Resveratrol, a SIRT1 activator, attenuates aging-associated alterations in skeletal muscle and heart in mice.

Aging is associated with impairment of multiple organs, including skeletal muscle and heart. In this study, we investigated whether resveratrol, an activator of an NAD+-dependent protein deacetylase Sirtuin-1 (SIRT1), attenuates age-related sarcopenia and cardiomyocyte hypertrophy in mice. Treatment of mice with resveratrol (0.4 g/kg diet) from 28 weeks of age for 32 weeks prevented aging-associated shortening of rotarod riding time. In the tibialis anterior (TA) muscle, histogram analysis showed that the atrophic muscle was increased in 60-week-old (wo) mice compared with 20-wo mice, which was attenuated by resveratrol. In the heart, resveratrol attenuated an aging-associated increase in the cardiomyocyte diameter. Acetylated proteins were increased and autophagic activity was reduced in the TA muscle of 60-wo mice compared with those of 20-wo mice. Resveratrol treatment reduced levels of acetylated proteins and restored autophagic activity in the TA muscle. Aging-related reduction in myocardial autophagy was also suppressed by resveratrol. Skeletal muscle-specific SIRT1 knockout mice showed increases in acetylated proteins and atrophic muscle fibers and reduced autophagic activity in the TA muscle. These results suggest that activation of SIRT1 by treatment with resveratrol suppresses sarcopenia and cardiomyocyte hypertrophy by restoration of autophagy in mice.

Enrichment of Neurodegenerative Microglia Signature in Brain-Derived Extracellular Vesicles Isolated from Alzheimer's Disease Mouse Models.

Extracellular vesicles (EVs) are secreted by any neural cells in the central nervous system for molecular clearance, cellular communications, and disease spread in multiple neurodegenerative diseases, including Alzheimer's disease (AD), although their exact molecular mechanism is poorly understood. We hypothesize that high-resolution proteomic profiling of EVs separated from animal models of AD would determine the composition of EV contents and their cellular origin. Here, we examined recently developed transgenic mice (CAST.APP/PS1), which express familial AD-linked mutations of amyloid precursor protein (APP) and presenilin-1 (PS1) in the CAST/EiJ mouse strain and develop hippocampal neurodegeneration. Quantitative proteomics analysis of EVs separated from CAST.APP/PS1 and age-matched control mice by tandem mass tag-mass spectrometry identified a total of 3444 unique proteins, which are enriched in neuron-, astrocyte-, oligodendrocyte-, and microglia-specific molecules. CAST.APP/PS1-derived EVs show significant enrichment of Psen1, APP, and Itgax and reduction of Wdr61, Pmpca, Aldh1a2, Calu, Anp32b, Actn4, and Ndufv2 compared to WT-derived EVs, suggesting the involvement of Aß-processing complex and disease-associated/neurodegenerative microglia (DAM/MGnD) in EV secretion. In addition, Itgax and Apoe, DAM/MGnD markers, in EVs show a positive correlation with Itgax and Apoe mRNA expression from brain tissue in CAST.APP/PS1 mice. These datasets indicate the significant contribution of Aß plaque and neurodegeneration-induced DAM/MGnD microglia for EV secretion in CAST.APP/PS1 mice and shed light on understanding AD pathogenesis.

SIRT1 decelerates morphological processing of oligodendrocyte cell lines and regulates the expression of cytoskeleton-related oligodendrocyte proteins.

SIRT1 is involved in the regulation of a variety of biological processes such as metabolism, stress response, autophagy and differentiation. Although progenitor cells of oligodendrocytes (OPCs) express high level of SIRT1, its function on differentiation is unknown. Because we have shown that SIRT1 plays a pivotal role in differentiation of neural precursor cells, we hypothesized that SIRT1 may also participate in the differentiation of oligodendrocytes (OLGs). We examined whether SIRT1 was expressed in two human oligodendrocyte cell lines: KG-1-C and MO 3.13 OLG. Transfection of cell lines with SIRT1-siRNA and SIRT2-siRNA promoted the extension of cellular processes. SIRT1-siRNA and SIRT2-siRNA increased acetyl-α-tubulin level, conversely, over expression of SIRTs resulted in decreased the ratio of acetyl-α-tubulin to α-tubulin. We also found knockdown of SIRT1 and SIRT2 induced overexpression of ßIV-tubulin and tubulin polymerization promoting protein (TPPP) (OLG-specific cytoskeleton-related molecules) that distributed widely in cell bodies. Taken together, SIRT1 may play a role in oligodenroglial differentiation and myelinogenesis.

