Effects of Obesity in Old Age on the Basement Membrane of Skeletal Muscle in Mice.

Obesity and aging are known to affect the skeletal muscles. Obesity in old age may result in a poor basement membrane (BM) construction response, which serves to protect the skeletal muscle, thus making the skeletal muscle more vulnerable. In this study, older and young male C57BL/6J mice were divided into two groups, each fed a high-fat or regular diet for eight weeks. A high-fat diet decreased the relative gastrocnemius muscle weight in both age groups, and obesity and aging individually result in a decline in muscle function. Immunoreactivity of collagen IV, the main component of BM, BM width, and BM-synthetic factor expression in young mice on a high-fat diet were higher than that in young mice on a regular diet, whereas such changes were minimal in obese older mice. Furthermore, the number of central nuclei fibers in obese older mice was higher than in old mice fed a regular diet and young mice fed a high-fat diet. These results suggest that obesity at a young age promotes skeletal muscle BM formation in response to weight gain. In contrast, this response is less pronounced in old age, suggesting that obesity in old age may lead to muscle fragility.

Poly(I:C) promotes neurotoxic amyloid ß accumulation through reduced degradation by decreasing neprilysin protein levels in astrocytes.

Inflammation associated with viral infection of the nervous system has been involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer's disease (AD) and multiple sclerosis. Polyinosinic:polycytidylic acid (poly[I:C]) is a Toll-like receptor 3 (TLR3) agonist that mimics the inflammatory response to systemic viral infections. Despite growing recognition of the role of glial cells in AD pathology, their involvement in the accumulation and clearance of amyloid ß (Aß) in the brain of patients with AD is poorly understood. Neprilysin (NEP) and insulin-degrading enzyme (IDE) are the main Aß-degrading enzymes in the brain. This study investigated whether poly(I:C) regulated Aß degradation and neurotoxicity by modulating NEP and IDE protein levels through TLR3 in astrocytes. To this aim, primary rat primary astrocyte cultures were treated with poly(I:C) and inhibitors of the TLR3 signaling. Protein levels were assessed by Western blot. Aß toxicity to primary neurons was measured by lactate dehydrogenase release. Poly(I:C) induced a significant decrease in NEP levels on the membrane of astrocytes as well as in the culture medium. The degradation of exogenous Aß was markedly delayed in poly(I:C)-treated astrocytes. This delay significantly increased the neurotoxicity of exogenous Aß1-42. Altogether, these results suggest that viral infections induce Aß neurotoxicity by decreasing NEP levels in astrocytes and consequently preventing Aß degradation.

Fatty Acid-Binding Proteins Aggravate Cerebral Ischemia-Reperfusion Injury in Mice.

Fatty acid-binding proteins (FABPs) regulate the intracellular dynamics of fatty acids, mediate lipid metabolism and participate in signaling processes. However, the therapeutic efficacy of targeting FABPs as novel therapeutic targets for cerebral ischemia is not well established. Previously, we synthesized a novel FABP inhibitor, i.e., FABP ligand 6 [4-(2-(5-(2-chlorophenyl)-1-(4-isopropylphenyl)-1H-pyrazol-3-yl)-4-fluorophenoxy)butanoic acid] (referred to here as MF6). In this study, we analyzed the ability of MF6 to ameliorate transient middle cerebral artery occlusion (tMCAO) and reperfusion-induced injury in mice. A single MF6 administration (3.0 mg/kg, per os) at 0.5 h post-reperfusion effectively reduced brain infarct volumes and neurological deficits. The protein-expression levels of FABP3, FABP5 and FABP7 in the brain gradually increased after tMCAO. Importantly, MF6 significantly suppressed infarct volumes and the elevation of FABP-expression levels at 12 h post-reperfusion. MF6 also inhibited the promotor activity of FABP5 in human neuroblastoma cells (SH-SY5Y). These data suggest that FABPs elevated infarct volumes after ischemic stroke and that inhibiting FABPs ameliorated the ischemic injury. Moreover, MF6 suppressed the inflammation-associated prostaglandin E2 levels through microsomal prostaglandin E synthase-1 expression in the ischemic hemispheres. Taken together, the results imply that the FABP inhibitor MF6 can potentially serve as a neuroprotective therapeutic for ischemic stroke.

Protein kinases A and C regulate amyloid-ß degradation by modulating protein levels of neprilysin and insulin-degrading enzyme in astrocytes.

