Repositioning of Anti-Diabetic Drugs against Dementia: Insight from Molecular Perspectives to Clinical Trials.

Insulin resistance as a hallmark of type 2 DM (T2DM) plays a role in dementia by promoting pathological lesions or enhancing the vulnerability of the brain. Numerous studies related to insulin/insulin-like growth factor 1 (IGF-1) signaling are linked with various types of dementia. Brain insulin resistance in dementia is linked to disturbances in Aß production and clearance, Tau hyperphosphorylation, microglial activation causing increased neuroinflammation, and the breakdown of tight junctions in the blood-brain barrier (BBB). These mechanisms have been studied primarily in Alzheimer's disease (AD), but research on other forms of dementia like vascular dementia (VaD), Lewy body dementia (LBD), and frontotemporal dementia (FTD) has also explored overlapping mechanisms. Researchers are currently trying to repurpose anti-diabetic drugs to treat dementia, which are dominated by insulin sensitizers and insulin substrates. Although it seems promising and feasible, none of the trials have succeeded in ameliorating cognitive decline in late-onset dementia. We highlight the possibility of repositioning anti-diabetic drugs as a strategy for dementia therapy by reflecting on current and previous clinical trials. We also describe the molecular perspectives of various types of dementia through the insulin/IGF-1 signaling pathway.

Melatonin and Exercise Counteract Sarcopenic Obesity through Preservation of Satellite Cell Function.

Sarcopenic obesity (SO) is characterized by atrophic skeletal muscle impairment (sarcopenia) and obesity, which is associated with adverse outcomes of morbidity and mortality in elderly people. We investigated the effects of melatonin and exercise training on SO in 32-week-old senescence-accelerated mouse-prone-8 (SAMP8) mice fed a normal diet or a high-fat diet for 16 weeks. Melatonin, exercise, or melatonin and exercise for 8 weeks displayed reductions in the SO-induced impairment of skeletal muscle function and atrophy. Specifically, a decrease in mitochondrial calcium retention capacity in skeletal muscles observed in the HFD-con group was attenuated in melatonin and/or exercise intervention groups. More importantly, HFD-con mice displayed a lower number of Pax7+ satellite cells (SCs) and higher expression of p16ink than P8ND mice, which were attenuated by melatonin and/or exercise interventions. The cellular senescence in SC-derived primary myoblasts from HFD-con mice was significantly attenuated in myoblasts from the melatonin and/or exercise groups, which was reproduced in a senescence model of H2O2-treated C2C12 myoblasts. Our results suggest that melatonin and exercise training attenuate SO-induced skeletal muscle dysfunction, at least in part, through preserving the SC pool by inhibiting cellular senescence and attenuating mitochondrial dysfunction.

Circulating micro-RNAs Differentially Expressed in Korean Alzheimer's Patients With Brain Aß Accumulation Activate Amyloidogenesis.

BACKGROUND: Roles for extracellular vesicles (EVs) enriched with micro-RNAs (miRNAs) have been proposed in Alzheimer's disease (AD) pathogenesis, leading to the discovery of blood miRNAs as AD biomarkers. However, the diagnostic utility of specific miRNAs is not consistent. This study aimed to discover blood miRNAs that are differentially expressed in Korean AD patients, evaluate their clinical performance, and investigate their role in amyloidogenesis. METHODS: We discovered miRNAs differentially expressed in AD (N = 8) from cognitively normal participants (CN, N = 7) or Parkinson's disease (PD) patients (N = 8). We evaluated the clinical performance of these miRNAs in plasma of subgroup (N = 99) and in plasma EVs isolated from the total cohort (N = 251). The effects of miRNAs on amyloidogenesis and on the regulation of their target genes were investigated in vitro. RESULTS: Among 17 upregulated and one downregulated miRNAs in AD (>twofold), miR-122-5p, miR-210-3p, and miR-590-5p were differentially expressed compared with CN or PD. However, the diagnostic performance of the selected plasma or EV miRNAs in total participants were limited (area under the curve < 0.8). Nevertheless, levels of 3 miRNAs in plasma or plasma EVs of participants who were amyloid positron emission tomography (Aß-PET) positive were significantly higher than those from the Aß-PET negative participants (p < .05). The selected miRNAs induced Aß production (p < .05) through activation of ß-cleavage of amyloid precursor protein (CTF-ß; p < .01), and downregulated their target genes (ADAM metallopeptidase domain 10, Brain-derived neurotrophic factor, and Jagged canonical notch ligand 1; p < .05), which was further supported by pathway enrichment analysis of target genes of the miRNAs. CONCLUSION: In conclusion, despite of the limited diagnostic utility of selected miRNAs as plasma or plasma EV biomarkers, the discovered miRNAs may play a role in amyloidogenesis during AD onset and progression.