Different Antioxidative and Antiapoptotic Effects of Piceatannol and Resveratrol.

Resveratrol affords protection against reactive oxygen species (ROS)-related diseases via activation of SIRT1, an NAD+-dependent deacetylase. However, the low bioavailability of resveratrol limits its therapeutic applications. Since piceatannol is a hydroxyl analog of resveratrol with higher bioavailability, it could be an alternative to resveratrol. In this study, we compared the cytotoxicity, antioxidative activity, and mechanisms of cytoprotection of piceatannol with those of resveratrol. In C2C12 cells incubated with piceatannol, electrospray ionization mass spectrometry analysis showed that piceatannol was present in the intracellular fraction. A high concentration (50 µM) of piceatannol, but not resveratrol, induced mitochondrial depolarization and apoptosis. However, piceatannol at 10 µM inhibited the increase in mitochondrial ROS level induced by antimycin A, and this ROS reduction was greater than that by resveratrol. The reduction in hydrogen peroxide-induced ROS by piceatannol was also greater than that by resveratrol or vitamin C. Piceatannol reduced antimycin A-induced apoptosis more than did resveratrol. SIRT1 knockdown abolished the antiapoptotic activity of resveratrol, whereas it blocked only half of the antiapoptotic activity of piceatannol. Piceatannol, but not resveratrol, induced heme oxygenase-1 (HO1) expression, which was blocked by knockdown of the transcription factor NRF2, but not by SIRT1 knockdown. HO1 knockdown partially blocked the reduction of ROS by piceatannol. Furthermore, the antiapoptotic action of piceatannol was abolished by HO1 knockdown. Our results suggest that the therapeutic dose of piceatannol protects cells against mitochondrial ROS more than does resveratrol via SIRT1- and NRF2/HO1-dependent mechanisms. The activation of NRF2/HO1 could be an advantage of piceatannol compared with resveratrol for cytoprotection. SIGNIFICANCE STATEMENT: This study showed that piceatannol and resveratrol were different in cytotoxicity, oxidant-scavenging activities, and mechanisms of cytoprotection. Protection by piceatannol against apoptosis induced by reactive oxygen species was superior to that by resveratrol. In addition to the sirtuin 1-dependent pathway, piceatannol exerted nuclear factor erythroid 2-related factor 2/heme oxygenase-1-mediated antioxidative and antiapoptotic effects, which could be an advantage of piceatannol compared with resveratrol.

Aging-associated inflammation and fibrosis in arachnoid membrane.

BACKGROUND: The physiological and pathological significance of the arachnoid membrane (AM) is still unknown. In this study, we investigated various characteristics of the AM, focusing on the influence of inflammation and fibrosis. METHODS: Small pieces of AM sample were obtained during neurosurgical procedures from 74 cases. The clinical and pathological characteristics of the hyperplastic AM group (≥ 50 µm) and the non-hyperplastic AM group (< 50 µm) were compared. Then, potential correlations between AM thickness and clinical characteristics were analyzed. Moreover, VEGFα, TGFß, and TGFα levels were quantitated by real time PCR. Then, the potential correlations between AM thickness and these inflammatory or anti-inflammatory markers, and the influence of the original disease were calculated. RESULTS: The median age of the patients in hyperplastic AM group was significantly older than that of the non-hyperplastic AM group. Moreover, the number of fibroblasts, CD68+ cells, CD86+ cells, and CD206+ cells in the hyperplastic AM group was significantly higher than that in the non-hyperplastic AM group. The AM thickness was significantly correlated to age and number of fibroblasts, CD68+ cells, CD86+ cells, and CD206+ cells. The thickness of the AM was significantly correlated to the messenger RNA expression levels of VEGFα (ρ = 0.337), and the VEGFα expression levels were significantly correlated with TGFß and TNFα. CONCLUSIONS: The AM hyperplasia was influenced by aging and could be a result of inflammation and fibrosis through cytokine secretion from the inflammatory cells and fibroblasts in the AM.