The pathology of sporadic Alzheimer's disease is hallmarked by altered signal transduction via the neurotransmitter receptor-G-protein-mediated protein kinase A (PKA) and protein kinase C (PKC) pathways. Because the accumulation of amyloid-ß (Aß) depends on its rates of synthesis and clearance, the metabolic pathway of Aß in the brain and the entire body warrants exploration. The two major enzymes involved in Aß degradation in the brain are believed to be the neprilysin and insulin-degrading enzyme (IDE). This study investigated whether PKA and PKC regulate the degradation of Aß by modulating the protein levels of neprilysin and IDE in astrocytes. Activation of PKA induced a significant decrease in neprilysin protein levels in cultured astrocytes, whereas activation of PKC induced a significant decrease in the protein level of neprilysin and an increase in the protein level of IDE. Following activation of PKC, the reduction of neprilysin was achieved by its secretion into the culture media. Moreover, PKA-activated astrocytes significantly delayed the degradation of exogenous Aß, whereas PKC-activated astrocytes significantly facilitated its degradation. These results suggest that PKA and PKC regulate Aß degradation in astrocytes through a decrease in the protein level of neprilysin and an increase in neprilysin secretion and protein levels of IDE, respectively.

Protective effects of butein on corticosterone-induced cytotoxicity in Neuro2A cells.

A functional understanding of the relationship between glucocorticoids and neuronal apoptosis induced by the production of reactive oxygen species (ROS) may lead to a novel strategy for the treatment or prevention of depression. Previous reports suggest that butein, a type of flavonoids, may be a potent candidate against depression-related neuronal cell apoptosis caused by oxidative stress; however, the protective effects of butein on damaged corticosterone (CORT)-treated neuronal cells has not been elucidated. In the present study, we examined the protective effect of butein on CORT-induced cytotoxicity and neurite growth during cell differentiation of mouse neuroblastoma Neuro2A (N2A) cells. Moreover, the effect on cultured cells by high concentrations of butein was confirmed. Our results demonstrate that CORT treatment significantly decreases cell viability and induces cell death. CORT was suggested to induce apoptosis via mitochondrial dysfunction and caspase-3 activation; this apoptosis may be attributed to DNA damage by ROS generation, found in this study to be significantly inhibited by pretreatment with butein. We found that CORT produced significant growth suppression of retinoic acid-induced neurite outgrowth in N2A cells; however, butein significantly increased neurite length and induced dose-dependent apoptotic cytotoxicity in N2A cells. This study suggests that low concentration of butein can prevent CORT-induced cytotoxicity in N2A cells, and provides preliminary results supporting some of the beneficial roles of butein in neuroprotection.

Microsomal prostaglandin E synthase-1 is a critical factor in dopaminergic neurodegeneration in Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder of uncertain pathogenesis characterized by the loss of nigrostriatal dopaminergic neurons. Although increased production of prostaglandin E2 (PGE2) has been implicated in tissue damage in several pathological settings, the role of microsomal prostaglandin E synthase-1 (mPGES-1), an inducible terminal enzyme for PGE2 synthesis, in dopaminergic neurodegeneration remains unclear. Here we show that mPGES-1 is up-regulated in the dopaminergic neurons of the substantia nigra of postmortem brain tissue from PD patients and in neurotoxin 6-hydroxydopamine (6-OHDA)-induced PD mice. The expression of mPGES-1 was also up-regulated in cultured dopaminergic neurons stimulated with 6-OHDA. The genetic deletion of mPGES-1 not only abolished 6-OHDA-induced PGE2 production but also inhibited 6-OHDA-induced dopaminergic neurodegeneration both in vitro and in vivo. Nigrostriatal projections, striatal dopamine content, and neurological functions were significantly impaired by 6-OHDA administration in wild-type (WT) mice, but not in mPGES-1 knockout (KO) mice. Furthermore, in cultured primary mesencephalic neurons, addition of PGE2 to compensate for the deficiency of 6-OHDA-induced PGE2 production in mPGES-1 KO neurons recovered 6-OHDA toxicity to almost the same extent as that seen in WT neurons. These results suggest that induction of mPGES-1 enhances 6-OHDA-induced dopaminergic neuronal death through excessive PGE2 production. Thus, mPGES-1 may be a valuable therapeutic target for treatment of PD.

Insulin-signaling Pathway Regulates the Degradation of Amyloid ß-protein via Astrocytes.