Alzheimer's cerebrospinal biomarkers from Lumipulse fully automated immunoassay: concordance with amyloid-beta PET and manual immunoassay in Koreans : CSF AD biomarkers measured by Lumipulse in Koreans.

BACKGROUND: Alzheimer's disease (AD) cerebrospinal fluid (CSF) biomarker cutoffs from immunoassays with low interlaboratory variability in diverse ethnic groups are necessary for their use in clinics and clinical trials. With lack of cutoffs from fully automated immunoassay platforms in diverse races, the aim of this study is to evaluate the clinical utility of CSF AD biomarkers from the Lumipulse fully automated immunoassay based on ß-amyloid (Aß) positron emission tomography (PET) status comparing with these from two manual immunoassays, in Koreans. METHODS: Among 331 Korean participants enrolled from a prospective, 3-year longitudinal observational study of the validation cohort of Korean Brain Aging Study for the Early Diagnosis and Prediction of AD, 139 (29 CN, 58 SCD, 29 MCI, and 23 AD) provided CSF and 271 underwent baseline amyloid PET (n = 128 with overlapping CSF and Aß-PET, and 143 without CSFs). Three annual cognitive and neuropsychiatric function tests were conducted. Aß42, Aß40, total-tau, and phosphorylated-tau181 were measured by Lumipulse fully automated immunoassay and two manual immunoassays (INNO-BIA AlzBio3, INNOTEST). Clinical utility of CSF biomarker cutoffs, based on 128 participants with Aß-PET, was evaluated. RESULTS: Cognitive and neuropsychological scores differed significantly among the groups, with descending performance among CN>SCD>MCI>AD. Biomarker levels among immunoassays were strongly intercorrelated. We determined the Aß-PET status in a subgroup without CSF (n = 143), and then when we applied CSF biomarker cutoffs determined based on the Aß-PET status, the CSF biomarkers (cutoffs of 642.1 pg/mL for Aß42, 0.060 for Aß42/Aß40, 0.315 for t-tau/Aß42, and 0.051 for p-tau/Aß42, respectively) showed good agreement with Aß-PET (overall AUC ranges of 0.840-0.898). Use of the Aß-PET-based CSF cutoffs showed excellent diagnostic discrimination between AD and CN (Aß42, Aß42/Aß40, t-tau/Aß42, and p-tau/Aß42) with overall AUC ranges of 0.876-0.952. During follow-up, participants with AD-like CSF signature determined by Aß-PET-based cutoffs from Lumipulse showed rapid progression of cognitive decline in 139 subjects, after adjustment for potential confounders, compared with those with a normal CSF signature. CONCLUSION: CSF AD biomarkers measured by different immunoassay platforms show strong intercorrelated agreement with Aß-PET in Koreans. The Korean-specific Aß-PET-based CSF biomarker cutoffs measured by the Lumipulse assay strongly predicts progression of cognitive decline. The clinical utility of CSF biomarkers from fully-automated immunoassay platforms should be evaluated in larger, more diverse cohorts.

Sirtuin 1-dependent regulation of high mobility box 1 in hypoxia-reoxygenated brain microvascular endothelial cells: roles in neuronal amyloidogenesis.