A novel lamin A/C gene mutation causing spinal muscular atrophy phenotype with cardiac involvement: report of one case.

BACKGROUND: Mutations of the lamin A/C gene have been associated with several diseases such as Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy and Charcot-Marie-Tooth disease, referred to as laminopathies. Only one report of spinal muscular atrophy and cardiomyopathy phenotype with lamin A/C gene mutations has been published. The concept that lamin A/C gene mutations cause spinal muscular atrophy has not been established. CASE PRESENTATION: We report a man aged 65 years who presented with amyotrophy of lower limbs, arrhythmia and cardiac hypofunction. He showed gait disturbance since childhood, and his family showed similar symptoms. Neurological and electrophysiological findings suggested spinal muscular atrophy type 3. Gene analysis of lamin A/C gene showed a novel nonsense mutation p.Q353X (c.1057C > T). Further investigations revealed that he and his family members had cardiac diseases including atrioventricular block. CONCLUSIONS: We report the first Japanese case of spinal muscular atrophy phenotype associated with lamin A/C mutation. When a patient presents a spinal muscular atrophy phenotype and unexplained cardiac disease, especially when the family history is positive, gene analysis of lamin A/C gene should be considered.

Transcriptional dysregulation of autophagy in the muscle of a mouse model of Duchenne muscular dystrophy.

It has been reported that autophagic activity is disturbed in the skeletal muscles of dystrophin-deficient mdx mice and patients with Duchenne muscular dystrophy (DMD). Transcriptional regulations of autophagy by FoxO transcription factors (FoxOs) and transcription factor EB (TFEB) play critical roles in adaptation to cellular stress conditions. Here, we investigated whether autophagic activity is dysregulated at the transcription level in dystrophin-deficient muscles. Expression levels of autophagy-related genes were globally decreased in tibialis anterior and soleus muscles of mdx mice compared with those of wild-type mice. DNA microarray data from the NCBI database also showed that genes related to autophagy were globally downregulated in muscles from patients with DMD. These downregulated genes are known as targets of FoxOs and TFEB. Immunostaining showed that nuclear localization of FoxO1 and FoxO3a was decreased in mdx mice. Western blot analyses demonstrated increases in phosphorylation levels of FoxO1 and FoxO3a in mdx mice. Nuclear localization of TFEB was also reduced in mdx mice, which was associated with elevated phosphorylation levels of TFEB. Collectively, the results suggest that autophagy is disturbed in dystrophin-deficient muscles via transcriptional downregulation due to phosphorylation-mediated suppression of FoxOs and TFEB.

[Clinical characteristics of seizure-predominant autoimmune encephalitis and utility of anti-neuronal antibody scores for early treatment].

We analyzed 20 patients diagnosed with autoimmune neurological diseases with seizure predominance. In these patients, we examined the usefulness of Antibody Prevalence in Epilepsy and Encephalopathy (APE2) score and Antibodies Contributing to Focal Epilepsy Signs and Symptoms (ACES) score in autoimmune encephalitis (AE) for facilitating early treatment. APE2 score was positive in 19 of 20 patients. ACES score was positive in 15 of 20 patients, and 4 of 5 of the patients with negative ACES score did not have AE. Comprehensive assessment including the use of the above scores is desirable in the early stage of AE.

SIRT1 in the cardiomyocyte counteracts doxorubicin-induced cardiotoxicity via regulating histone H2AX.

