher mouse OL precursor cells proliferated normally, but their differentiation and survival were intrinsically defective. These defects were associated with aberrant accumulation of endogenous psychosine (galactosylsphingosine) and reduced activation of the Erk1/2 and Akt/mTOR pathways before apoptotic cell death. Collectively, our results demonstrate that GALC deficiency in developing KD OLs profoundly affects their differentiation and maturation, indicating the critical contribution of OL dysfunction to KD pathogenesis.

SAMP8 mice as a neuropathological model of accelerated brain aging and dementia: Toshio Takeda's legacy and future directions.

Senescence accelerated mice P8 (SAMP8) show significant age-related deteriorations in memory and learning ability in accordance with early onset and rapid advancement of senescence. Brains of SAMP8 mice reveal an age-associated increase of PAS-positive granular structures in the hippocampal formation and astrogliosis in the brain stem and hippocampus. A spongy degeneration in the brain stem appears at 1 month of age and reaches a maximum at 4-8 months. In addition, clusters of activated microglia also appear around the vacuoles in the brain stem. ß/A4(Aß) protein-like immunoreactive granular structures are observed in various regions and increase in number markedly with age. Other age-associated histological changes include cortical atrophy, neuronal cell loss in locus coeruleus and lateral tegmental nuclei, intraneuronal accumulation of lipopigments in Purkinje cells and eosinophilic inclusion bodies in thalamic neurons. A blood-brain barrier dysfunction and astrogliosis are also prominent with advancing age in the hippocampus. These changes are generally similar to the pathomorphology of aging human brains and characterized by their association with some specific glioneuronal reactions. As for the hallmarks of Alzheimer brains, tau morphology has not yet been confirmed regardless of the age-related increase in phosphorylated tau in SAMP8 mice brains, but early age-related Aß deposition in the hippocampus has recently been published. SAMP8 mice are, therefore, not only a senescence-accelerated model but also a promising model for Alzheimer's disease and other cognitive disorders.

Novel frame-shift mutation in Slc5a2 encoding SGLT2 in a strain of senescence-accelerated mouse SAMP10.

The senescence-accelerated mouse prone10 (SAMP10) strain, a model of aging, exhibits cognitive impairments and cerebral atrophy. We noticed that SAMP10/TaSlc mice, a SAMP10 substrain, have developed persistent glucosuria over the past few years. In the present study, we characterized SAMP10/TaSlc mice and further identified a spontaneous mutation in the Slc5a2 gene encoding sodium-glucose co-transporter (SGLT) 2. The mean concentration of urine glucose was high in SAMP10/TaSlc mice and increased further with advancing age, whereas other strains of senescence-accelerated mice, including SAMP1/SkuSlc, SAMP6/TaSlc and SAMP8/TaSlc or normal aging control SAMR1/TaSlc mice, exhibited no detectable glucose in urine. SAMP10/TaSlc mice consumed increasing amounts of food and water compared to SAMR1/TaSlc mice, suggesting the compensation of polyuria and the loss of glucose. Oral glucose tolerance tests showed decreased glucose reabsorption in the kidney of SAMP10/TaSlc mice. In addition, blood glucose levels decreased in an age-dependent fashion. The kidney was innately larger than that of control mice with no histological alterations. We examined the expression levels of glucose transporters in the kidney. Among SGLT1, SGLT2, glucose transporter (GLUT) 1 and GLUT2, we found a significant decrease only in the level of SGLT2. DNA sequencing of SGLT2 in SAMP10/TaSlc mice revealed a single nucleotide deletion of guanine at 1236, which resulted in a frameshift mutation that produced a truncated protein. We designate this strain as SAMP10/TaSlc-Slc5a2(slc) (SAMP10-ΔSglt2). Recently, SGLT2 inhibitors have been demonstrated to be effective for the treatment of patients with type 2 diabetes (T2D). SAMP10-ΔSglt2 mice may serve as a unique preclinical model to study the link between aging-related neurodegenerative disorders and T2D.

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.

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

BACKGROUND: Senescence-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. RESULTS: To 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. CONCLUSIONS: Our data indicate that different combinations of mutations in disease-causing genes may be responsible for the various phenotypes of SAMP strains.

Spontaneous occurrence of photoageing-like phenotypes in the dorsal skin of old SAMP1 mice, an oxidative stress model.