Hypoxia-reperfusion injury is one of the major risk factors for neurodegeneration. However, it is unclear whether ischaemic damage in brain microvascular endothelial cells plays roles in neurodegeneration, particularly in the amyloidogenic changes contributing to the development of Alzheimer's disease (AD) pathologies. Therefore, we investigated the roles of hypoxia-reoxygenation (H/R)-induced release of high mobility group box protein 1 (HMGB1), a risk molecule for AD pathogenesis in the ischaemic damaged brain, from human brain microvascular endothelial cells (HBMVECs) in neuronal amyloid-beta (Aß) production. H/R increased nuclear-cytosolic translocation and secretion of HMGB1 in HBMVECs, along with increased permeability and HMGB1-dependent p-c-Jun activation. In addition, H/R increased the expression of Sirtuin 1 (Sirt1), coincident with an increase of intracellular Sirt1-HMGB1 binding in HBMVECs. H/R increased the acetylation of HMGB1 and extracellular secretion, which was significantly inhibited by Sirt1 overexpression. Furthermore, Sirt1 contributed to autophagy-mediated endogenous HMGB1 degradation. More importantly, treatment of neuronal cells with conditioned medium from H/R-stimulated HBMVECs (H/R-CM) activated their amyloidogenic pathways. The neuronal amyloidogenic changes (i.e. increased levels of extracellular Aß40 and Aß42) by H/R-CM from HBMVECs were further increased by Sirt1 inhibition, which was significantly suppressed by neutralization of the HMGB1 in H/R-CM. Collectively, our results suggest that HMGB1 derived from H/R-stimulated HBMVECs contributes to amyloidogenic pathways in neurons playing roles in the pathogenesis of AD, which are regulated by endothelial Sirt1.

Effects of Aerobic Exercise on Tau and Related Proteins in Rats with the Middle Cerebral Artery Occlusion.

Although Alzheimer's disease (AD)-like pathology is frequently found in patients with post-stroke dementia, little is known about the effects of aerobic exercise on the modifications of tau and related proteins. Therefore, we evaluated the effects of aerobic exercise on the phosphorylation and acetylation of tau and the expressions of tau-related proteins, after middle cerebral artery occlusion (MCAO) stroke. Twenty-four Sprague-Dawley rats with MCAO infarction were used in this study. The rehabilitation group (RG) received treadmill training 40 min/day for 12 weeks, whereas the sedentary group (SG) did not receive any type of training. Functional tests, such as the single pellet reaching task, rotarod, and radial arm maze tests, were performed monthly for 3 months. In ipsilateral cortices in the RG and SG groups, level of Ac-tau was lower in the RG, whereas levels of p-tauS396, p-tauS262, and p-tauS202/T205 were not significantly lower in the RG. Level of phosphorylated glycogen synthase kinase 3-beta Tyr 216 (p-GSK3ßY216) was lower in the RG, but levels of p-AMPK and phosphorylated glycogen synthase kinase 3-beta Ser 9 (p-GSK3ßS9) were not significantly lower. Levels of COX-2 and BDNF were not significantly different between the two groups, while SIRT1 significantly decreased in ipsilateral cortices in RG. In addition, aerobic training also improved motor, balance, and memory functions. Rehabilitation with aerobic exercise inhibited tau modification, especially tau acetylation, following infarction in the rat MCAO model, which was accompanied with the improvement of motor and cognitive functions.

Effects of Aerobic Exercise on Tau and Related Proteins in Rats with Photochemically-Induced Infarction.

BACKGROUND: Recent evidence indicates brain ischemia is associated with accumulations of abnormal tau and related proteins. However, the effects of aerobic training on these proteins have not been evaluated. OBJECTIVE: We aimed to evaluate the effect of aerobic exercise on the phosphorylation and acetylation of tau and on the expressions of tau related proteins in a rat stroke model and to compare the effects of aerobic exercise with those observed in our previous study on task specific training (TST). METHODS: Twenty-four Sprague- Dawley rats with photothrombotic cortical infarction were used in the current study. The rehabilitation group (RG) received treadmill training 40 min/day for 28 days, whereas the sedentary group (SG) did not receive any type of training. Functional tests such as the single pellet reaching task, rotarod, and radial arm maze tests were performed weekly for 4 weeks post-infarction. RESULTS: Levels of p-taus396 and p-AMPK were found to be lower in ipsilateral cortices in the RG than in the SG (p < 0.05). Levels of p-taus262, Ac-tau, p-GSK3ßS9, p-Akt, p-Sin1, and p-P70-S6K were significantly lower in ipsilateral than in contralateral cortices in the RG (p < 0.05). Aerobic training also improved motor, balance, and memory functions. CONCLUSION: Aerobic training inhibited the phosphorylation and acetylation of tau and modulated the expressions of tau related proteins after stroke by modifying the p70-S6K pathway and p-AMPK. By comparison with our previous study on the effects of TST, we have evidence to suggest that TST and aerobic exercise differ, although both types of rehabilitation inhibit tau phosphorylation and acetylation.