AIMS: Cardiotoxicity by doxorubicin predicts worse prognosis of patients. Accumulation of damaged DNA has been implicated in doxorubicin-induced cardiotoxicity. SIRT1, an NAD+-dependent histone/protein deacetylase, protects cells by deacetylating target proteins. We investigated whether SIRT1 counteracts doxorubicin-induced cardiotoxicity by mediating Ser139 phosphorylation of histone H2AX, a critical signal of the DNA damage response. METHODS AND RESULTS: Doxorubicin (5 mg/kg per week, x4) was administered to mice with intact SIRT1 (Sirt1f/f) and mice that lack SIRT1 activity in cardiomyocytes (Sirt1f/f;MHCcre/+). Reductions in left ventricular fractional shortening and ejection fraction by doxorubicin treatment were more severe in Sirt1f/f;MHCcre/+ than in Sirt1f/f. Myocardial expression level of type-B natriuretic peptide was 2.5-fold higher in Sirt1f/f;MHCcre/+ than in Sirt1f/f after doxorubicin treatment. Sirt1f/f;MHCcre/+ showed larger fibrotic areas and higher nitrotyrosine levels in the heart after doxorubicin treatment. Although doxorubicin-induced DNA damage evaluated by TUNEL staining was enhanced in Sirt1f/f;MHCcre/+, the myocardium from Sirt1f/f;MHCcre/+ showed blunted Ser139 phosphorylation of H2AX by doxorubicin treatment. In H9c2 cardiomyocytes, SIRT1 knockdown attenuated Ser139 phosphorylation of H2AX, increased DNA damage, and enhanced caspase-3 activation under doxorubicin treatment. Immunostaining revealed that acetylation level of H2AX at Lys5 was higher in hearts from Sirt1f/f;MHCcre/+. In H9c2 cells, acetyl-Lys5-H2AX level was increased by SIRT1 knockdown and reduced by SIRT1 overexpression. Ser139 phosphorylation in response to doxorubicin treatment was blunted in a mutant H2AX with substitution of Lys5 to Gln (K5Q) that mimics acetylated lysine compared with that in wild-type H2AX. Expression of K5Q-H2AX as well as S139A-H2AX, which cannot be phosphorylated at Ser139, augmented doxorubicin-induced caspase-3 activation. Treatment of mice with resveratrol, a SIRT1 activator, attenuated doxorubicin-induced cardiac dysfunction, which was associated with a reduction in acetyl-Lys5-H2AX level and a preserved phospho-Ser139-H2AX level. CONCLUSION: These findings suggest that SIRT1 counteracts doxorubicin-induced cardiotoxicity by mediating H2AX phosphorylation through its deacetylation in cardiomyocytes.

Activation of SIRT1 promotes membrane resealing via cortactin.

Muscular dystrophies are inherited myopathic disorders characterized by progressive muscle weakness. Recently, several gene therapies have been developed; however, the treatment options are still limited. Resveratrol, an activator of SIRT1, ameliorates muscular function in muscular dystrophy patients and dystrophin-deficient mdx mice, although its mechanism is still not fully elucidated. Here, we investigated the effects of resveratrol on membrane resealing. We found that resveratrol promoted membrane repair in C2C12 cells via the activation of SIRT1. To elucidate the mechanism by which resveratrol promotes membrane resealing, we focused on the reorganization of the cytoskeleton, which occurs in the early phase of membrane repair. Treatment with resveratrol promoted actin accumulation at the injured site. We also examined the role of cortactin in membrane resealing. Cortactin accumulated at the injury site, and cortactin knockdown suppressed membrane resealing and reorganization of the cytoskeleton. Additionally, SIRT1 deacetylated cortactin and promoted the interaction between cortactin and F-actin, thus possibly enhancing the accumulation of cortactin at the injury site. Finally, we performed a membrane repair assay using single fiber myotubes from control and resveratrol-fed mice, where the oral treatment with resveratrol promoted membrane repair ex vivo. These findings suggest that resveratrol promotes membrane repair via the SIRT1/cortactin axis.

Downregulation of IGFBP5 contributes to replicative senescence via ERK2 activation in mouse embryonic fibroblasts.