Alzheimer's disease (AD) has been considered as a metabolic dysfunction disease associated with impaired insulin signaling. Determining the mechanisms underlying insulin signaling dysfunction and resistance in AD will be important for its treatment. Impaired clearance of amyloid-ß peptide (Aß) significantly contributes to amyloid accumulation, which is typically observed in the brain of AD patients. Reduced expression of important Aß-degrading enzymes in the brain, such as neprilysin (NEP) and insulin-degrading enzyme (IDE), can promote Aß deposition in sporadic late-onset AD patients. Here, we investigated whether insulin regulates the degradation of Aß by inducing expression of NEP and IDE in cultured astrocytes. Treatment of astrocytes with insulin significantly reduced cellular NEP levels, but increased IDE expression. The effects of insulin on the expression of NEP and IDE involved activation of an extracellular signal-regulated kinase (ERK)-mediated pathway. The reduction in cellular NEP levels was associated with NEP secretion into the culture medium, whereas IDE was increased in the cell membranes. Moreover, insulin-treated astrocytes significantly facilitated the degradation of exogenous Aß within the culture medium. Interestingly, pretreatment of astrocytes with an ERK inhibitor prior to insulin exposure markedly inhibited insulin-induced degradation of Aß. These results suggest that insulin exposure enhanced Aß degradation via an increase in NEP secretion and IDE expression in astrocytes, via activation of the ERK-mediated pathway. The inhibition of insulin signaling pathways delayed Aß degradation by attenuating alterations in NEP and IDE levels and competition with insulin and Aß. Our results provide further insight into the pathological relevance of insulin resistance in AD development.

Epigallocatechin gallate induces extracellular degradation of amyloid ß-protein by increasing neprilysin secretion from astrocytes through activation of ERK and PI3K pathways.

Amyloid-ß (Aß) production and clearance in the brain is a crucial focus of investigations into the pathogenesis of Alzheimer disease. Imbalance between production and clearance leads to accumulation of Aß. The important Aß-degrading enzymes in the brain are neprilysin (NEP) and insulin-degrading enzyme (IDE), and defective enzyme expression may facilitate Aß deposition in sporadic late-onset AD patients. It has been suggested that epigallocatechin gallate (EGCG), a member of the catechin family, might be an effective treatment for AD, because it has been shown to elevate NEP expression. Therefore, we examined whether catechins, which are functional components of common foods, could regulate the degradation of Aß by inducing NEP and IDE expression. We also investigated the role of catechins in activating intracellular signal transduction in astrocytes. Treatment of cultured rat astrocytes with EGCG significantly reduced the expression of NEP, but not IDE, in a concentration- and time-dependent manner. NEP expression in cultured astrocytes was suppressed by activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K), and reduced NEP expression was accompanied by an increase of NEP release into the extracellular space (culture medium). Moreover, culture medium from EGCG-treated astrocytes facilitated the degradation of exogenous Aß. These results suggest that EGCG may have a beneficial effect on AD by activating ERK-and PI3K-mediated pathways in astrocytes, thus increasing astrocyte secretion of NEP and facilitating degradation of Aß.

The Role of mPGES-1 in Inflammatory Brain Diseases.

Prostaglandin E2 (PGE2) has been thought to be an important mediator of inflammation in peripheral tissues, but recent studies clearly show the involvement of PGE2 in inflammatory brain diseases. In some animal models of brain disease, the genetic disruption and chemical inhibition of cyclooxygenase (COX)-2 resulted in the reduction of PGE2 and amelioration of symptoms, and it had been thought that PGE2 produced by COX-2 may be involved in the progression of injuries. However, COX-2 produces not only PGE2, but also some other prostanoids, and thus the protective effects of COX-2 inhibition, as well as severe side effects, may be caused by the inhibition of prostanoids other than PGE2. Therefore, to elucidate the role of PGE2, studies of microsomal prostaglandin E synthase-1 (mPGES-1), an inducible terminal enzyme for PGE2 synthesis, have recently been an active area of research. Studies from mPGES-1 deficient mice provide compelling evidence for its role in a variety of inflammatory brain diseases, such as ischemic stroke, Alzheimer's disease and epilepsy, and clues for developing new therapeutic treatments for brain diseases by targeting mPGES-1. Considering that COX inhibitors may non-selectively suppress the production of many types of prostanoids that are essential for normal physiological functioning of the brain and peripheral tissues, as well as induce gastro-intestinal, renal and cardiovascular complications, mPGES-1 inhibitors are expected to be injury-selective and have fewer side-effects when treating human brain diseases. Thus, this paper focuses on recent studies that have demonstrated the involvement of mPGES-1 in pathological brain diseases.

Astrocyte-mediated ischemic tolerance.