Experimental Models of Sarcopenia: Bridging Molecular Mechanism and Therapeutic Strategy.

Sarcopenia has been defined as a progressive decline of skeletal muscle mass, strength, and functions in elderly people. It is accompanied by physical frailty, functional disability, falls, hospitalization, and mortality, and is becoming a major geriatric disorder owing to the increasing life expectancy and growing older population worldwide. Experimental models are critical to understand the pathophysiology of sarcopenia and develop therapeutic strategies. Although its etiologies remain to be further elucidated, several mechanisms of sarcopenia have been identified, including cellular senescence, proteostasis imbalance, oxidative stress, and "inflammaging." In this article, we address three main aspects. First, we describe the fundamental aging mechanisms. Next, we discuss both in vitro and in vivo experimental models based on molecular mechanisms that have the potential to elucidate the biochemical processes integral to sarcopenia. The use of appropriate models to reflect sarcopenia and/or its underlying pathways will enable researchers to understand sarcopenia and develop novel therapeutic strategies for sarcopenia. Lastly, we discuss the possible molecular targets and the current status of drug candidates for sarcopenia treatment. In conclusion, the development of experimental models for sarcopenia is essential to discover molecular targets that are valuable as biochemical biomarkers and/or therapeutic targets for sarcopenia.

Enrichment of Exosome-Like Extracellular Vesicles from Plasma Suitable for Clinical Vesicular miRNA Biomarker Research.

Exosome-like extracellular vesicles (ELVs) contain biomolecules that have potential as diagnostic biomarkers, such as proteins, micro-RNAs (miRNAs), and lipids. However, it is difficult to enrich ELVs consistently with high yield and purity from clinical samples, which hampers the development of ELV biomarkers. This is particularly true for miRNAs in protein-rich plasma. Hence, we modified ELV isolation protocols of three commercially available polymer-precipitation-based kits using proteinase K (PK) treatment to quantify ELV-associated miRNAs in human plasma. We compared the yield, purity, and characteristics of enriched plasma ELVs, and measured the relative quantity of three selected miRNAs (miR-30c, miR-126, and miR-192) in ELVs using six human plasma samples. Compared with the original protocols, we demonstrated that ELVs can be isolated with PK treatment with high purity (i.e., lack of non-exosomal proteins and homogeneous size of vesicles) and yield (i.e., abundancy of exosomal markers), which were dependent on kits. Using the kit with the highest purity and yield with PK treatment, we successfully quantified ELV miRNAs (levels of 45%-65% in total plasma) with acceptable variability. Collectively, ELV enrichment using the modified easy-to-use method appears suitable for the analysis of miRNAs, although its clinical applicability needs to be confirmed in larger clinical studies.

In vivo bone regeneration by differently designed titanium membrane with or without surface treatment: a study in rat calvarial defects.

The current objective was to evaluate six groups of titanium membranes in a rat calvarial defect model, regarding the surface treatment with or without calcium-phosphate coating and surface topography with no, small, or large holes. Critical size defects (Ф = 8 mm, n = 42) were surgically created in rat calvaria, and then were treated by one of the six groups. Biopsies were obtained at 4 weeks (n = 5 per group) for micro-computed tomography and histomorphometric analyses. Fluorochrome bone markers were injected in two rats each group at 1 (Alizarin red), 3 (Calcein green) and 5 weeks (Oxytetracyclin yellow), followed by histological examination at 7 weeks to assess bone regeneration dynamic. At 4 weeks, the highest bone volume was observed in no-hole groups independent of surface treatment (p < 0.05). Treated groups with no-hole and large-hole membranes showed increased bone mineral density than with respective non-treated groups (p < 0.05). Histology exhibited an intimate bone formation onto the treated membranes, whereas non-treated ones demonstrated interposition of connective tissue, which was confirmed through bone contact percentages. The results suggest that occlusive membranes showed more bone formation than other perforated ones, and calcium-phosphate treatment induces intimate bone formation toward the membrane.