Insulin-like growth factor (IGF)-binding proteins (IGFBPs) are secretory proteins that regulate IGF signaling. In this study, we investigated the role of IGFBP5 in replicative senescence in embryonic mouse fibroblasts (MEFs). During passages according to the 3T3 method, MEFs underwent senescence after the 5th passage (P5) based on cell growth arrest, an increase in the number of cells positive for senescence-associated ß-galactosidase (SA-ß-GAL) staining, and upregulation of p16 and p19. In P8 MEFs, IGFBP5 mRNA level was markedly reduced compared with that in P2 MEFs. Downregulation of IGFBP5 via siRNA in P2 MEFs increased the number of SA-ß-GAL-positive cells, upregulated p16 and p19, and inhibited cell growth. Incubation of MEFs with IGFBP5 during serial passage increased the cumulative population doubling and decreased SA-ß-GAL positivity compared with those in vehicle-treated cells. IGFBP5 knockdown in P2 MEFs increased phosphorylation levels of ERK1 and ERK2. Silencing of ERK2, but not that of ERK1, blocked the increase in the number of SA-ß-GAL-positive cells in IGFBP5-knockdown cells. The reduction in the cell number and upregulation of p16 and p21 in IGFBP5-knockdown cells were attenuated by ERK2 knockdown. Our results suggest that downregulation of IGFBP5 during serial passage contributes to replicative senescence via ERK2 in MEFs.

Ubiquitin-dependent rapid degradation conceals a cell-protective function of cytoplasmic SIRT3 against oxidative stress.

SIRT3 is an NAD+-dependent protein deacetylase localized in mitochondria. Several studies reported localization of SIRT3 in the cytoplasm or nucleus, but data of these studies were not consistent. We detected expression of mitochondrial (SIRT3mt) and cytoplasmic (SIRT3ct) Sirt3 mRNAs in the mouse brain, and we also found SIRT3 immunostaining of mitochondria and cytoplasm in the brain and cultured neural cells. However, expression levels of SIRT3ct in COS cells transfected with SIRT3ct cDNA were much lower than those of SIRT3mt. We found that SIRT3ct but not SIRT3mt was promptly degraded by ubiquitin-dependent degradation, in which SIRT3ct degradation was mediated mainly by ubiquitination of NH2-terminal methionine and partly by that of lysine residues of SIRT3ct. SIRT3ct expression level was significantly enhanced by the treatment of cells with staurosporine or H2O2. H2O2 treatment promoted nuclear translocation of SIRT3ct and induced histone H3 deacetylation and superoxide dismutase 2 expression. Overexpression of SIRT3ct decreased cell death caused by H2O2 at levels similar to those achieved by overexpression of SIRT3mt. Knockdown of Sirt3 mRNA increased cell death caused by amyloid-ß (Aß), and overexpression of SIRT3ct suppressed the toxic function of Aß in PC12 cells. These results indicate that SIRT3ct promotes cell survival under physiological and pathological conditions.

Plaque associated microglia hyper-secrete extracellular vesicles and accelerate tau propagation in a humanized APP mouse model.

BACKGROUND: Recent studies suggest that microglia contribute to tau pathology progression in Alzheimer's disease. Amyloid plaque accumulation transforms microglia, the primary innate immune cells in the brain, into neurodegenerative microglia (MGnD), which exhibit enhanced phagocytosis of plaques, apoptotic neurons and dystrophic neurites containing aggregated and phosphorylated tau (p-tau). It remains unclear how microglia promote disease progression while actively phagocytosing pathological proteins, therefore ameliorating pathology. METHODS: Adeno-associated virus expressing P301L tau mutant (AAV-P301L-tau) was stereotaxically injected into the medial entorhinal cortex (MEC) in C57BL/6 (WT) and humanized APP mutant knock-in homozygote (AppNL-G-F) mice at 5 months of age. Mice were fed either chow containing a colony stimulating factor-1 receptor inhibitor (PLX5622) or control chow from 4 to 6 months of age to test the effect of microglia depletion. Animals were tested at 6 months of age for immunofluorescence, biochemistry, and FACS of microglia. In order to monitor microglial extracellular vesicle secretion in vivo, a novel lentiviral EV reporter system was engineered to express mEmerald-CD9 (mE-CD9) specifically in microglia, which was injected into the same region of MEC. RESULTS: Expressing P301L tau mutant in the MEC induced tau propagation to the granule cell layer of the hippocampal dentate gyrus, which was significantly exacerbated in AppNL-G-F mice compared to WT control mice. Administration of PLX5622 depleted nearly all microglia in mouse brains and dramatically reduced propagation of p-tau in WT and to a greater extent in AppNL-G-F mice, although it increased plaque burden and plaque-associated p-tau+ dystrophic neurites. Plaque-associated MGnD microglia strongly expressed an EV marker, tumor susceptibility gene 101, indicative of heightened synthesis of EVs. Intracortical injection of mE-CD9 lentivirus successfully induced microglia-specific expression of mE-CD9+ EV particles, which were significantly enhanced in Mac2+ MGnD microglia compared to Mac2- homeostatic microglia. Finally, consecutive intracortical injection of mE-CD9 lentivirus and AAV-P301L-tau into AppNL-G-F mice revealed encapsulation of p-tau in microglia-specific mE-CD9+ EVs as determined by super-resolution microscopy and immuno-electron microscopy. DISCUSSION: Our findings suggest that MGnD microglia hyper-secrete p-tau+ EVs while compacting Aß plaques and clearing NP tau, which we propose as a novel mechanistic link between amyloid plaque deposition and exacerbation of tau propagation in AppNL-G-F mice.