Skin photoageing is a complex, multifactorial process and both intrinsic and extrinsic factors may contribute to its pathogenesis. The ultraviolet-irradiated hairless mouse has been used as an animal model for photoageing, but this model mimics only the 'extrinsic' aspects. Here, we show that skin from old SAMP1 mice, a model for higher oxidative stress and senescence acceleration, exhibited histological and gene expression changes similar to those in human photoaged skin without ultraviolet irradiation. These changes include an increase in elastic fibre and glycosaminoglycan histologically, an upregulation of several proinflammatory cytokines and matrix metalloproteinases, and an increase in lipid peroxide. We propose that SAMP1 mice are a spontaneous animal model for photoageing caused by an exaggerated intrinsic mechanism, namely, higher oxidative status. This mouse model is useful to explore the link between oxidative stress and photoageing, and to evaluate the efficacy of antioxidants.

Selective localization of bone marrow-derived ramified cells in the brain adjacent to the attachments of choroid plexus.

Although the immune system modulates higher functions of the brain under non-inflammatory conditions, how immune cells interact with brain parenchymal cells remains to be determined. Using bone marrow chimeric mice in which the recipients' immune system was reconstituted by marrow cells derived from GFP-transgenic mice by syngeneic intra-bone marrow-bone marrow transplantation (IBM-BMT) and by intravenous (IV)-BMT, we examined the distribution, density and differentiation of donor-derived marrow cells in the brain parenchyma 2 weeks and 1, 4 and 8 months after BMT. Marrow-derived cells started to populate discrete brain regions from 1 to 4 months after BMT, exhibited ramified morphology and expressed Iba-1. The ramified marrow-derived cells were distributed in more brain regions and for a longer time after IBM-BMT than IV-BMT. Most of these discrete regions were adjacent to the attachments of choroid plexus that comprised thinned brain parenchyma consisting of astroglial processes in the narrow channel between the ependyma and pia. These specific portions of astroglial processes expressed fractalkine. In the choroid plexus stroma, not only Iba-1+ myeloid cells but also non-myeloid CXCL12-expressing cells were of bone marrow-origin. Transcripts of fractalkine, CXCL12 and their related molecules such as CX3CR1, ADAM10 and CXCR4 were detected in the tissue consisting of the choroid plexus, the attachments and adjacent brain parenchyma. Thus, bone marrow cells selectively enter the discrete brain regions adjacent to the attachments of choroid plexus and differentiate into ramified myeloid cells. Fractalkine in the attachments of choroid plexus and CXCL12 in the choroid plexus stroma may be involved in these brain-immune interactions.

Proteomic identification of hippocampal proteins vulnerable to oxidative stress in excitotoxin-induced acute neuronal injury.

Excitotoxicity is involved in seizure-induced acute neuronal death, hypoxic-ischemic encephalopathy, and chronic neurodegenerative conditions such as Alzheimer's disease. Although oxidative stress has been implicated in excitotoxicity, the target proteins of oxidative damage during the course of excitotoxic cell death are still unclear. In the present study, we performed 2D-oxyblot analysis and mass spectrometric amino acid sequencing to identify proteins that were vulnerable to oxidative damage in the rat hippocampus during kainic acid (KA)-induced status epilepticus. We first investigated the time course in which oxidative protein damage occurred using immunohistochemistry. Carbonylated proteins, a manifestation of protein oxidation, were detected in hippocampal neurons as early as 3h after KA administration. Immunoreactivity for 8-hydroxy-2'-deoxyguanosine (8-OHdG) was also elevated at the same time point. The increase in oxidative damage to proteins and DNA occurred concomitantly with the early morphological changes in KA-treated rat hippocampus, i.e., changes in chromatin distribution and swelling of rough endoplasmic reticulum and mitochondria, which preceded the appearance of morphological features of neuronal death such as pyknotic nuclei and hypereosinophilic cytoplasm. Proteomic analysis revealed that several hippocampal proteins were consistently carbonylated at this time point, including heat shock 70kDa protein 4, valosin-containing protein, mitochondrial inner membrane protein (mitofilin), α-internexin, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (14-3-3 protein). We propose that oxidative damage to these proteins may be one of the upstream events in the molecular pathway leading to excitotoxic cell death in KA-treated rat hippocampus, and these proteins may be targets of therapeutic intervention for seizure-induced neuronal death.

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.

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.

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.

Proteomic analysis of aging brain in SAMP10 mouse: a model of age-related cerebral degeneration.

Senescence-accelerated mouse prone 10 (SAMP10) strain is a model of age-related neurodegeneration in the limbic forebrain. To investigate changes in protein expression profiles involved in neurodegeneration, we performed two-dimensional fluorescence difference gel electrophoresis and compared protein expression in the limbic and non-limbic forebrains of SAMP10 and control mice at various ages. Among protein spots in which patterns of aging in expression in the limbic forebrain differed between SAMP10 and control, we identified three proteins by mass spectrometry: pyridoxal phosphate phosphatase (PLPP), collapsin response mediator protein 2 (CRMP-2) and alpha-internexin. Expression of PLPP was increased in the limbic forebrain of 3-month-old SAMP10 mice. Levels of CRMP-2 and phosphorylated alpha-internexin were increased in the limbic forebrain of SAMP10 mice at age 8 months and remained high until 14 months. Western blot revealed elevation in the level of phosphorylated CRMP-2 and the ratio of phosphorylation of alpha-internexin. Immunohistochemistry revealed that alpha-internexin was chiefly distributed in axons. Aging in SAMP10 mice was associated with abnormality of PLPP, CRMP-2 and alpha-internexin, all of which are known to be involved in brain cytoskeleton formation and associated with acute and chronic neurodegenerative conditions. These proteins are promising targets for further investigation of the mechanisms underlying brain aging.