Roles of myokines in exercise-induced improvement of neuropsychiatric function.

Exercise is a well-known non-pharmacological intervention to improve brain functions, including cognition, memory, and motor coordination. Contraction of skeletal muscles during exercise releases humoral factors that regulate the whole-body metabolism via interaction with other non-muscle organs. Myokines are muscle-derived effectors that regulate body metabolism by autocrine, paracrine, or endocrine action and were reportedly suggested as "exercise factors" that can improve the brain function. However, several aspects remain to be elucidated, namely the specific activities of myokines related to the whole-body metabolism or brain function, the mechanisms of regulation of other organs or cells, the sources of "exercise factors" that regulate brain function, and their mechanisms of interaction with non-muscle organs. In this paper, we present the physiological functions of myokines secreted by exercise, including regulation of the whole-body metabolism by interaction with other organs and adaptation of skeletal muscles to exercise. In addition, we discuss the functions of myokines that possibly contribute to exercise-induced improvement of brain function. Among several myokines, brain-derived neurotrophic factor (BDNF) is the most studied myokine that regulates adult neurogenesis and synaptic plasticity. However, the source of circulating BDNF and its upstream effector, insulin-like growth factor (IGF-1), and irisin and the effect size of peripheral BDNF, irisin, and IGF-1 released after exercise should be further investigated. Recently, cathepsin B has been reported to be secreted from skeletal muscles and upregulate BDNF following exercise, which was associated with improved cognitive function. We reviewed the level of evidence for the effect of myokine on the brain function. Level of evidence for the association of the change in circulating myokine following exercise and improvement of neuropsychiatric function is lower than the level of evidence for the benefit of exercise on the brain. Therefore, more clinical evidences for the association of myokine release after exercise and their effect on the brain function are required. Finally, we discuss the effect size of the action of myokines on cognitive benefits of exercise, in addition to other contributors, such as improvement of the cardiovascular system or the effect of "exercise factors" released from non-muscle organs, particularly in patients with sarcopenia.

Lactate Overload Inhibits Myogenic Activity in C2C12 Myotubes.

Lactate (LA), an endogenous metabolite produced from pyruvate, can accumulate in skeletal muscle in certain conditions including major diseases, as well as during intensive exercise. Using differentiated C2C12 myotubes, we evaluated the early (1-h) and delayed (24-h) effects of LA (8 mM) on mechanisms involved in myogenesis or muscle atrophy, including 5'-adenosine monophosphate-activated protein kinase (AMPK)-mediated inhibition of protein synthesis through the mTOR/P70-S6K pathway, Akt-mediated inhibition of expression of the MAFbx atrophic factor by FOXO3a and expression of the myogenic transcription factors, MyoD, myogenin and myosin heavy chain. Although the early effects of LA overload were not significant on myogenic or atrophic mechanisms, LA treatment for 24 h significantly activated atrophic mechanisms but suppressed myogenesis in myotubes. In addition, LA overload for 24 h significantly suppressed the expression of Sirtuin 1 and peroxisome proliferator-activated receptor gamma coactivator-1 alpha. Consistent with LA-induced activation of atrophic mechanisms, the diameter of C2C12 myotubes treated with LA for 24 h, but not for 1 h, was significantly lower than in control myotubes. Thus, a sustained, but not a transient, LA overload could induce muscle atrophy through the regulation of AMPK- and Akt-mediated pathways, although further in vivo studies are needed to confirm this.

Roles of Exosome-Like Vesicles Released from Inflammatory C2C12 Myotubes: Regulation of Myocyte Differentiation and Myokine Expression.