Transcriptional activation of NAD+-dependent protein deacetylase SIRT1 by nuclear receptor TLX.

An orphan nuclear receptor TLX is a transcriptional repressor that promotes the proliferation and self-renewal of neural precursor cells (NPCs). SIRT1, an NAD(+)-dependent protein deacetylase, is highly expressed in the NPCs and participates in neurogenesis. Here, we found that TLX colocalized with SIRT1 and knockdown of TLX by small interfering RNAs decreased SIRT1 levels in NPCs. TLX increased the SIRT1 expression by binding to the newly identified TLX-activating element in the SIRT1 gene promoter in HEK293 cells. Thus, TLX is an inducer of SIRT1 and may contribute to neurogenesis both as a transactivator and as a repressor.

SIRT1 deficiency interferes with membrane resealing after cell membrane injury.

Activation of SIRT1, an NAD+-dependent protein deacetylase, ameliorates muscular pathophysiology of δ-sarcoglycan-deficient TO-2 hamsters and dystrophin-deficient mdx mice. We found that SIRT1 was highly expressed beneath the cellular membranes of muscle cells. To elucidate functional roles of SIRT1 on muscles, skeletal muscle-specific SIRT1 knockout mice (SIRT1-MKO) were generated. SIRT1-MKO mice showed muscular pathology similar to mild muscular dystrophies with increased numbers of centrally nucleated small myofibers and decreased numbers of middle-sized (2000-3001 µm2) myofibers compared to those of wild-type (WT) mice. Accordingly, SIRT1-MKO mice showed significantly decreased exercise capacity in treadmill and inverted hanging tests with higher levels of serum creatine kinase activities compared with those in WT mice. Evans blue dye uptake after exercise was greater in the muscles of SIRT1-MKO than those of WT mice, suggesting membrane fragility in SIRT1-MKO mice. Because SIRT1 was dominantly localized beneath the membranes of muscular cells, SIRT1 may have a new role in the membranes. We found that levels of fluorescent FM1-43 dye intake after laser-induced membrane disruption in C2C12 cells were significantly increased by SIRT1 inhibitors or Sirt1-siRNA compared with those of control cells. Inhibition of SIRT1 or SIRT1-knockdown severely disturbed the dynamic aggregation of membrane vesicles under the injured site but did not affect expression levels of membrane repair proteins. These data suggested that SIRT1 had a critical role in the resealing of membrane-ruptured muscle cells, which could affect phenotypes of SIRT1-MKO mice. To our knowledge, this report is the first to demonstrate that SIRT1 affected plasma-membrane repair mechanisms.

Transplantation of Mesenchymal Stem Cells Improves Amyloid-ß Pathology by Modifying Microglial Function and Suppressing Oxidative Stress.