Quantitative trait locus on chromosome X affects bone loss after maturation in mice.

Genetic programming is known to affect the peak bone mass and bone loss after maturation. However, little is known about how polymorphic genes on chromosome X (Chr X) modulate bone loss after maturation. We previously reported a quantitative trait locus (QTL) on Chr X, designated Pbd3, which had a suggestive linkage to bone mass, in male SAMP2 and SAMP6 mice. In this study, we aimed to clarify the effects of Pbd3 on the skeletal phenotype. We generated a congenic strain, P2.P6-X, carrying a 45.6-cM SAMP6-derived Chr X interval on a SAMP2 genetic background. The effects of Pbd3 on the bone phenotype were determined by microcomputed tomography (microCT), whole-body dual-energy X-ray absorptiometry (DXA), serum bone turnover markers, and histomorphometric parameters. Both the bone area fraction (BA/TA) on microCT and whole-body DXA revealed reduced bone loss in P2.P6-X compared with that in SAMP2. The serum concentrations of bone turnover markers at 4 months of age were significantly lower in P2.P6-X than in SAMP2, but did not differ at 8 months of age. These results were observed in female mice, but not in male mice. In conclusion, a QTL within a segregated 45.6-cM interval on Chr X is sex-specifically related to the rate of bone loss after maturation.

Eosin-shadow method: a selective enhancement of light-microscopic visualization of pancreatic zymogen granules on hematoxylin-eosin sections.

We developed a novel method for enhancing light-microscopic visualization of pancreatic zymogen granules in a selective manner on hematoxylin and eosin-stained sections. By using an absorption filter that transmits light with wavelength from 510 to 550 nm, corresponding to the narrow absorption spectrum of eosin, only eosinophilic tissue and cellular components were remarkably highlighted as distinct shadows against lighter background consisting of basophilic components. Using a pair of mirror sections of the pancreas, immunocytochemistry with anti-amylase antibody confirmed that the shadows observed through the filter represented zymogen granules. Immersion in formalin for 36 h at room temperature was the optimal fixation condition. Here we designate the procedure as the "eosin-shadow method" and propose that this technique is convenient and useful to help investigators identify zymogen granules more easily in routine pathological examination and histological studies.

Risk of fall for individuals with intellectual disability.

Our aim was to identify risk factors for falling and establish a method to assess risk for falls in adults with intellectual disabilities. In a cross-sectional survey of 144 Japanese adults, we found that age, presence of epilepsy, and presence of paretic conditions were independent risk factors. The Tinetti balance and gait instrument was successfully administered to this population and resulted in high diagnostic accuracy (sensitivity 88.9%, specificity 91.9%) for identifying individuals at risk when the cutoff score was set at 25. Participants whose balance and gait deteriorated showed a decrease in the Tinetti score of at least 2 points per year. Thus, the Tinetti instrument may be an effective tool to detect an increased risk of fall in this population.

Effect of dietary unsaturated fatty acids on senile amyloidosis in senescence-accelerated mice.

Effects of dietary oils on aging were investigated in senescence-accelerated mice. For 26 weeks, mice were fed purified diets containing 4% olive oil, safflower oil, perilla oil, or fish oil. Serum total, high-density lipoprotein cholesterol, and apolipoprotein A-II (ApoA-II) were significantly lower in the fish oil group than in the perilla oil group, and these were significantly lower than in the olive oil or safflower oil group. The olive oil and safflower oil groups had significantly fewer ApoA-II amyloid fibril (AApoAII) deposits and anti-single-strand DNA (ssDNA) antibodies than the fish oil or perilla oil group, and the fish oil diet induced significantly more AApoAII deposits and anti-ssDNA antibodies than did the perilla oil diet. Survival decreased earlier in the fish oil group than in the other groups (as seen in the survival curve). The results suggest that greater the degree of unsaturation of dietary fatty acids, greater is the tendency for accelerated senescence.

Psycho-educational Horseback Riding to Facilitate Communication Ability of Children with Pervasive Developmental Disorders.