BACKGROUND/AIMS: The complicated differentiation processes of cells in skeletal muscle against inflammation that induce muscle atrophy are not fully elucidated. Given that skeletal muscle is a secretory organ, we evaluated the effects of inflammation on myogenic signals and myokine expression, and the roles of inflammatory exosomes released by myotubes in myogenic differentiation. METHODS: Inflammation was induced by treatment of fully differentiated C2C12 myotubes with a cytokine mixture of TNF-α and INF-γ. Exosome-like vesicles (ELVs) were isolated from conditioned media of control or inflamed myotubes and incubated with myoblasts. The expression of molecular switches that contribute to myogenic differentiation, including several kinases, their downstream targets, and myokines, were evaluated using immunoblot analysis in inflamed myotubes and in myoblasts treated with ELVs. RESULTS: Inflammation activated molecular mechanisms contributing to muscle atrophy, including AMPK, p-38 MAPK and JNK, while inhibiting Akt-mediated myogenic signals. In addition, inflammation induced myostatin expression with suppression of a myostatin-counteracting myokine, decorin. Well-characterized ELVs released from inflamed myotubes induced myoblast inflammation and inhibited myogenic mechanisms while stimulating atrophic signals. CONCLUSION: Inflammation of skeletal muscle induces muscle atrophy via multiple mechanisms, including the regulation of myokines and kinases. Inflammatory ELVs are likely to contribute to inflammation-induced muscle atrophy.

High Hydrostatic Pressure Extract of Ginger Exerts Antistress Effects in Immobilization-Stressed Rats.

Stress contributes to physiological changes such as weight loss and hormonal imbalances. The aim of the present study was to investigate antistress effects of high hydrostatic pressure extract of ginger (HPG) in immobilization-stressed rats. Male Sprague-Dawley rats (n = 24) were divided into three groups as follows: control (C), immobilization stress (2 h daily, for 2 weeks) (S), and immobilization stress (2 h daily, for 2 weeks) plus oral administration of HPG (150 mg/kg body weight/day) (S+G). Immobilization stress reduced the body weight gain and thymus weight by 50.2% and 31.3%, respectively, compared to the control group. The levels of serum aspartate transaminase, alanine transaminase, and corticosterone were significantly higher in the stress group, compared to the control group. Moreover, immobilization stress elevated the mRNA levels of tyrosine hydroxylase (Th), dopamine beta-hydroxylase (Dbh), and cytochrome P450 side-chain cleavage (P450scc), which are related to catecholamine and corticosterone synthesis in the adrenal gland. HPG administration also increased the body weight gain and thymus weight by 12.7% and 16.6%, respectively, compared to the stress group. Furthermore, the mRNA levels of Th, Dbh, phenylethanolamine-N-methyltransferase, and P450scc were elevated by the HPG treatment when compared to the stress group. These results suggest that HPG would have antistress effects partially via the reversal of stress-induced physiological changes and suppression of mRNA expression of genes related to corticosterone and catecholamine synthetic enzymes.

Effects of task-specific rehabilitation training on tau modification in rat with photothrombotic cortical ischemic damage.

Although stroke elicits progressive cognitive decline and is a leading cause of dementia, molecular interplay between stroke and Alzheimer's disease (AD) pathology has not been fully elucidated. Furthermore, studies on the effects of post-stroke rehabilitation on AD pathology are limited. We evaluated the acute effect of stroke on tau modification, and the molecular effects of task-specific training (TST) on tau modification using a model of photochemically-induced thrombosis (PIT)-induced cortical infarction. Following PIT in the dominant side of sensorimotor cortex, the rehabilitation group received 4-weeks of TST rehabilitation once daily by single pellet reaching training, whereas the sedentary control group did not received any type of training. Cortical expression levels of proteins related to tau modification were evaluated on post-stroke day 1 (PSD1) and 28; functional tests were also evaluated performed every week. The expression levels of acetyl-tau, phosphorylated-tau (p-tau), cyclooxygenase-2 and Akt-mTORC1-p70S6K pathway in infarcted cortices on PSD1 were significantly greater, whereas the expression levels of p-AMPK were significantly lower than in the paired contralateral sides. TST rehabilitation for 4 weeks greatly improved functional motor performance but not memory, which concurred with the down-regulations of ipsilateral p-AMPK, cyclooxygenase-2, Akt-mTORC1-p70S6K pathway, and p-tau in rehabilitation group. PIT-induced cortical infarction was found to induce cortical tau modification through the Akt-mTORC1-p70S6K activation, and to suppress the expression of AMPK-related proteins. TST rehabilitation greatly improved motor function, but not memory, and suppressed p-tau expression and neuroinflammation. Nevertheless, the role of TST-mediated regulation of tau hyperphosphorylation required further clarification.

Chronological changes in the expression of phosphorylated tau and 5­AMP­activated protein kinase in the brain of senescence­accelerated P8 mice.