Mesenchymal stem cells (MSC) are increasingly being studied as a source of cell therapy for neurodegenerative diseases, and several groups have reported their beneficial effects on Alzheimer's disease (AD). In this study using AD model mice (APdE9), we found that transplantation of MSC via the tail vein improved spatial memory in the Morris water maze test. Using electron paramagnetic resonance imaging to evaluate the in vivo redox state of the brain, we found that MSC transplantation suppressed oxidative stress in AD model mice. To elucidate how MSC treatment ameliorates oxidative stress, we focused on amyloid-ß (Aß) pathology and microglial function. MSC transplantation reduced Aß deposition in the cortex and hippocampus. Transplantation of MSC also decreased Iba1-positive area in the cortex and reduced activated ameboid shaped microglia. On the other hand, MSC transplantation accelerated accumulation of microglia around Aß deposits and prompted microglial Aß uptake and clearance as shown by higher frequency of Aß-containing microglia. MSC transplantation also increased CD14-positive microglia in vivo, which play a critical role in Aß uptake. To confirm the effects of MSC on microglia, we co-cultured the mouse microglial cell line MG6 with MSC. Co-culture with MSC enhanced Aß uptake by MG6 cells accompanied by upregulation of CD14 expression. Additionally, co-culture of MG6 cells with MSC induced microglial phenotype switching from M1 to M2 and suppressed production of proinflammatory cytokines. These data indicate that MSC treatment has the potential to ameliorate oxidative stress through modification of microglial functions, thereby improving Aß pathology in AD model mice.

Early administration of galantamine from preplaque phase suppresses oxidative stress and improves cognitive behavior in APPswe/PS1dE9 mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a common neurodegenerative disease that progressively impairs memory and cognition. Deposition of amyloid-ß (Aß) peptides is the most important pathophysiological hallmark of AD. Oxidative stress induced by generation of reactive oxygen species (ROS) is a prominent phenomenon in AD and known to occur early in the course of AD. Several reports suggest a relationship between change in redox status and AD pathology including progressive Aß deposition, glial cell activation, and inflammation. Galantamine is an acetylcholinesterase inhibitor and has been reported to have an oxidative stress inhibitory function. In the present study, galantamine was administered orally to AD model mice from before the appearance of Aß plaques (preplaque phase), and in vivo change in redox status of the brain was measured using electron paramagnetic resonance (EPR) imaging. Administration of galantamine from the preplaque phase ameliorated memory decline in Morris water maze test and novel object recognition test. Monitoring of the redox status of the brain using EPR imaging showed that galantamine treatment improved the unbalanced redox state. Additionally, galantamine administration enhanced microglial function to promote Aß clearance, reducing the Aß-positive area in the cortex and amount of insoluble Aß in the brain. In contrast, galantamine treatment from the preplaque phase suppressed the production of proinflammatory cytokines through neurotoxic microglial activity. Therefore, galantamine administration from the preplaque phase may have the potential of clinical application for the prevention of AD. In addition, our results demonstrate the usefulness of EPR imaging for speedy and quantitative evaluation of the efficacy of disease-modifying drugs for AD.

CD14 and Toll-Like Receptor 4 Promote Fibrillar Aß42 Uptake by Microglia Through A Clathrin-Mediated Pathway.

We previously demonstrated that microglia play an essential role in clearance of amyloid-ß (Aß) in Alzheimer's disease (AD)-like pathology. Our prior work also showed that several receptors expressed on microglia participated in Aß phagocytosis. However, clathrin-mediated endocytosis (CME), which is associated with production and release of Aß in neurons, has received much less attention in the context of microglial Aß uptake. To elucidate the detailed mechanisms of microglial Aß uptake pathways, we focused on CD14 and Toll-like receptor 4 (TLR4), which have been shown to mediate fibrillar Aß1 - 42 (fAß42) phagocytosis in microglia. CD14 has also been known to control lipopolysaccharide-induced internalization of TLR4 in a clathrin-dependent manner. However, it remains unclear whether CD14 and TLR4 engage in CME in microglial fAß42 uptake, including whether CD14 interacts with TLR4 in the process. In the present study, we found that CD14-positive microglia increased in an age-dependent manner in the cortex of AD model mice. Immunostaining showed that CD14 interacted with TLR4 to internalize fAß42 in the mouse microglial cell line MG6. Knock-down of CD14 and TLR4 in MG6 cells significantly reduced intracellular fAß42, showing their involvement in fAß42 uptake. We also found that clathrin participated in fAß42 uptake by MG6 cells. Furthermore, CD14 and TLR4 mediated fAß42 uptake via clathrin-dependent mechanisms. These results indicate that CD14 and TLR4 participate not only in phagocytosis but also in clathrin-dependent fAß42 internalization in microglia. These findings provide novel molecular understanding of microglial fAß42 uptake, which could be of therapeutic relevance for AD.