In this study, we applied a novel psycho-educational horseback riding (PEHR) program to the treatment of four Japanese children with pervasive developmental disorders (PDD) in order to facilitate the acquisition of verbal and nonverbal communication skills. The behavioral changes in each child were evaluated using a psychological and behavioral scale. The scale for evaluating the effect of Human-Equips-Interaction on Mental activity (HEIM scale) was designed to assess the behavioral improvement of children based on the following 10 items: Human relationships, Imitation, Emotional expression, Sudden physical movement, Fixative behavior, Adaptation to change, Visual response, Fear or nervousness, and Verbal and nonverbal communication. After taking part in the PEHR program for several months, all subjects showed remarkably improved HEIM scores and marked improvements were observed in eye contact with others (instructors, side walkers, and leaders) in the riding area. A statistical difference was found in items 1, 2, 3, 6, 7, 8, and 9. However, no statistical difference was found in items 4, 5, and 10. As the program progressed, the children showed enhanced verbal and nonverbal communication skills, and became more expressive in their emotional and empathetic interaction with their parents. These observations suggest that the normal functioning of pleasurable emotions and empathy may facilitate further improvements in joint attention, imitation and empathy, and may result in successful verbal expression by PDD children. Therefore, horseback riding can play a very important role in the psycho-educational support required for the communication ability of PDD children.

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.

Neuronal toxicity of expanded polyglutamine depends on intracellular distribution in addition to the expression level.

The proteins that accumulate in pathologic lesions of neurodegenerative disorders are thought to be closely associated with neuronal cell damage. However, whether or not the formation of cytoplasmic or nuclear inclusions by expanded polyglutamine (polyQ) is directly toxic to neurons has been controversial to date. We prepared a culture model system in which polyQ tracts were transfected into Neuro2a, cells of neuronal origin, to study novel factors involved in cell toxicity of polyQ tracts to neuronal cells. Pathogenic polyQ tracts of 79 repeats (Q79C) when expressed in cytoplasm of Neuro2a cells changed in their intracellular distribution patterns from homogeneous, via punctate aggregates, to massive aggregates with incubation time. Some polyQ tracts formed nuclear inclusions. Cytoplasmic massive aggregates of Q79C tended to be associated with apoptotic fate of Neuro2a cells. Cells exhibiting cytoplasmic massive inclusions had the highest expression level of polyQ tracts among cells with four patterns of intracellular distribution. The elevation in the expression levels of polyQ tracts was not due to the difference in the initial transfection efficiency. When compared among cells expressing polyQ tracts at similar levels, damages were most remarkable in cells with cytoplasmic massive aggregate in terms of shrunken cellular and nuclear sizes. Cells with the other patterns of polyQ tract distribution such as cytoplasmic homogeneous, cytoplasmic punctate and nuclear inclusions were relatively spared. These data suggest that the severity of cell damages depends on the type of intracellular distribution of polyQ tracts, in addition to the expression level of polyQ tracts.

Reduced expression of MAb6B4 epitopes on chondroitin sulfate proteoglycan aggrecan in perineuronal nets from cerebral cortices of SAMP10 mice: a model for age-dependent neurodegeneration.

The accelerated senescence-prone SAMP10 mouse strain is a model for age-dependent neurodegeneration and is characterized by brain atrophy and deficits in learning and memory. Because perineuronal nets play an important role in the synaptic plasticity of adult brains, we examined the distributions of molecules that constitute perineuronal nets in SAMP10 mouse brain samples and compared them with those in control SAMR1 mouse samples. Proteoglycan-related monoclonal antibody 6B4 (MAb6B4) clearly immunostained perineuronal nets in SAMR1 mice cortices, but the corresponding immunostaining in SAMP10 mice was very faint. MAb6B4 recognizes phosphacan/PTPzeta in immature brains. However, this antibody recognized several protein bands, including a 400-kDa core glycoprotein from chondroitin sulfate proteoglycan in homogenates of mature cortices from SAMR1 mice. The 400-kDa band was also recognized by antiaggrecan antibodies. The aggrecan core glycoprotein band was also detectable in samples from SAMP10 mice, but this glycoprotein was faintly immunostained by MAb6B4. Because MAb6B4 recognized the same set of protein bands that the monoclonal antibody Cat-315 recognized in mature cerebral cortices of SAMR1 mice, the MAb6B4 epitope appears to be closely related to that of Cat-315 and presumably represents a novel type of oligosaccharide that attaches to aggrecans. The Cat-315 epitope colocalized with aggrecan in perineuronal nets from SAMR1 mouse brain samples, whereas its expression was prominently reduced in SAMP10 mouse brain samples. The biological significance of the MAb6B4/Cat-315 epitope in brain function and its relationship to the neurodegeneration and learning disabilities observed in SAMP10 mice remain to be elucidated.