Senescence-accelerated mouse prone 8 (SAMP8), a non­transgenic animal model used for researching sporadic Alzheimer's disease (AD), presents AD pathologies and overall dysregulation in brain energy metabolism, which is one of the early pathogenic characteristics of AD. In the present study, the authors examined chronological changes in the expression patterns of phosphorylated tau and of proteins related to energy metabolism to evaluate the association of tau phosphorylation and metabolism, using young­ (2­months­old), middle­ (5­months­old) and old­aged (10­months­old) SAMP8. The levels of phosphorylated 5'­AMP activated protein kinase at Thr172 (p­AMPK) and phosphorylated glycogen synthase kinase 3ß (p­GSK3ßS9) in the cortex of SAMP8 at 2 months were significantly higher than those in senescence­accelerated mouse resistant 1 (SAMR1). The differences were not detected at 5 and 10 months of age, which were concurrent with the changes in levels of phosphorylated tau at Ser396 (p­tauS396), but not with p­tauS262. The level of p­tauS262 was considerably higher in the cortex of middle­aged SAMP8 when compared with that of SAMR1 and sustained in old­aged SAMP8, but not in the young cortex. The levels of cortical sirtuin1 (Sirt1) and insulin receptor substrate 1 (IRS­1) expression of young SAMP8 were significantly lower, when compared with those in SAMR1. However, in the hippocampus of SAMP8, the patterns of chronological changes and levels of p­tau, p­AMPK, Sirt1 and IRS­1 relative to SAMR1 were different from those in the cortex. Taken together, the results suggested that regulation of tau phosphorylation via the AMPK­GSK3ß pathway concurrent with dysregulation of energy metabolism may precede the pathological tau hyperphosphorylation in the cortex of SAMP8, and that the regulation of AMPK­GSK3ß­mediated tau phosphorylation may be dependent on phosphor­epitope in tau or the region of brain.

Comparison of Guided Bone Regeneration Between Surface-Modified and Pristine Titanium Membranes in a Rat Calvarial Model.

PURPOSE: The objectives of this study were to evaluate bioactivity of a titanium membrane with anodization, cyclic precalcification, and heat (APH) treatment (APHTM), and to compare APHTM and nontreated titanium membrane (NTTM) in guided bone regeneration using histologic analysis and microcomputed tomography (micro-CT). MATERIALS AND METHODS: APHTM samples were prepared and immersed in simulated body fluid for 2 days, then observed using field-emission scanning electron microscopy, followed by an analysis of calcium and phosphate precipitation using an energy dispersive x-ray spectroscopy. For the in vivo experiment, critical-size defects were created in rat calvaria (diameter, 8 mm) and treated with either APHTM or NTTM (n = 14 each). Biopsies were performed at 2 and 4 weeks for histologic analysis (n = 3 per group each time). Fluorochrome bone markers were injected in three rats in each group at 3 (alizarin red) and 5 weeks (calcein green), followed by histologic examination at 7 weeks. Micro-CT was performed at 8 weeks (n = 5 per group). RESULTS: APHTM exhibited high bioactivity, characterized by dense nano-sized flakelike crystals throughout the membrane and an increase in the calcium-phosphate concentrations after 2-day immersion in simulated body fluid. At 2 and 4 weeks, APHTM samples showed an intimate bone formation onto the membrane, whereas NTTM samples demonstrated interposition of connective tissue between the membrane and newly formed bone. The same pattern was found in the fluorescent study. The micro-CT analysis revealed significantly lower bone volume but higher bone mineral density in the APHTM samples than in the NTTM samples (P < .05). CONCLUSION: The results suggest that APH treatment on titanium membrane promotes intimate bone formation toward the membrane, thus increasing structural durability for bone regeneration. Further research is warranted to confirm the results found in these in vitro and in vivo experiments.

Effects of Eicosapentaenoic Acid and Docosahexaenoic Acid on Mitochondrial DNA Replication and PGC-1α Gene Expression in C2C12 Muscle Cells.