Evaluation of Mitochondrial Oxidative Stress in the Brain of a Transgenic Mouse Model of Alzheimer's Disease by in vitro Electron Paramagnetic Resonance Spectroscopy.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases responsible for progressive dementia. Deposition of amyloid-ß (Aß) in the brain is the most important pathophysiological hallmark of AD. In addition, recent evidence indicates that reactive oxygen species (ROS) derived from mitochondria contribute to progression of AD pathology. We thus hypothesized that Aß accumulates and oxidative stress increases in the brain mitochondria of a transgenic mouse model of AD (APdE9). We measured the quantity of Aß and the activity of the antioxidant enzyme superoxide dismutase (SOD) in brain mitochondrial fractions prepared from APdE9 and wild-type (WT) mice aged 6, 9, 15, and 18 months. We also quantified the age-related changes in redox status in the mitochondrial fractions obtained from both APdE9 and WT mouse brains by electron paramagnetic resonance (EPR) spectrometry using a paramagnetic nitroxide "Mito-Tempo" [(2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride monohydrate] as a mitochondria-targeted redox-sensitive probe. In APdE9 mice, Aß accumulated in brain mitochondria earlier than in the non-mitochondrial fraction of the brain. Furthermore, increased oxidative stress was demonstrated in brain mitochondria of APdE9 mice by in vitro SOD assay as well as EPR spectroscopy. EPR combined with a mitochondria-targeted redox-sensitive nitroxide probe is a potentially powerful tool to elucidate the etiology of AD and facilitate the development of new therapeutic strategies for AD.

Role of Suppressor of Cytokine Signaling 3 (SOCS3) in Altering Activated Microglia Phenotype in APPswe/PS1dE9 Mice.

In response to changes of the central nervous system environment, microglia are capable of acquiring diverse phenotypes for cytotoxic or immune regulation and resolution of injury. Alzheimer's disease (AD) pathology also induces several microglial activations, resulting in production of pro-inflammatory cytokines and reactive oxygen species or clearance of amyloid-ß (Aß) through phagocytosis. We previously demonstrated that microglial activation and increase in oxidative stress started from the middle age in APPswe/PS1dE9 mice, and hypothesized that M1 activation occurs in middle-aged AD mice by Aß stimulation. In the present study, we analyzed in vivo expressions of pro-inflammatory cytokines (M1 microglial markers), M2 microglial markers, and suppressor of cytokine signaling (SOCS) family, and examined the microglial phenotypic profile in APPswe/PS1dE9 mice. Then we compared the in vitro gene expression patterns of Aß- and lipopolysaccharide (LPS)-stimulated primary-cultured microglia. Microglia in APPswe/PS1dE9 mice exhibited an M1-like phenotype, expressing tumor necrosis factor α (TNFα) but not interleukin 6 (IL6). Aß-stimulated primary-cultured microglia also expressed TNFα but not IL6, whereas LPS-stimulated primary-cultured microglia expressed both pro-inflammatory cytokines. Furthermore, both microglia in APPswe/PS1dE9 mice and Aß-stimulated primary-cultured microglia expressed SOCS3. Reduction of SOCS3 expression in Aß-challenged primary-cultured microglia resulted in upregulation of IL6 expression. Our findings indicate that SOCS3 suppresses complete polarization to M1 phenotype through blocking IL6 production, and Aß-challenged primary-cultured microglia replicate the in vivo gene expression pattern of microglia in APPswe/PS1dE9 mice. Aß may induce the M1-like phenotype through blocking of IL6 by SOCS3.