Mitochondrial biogenesis is a complex process requiring coordinated expression of nuclear and mitochondrial genomes. The peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α) is a key regulator of mitochondrial biogenesis, and it controls mitochondrial DNA (mtDNA) replication within diverse tissues, including muscle tissue. The aim of this study was to investigate the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on mtDNA copy number and PGC-1α promoter activity in C2C12 muscle cells. mtDNA copy number and mRNA levels of genes related to mitochondrial biogenesis such as PGC-1α, nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (Tfam) were assayed by quantitative real-time PCR. The PGC-1α promoter from -970 to +412 bp was subcloned into the pGL3-basic vector, which includes a luciferase reporter gene. Both EPA and DHA significantly increased mtDNA copy number, dose and time dependently, and up-regulated mRNA levels of PGC-1α, NRF1, and Tfam. Furthermore, EPA and DHA stimulated PGC-1α promoter activity in a dose-dependent manner. These results suggest that EPA and DHA may modulate mitochondrial biogenesis, which was partially associated with increased mtDNA replication and PGC-1α gene expression in C2C12 muscle cells.

Activation of the 5'-AMP-Activated Protein Kinase in the Cerebral Cortex of Young Senescence-Accelerated P8 Mice and Association with GSK3ß- and PP2A-Dependent Inhibition of p-tau396 Expression.

The 5'-AMP-activated protein kinase (AMPK), which is a sensor of cellular energy, regulates neuronal survival and energy homeostasis. However, the roles of AMPK in the pathogenesis of Alzheimer's disease (AD) are unclear. The senescence-accelerated mouse prone 8 (SAMP8) strain is characterized by deficits in learning and memory, exhibits pathological characteristics of AD as early as 5 months of age, and is being increasingly recognized as a model of AD. Here, we investigated the relationship between AMPK activation and phosphorylation of the tau protein in the brain of young (2-month-old) SAMP8 animals and in differentiated SH-SY5Y cells. Upregulation of p-AMPK, p-ACC, and p-GSK3ßS9 and downregulation of p-tau396 and sirtuin 1 (Sirt1) were observed in the cerebral cortex of young SAMP8 mice compared with that of age-matched SAMR1 animals. The hippocampal levels of p-AMPK and p-tau396 in SAMP8 animals were not significantly different from those of SAMR1, whereas upregulation of p-GSK3ßS9 and downregulation of sirt1 was observed in the hippocampus of SAMP8 mice. Consistent with in vivo findings in the cortex, AMPK activation in SH-SY5Y cells upregulated p-GSK3ßS9 but downregulated p-tau396, whereas it had no significant effect on p-tau262 expression. In addition, the AMPK-mediated inhibition of p-tau396 expression was attenuated by okadaic acid, a protein phosphatase 2A (PP2A) inhibitor. Taken together, our data showed that AMPK activation inhibits p-tau396 expression in a GSK3ß- and PP2A-dependent manner, and suggest that differential regulation of tau phosphorylation in young SAMP8 mice by AMPK plays a compensatory role against accelerated senescence in this AD animal model.

The effect of APH treatment on surface bonding and osseointegration of Ti-6Al-7Nb implants: an in vitro and in vivo study.

This study investigated the effects of anodization-cyclic precalcification-heat (APH) treatment on the bonding ability of Ca-P coating to the parent metal and osseointegration of Ti-6Al-7Nb implants. Eighteen Ti-6Al-7Nb discs, 9 untreated and 9 APH-treated, were cultured with osteoblast cells in vitro, and the cellular differentiation ability was assayed at 1, 2, and 3 weeks. For in vivo testing, 28 Ti-6Al-7Nb implants (14 implants of each group) were inserted to rat tibias, and after each 4 and 6 weeks of implantation, bone bonding, and osseointegration were evaluated through removal torque and histological analysis. Osteoblast-culturing showed twice as much of the alkaline phosphatase activity on the treated surface at 3 weeks than on the untreated surface (p < 0.05). The treated implants exhibited higher removal torque values than the untreated ones (15.5 vs. 1.8 Ncm at 4 weeks and 19.7 vs. 2.6 Ncm at 6 weeks, p < 0.05). Moreover, the excellent bonding quality of coats was confirmed by the existence of cohesive fractures on the surface of removed APH implants (field emission scanning electron microscopy and histological observation). Within the limits of this study, it can be concluded that the APH treatment significantly enhanced osseointegration of the Ti-6Al-7Nb implant, with the stable bonding between the coating and the implant surface.