Preconditioning (PC) using a preceding sublethal ischemic insult is an attractive strategy for protecting neurons by inducing ischemic tolerance in the brain. Although the underlying molecular mechanisms have been extensively studied, almost all studies have focused on neurons. Here, using a middle cerebral artery occlusion model in mice, we show that astrocytes play an essential role in the induction of brain ischemic tolerance. PC caused activation of glial cells without producing any noticeable brain damage. The spatiotemporal pattern of astrocytic, but not microglial, activation correlated well with that of ischemic tolerance. Interestingly, such activation in astrocytes lasted at least 8 weeks. Importantly, inhibiting astrocytes with fluorocitrate abolished the induction of ischemic tolerance. To investigate the underlying mechanisms, we focused on the P2X7 receptor as a key molecule in astrocyte-mediated ischemic tolerance. P2X7 receptors were dramatically upregulated in activated astrocytes. PC-induced ischemic tolerance was abolished in P2X7 receptor knock-out mice. Moreover, our results suggest that hypoxia-inducible factor-1α, a well known mediator of ischemic tolerance, is involved in P2X7 receptor-mediated ischemic tolerance. Unlike previous reports focusing on neuron-based mechanisms, our results show that astrocytes play indispensable roles in inducing ischemic tolerance, and that upregulation of P2X7 receptors in astrocytes is essential.

[The role of prostaglandin E2 in stroke-reperfusion injury].

Although augmented prostaglandin E2 (PGE2) accumulation has been demonstrated at the lesion sites of rodent ischemia models, the role of postischemic PGE2 in neuronal survival has remained obscure. We recently identified the microsomal prostaglandin E synthase-1 (mPGES-1), an inducible terminal enzyme for prostaglandin E2 synthesis, as a critical factor in stroke-reperfusion injury. Co-induction of mPGES-1 and cyclooxygenase (COX)-2, an upstream enzyme for PGE2 production, was observed after brain ischemia. In mPGES-1 knockout (KO) mice, in which the postischemic PGE2 production in the cortex was completely absent, the ischemic injuries were less severe compared to those in wild-type (WT) mice. The ameliorated symptoms observed in KO mice after ischemia were reversed to almost the same severity as in the WT mice by intracerebroventricular injection of PGE2 into KO mice. The induction of mPGES-1 was also observed after glutamate exposure in cultured hippocampal slices. In mPGES-1 KO slices, glutamate-induced excitotoxicity was less severe compared to that in WT slices. Among the EP1-4 antagonists and agonists, only the EP3 antagonist attenuated and only the EP3 agonist augmented the glutamate-induced excitotoxicity. Furthermore, intraperitoneal injection of COX-2 inhibitor or EP3 antagonist reduced the ischemic injuries in WT mice, but not in mPGES-1 KO mice. In EP3 KO mice, the ischemic injuries were less severe compared to those in WT mice. These results suggest that mPGES-1 and COX-2 are co-induced by excessive glutamate in the ischemic brain and act together to exacerbate stroke injury through PGE2 production followed by activation of EP3 receptors.

NKCC1 knockdown decreases neuron production through GABA(A)-regulated neural progenitor proliferation and delays dendrite development.

Signaling through GABA(A) receptors controls neural progenitor cell (NPC) development in vitro and is altered in schizophrenic and autistic individuals. However, the in vivo function of GABA(A) signaling on neural stem cell proliferation, and ultimately neurogenesis, remains unknown. To examine GABA(A) function in vivo, we electroporated plasmids encoding short-hairpin (sh) RNA against the Na-K-2Cl cotransporter NKCC1 (shNKCC1) in NPCs of the neonatal subventricular zone in mice to reduce GABA(A)-induced depolarization. Reduced GABA(A) depolarization identified by a loss of GABA(A)-induced calcium responses in most electroporated NPCs led to a 70% decrease in the number of proliferative Ki67(+) NPCs and a 60% reduction in newborn neuron density. Premature loss of GABA(A) depolarization in newborn neurons resulted in truncated dendritic arborization at the time of synaptic integration. However, by 6 weeks the dendritic tree had partially recovered and displayed a small, albeit significant, decrease in dendritic complexity but not total dendritic length. To further examine GABA(A) function on NPCs, we treated animals with a GABA(A) allosteric agonist, pentobarbital. Enhancement of GABA(A) activity in NPCs increased the number of proliferative NPCs by 60%. Combining shNKCC1 and pentobarbital prevented the shNKCC1 and the pentobarbital effects on NPC proliferation, suggesting that these manipulations affected NPCs through GABA(A) receptors. Thus, dysregulation in GABA(A) depolarizing activity delayed dendritic development and reduced NPC proliferation resulting in decreased neuronal density.

Inhibition of prostaglandin E2 EP3 receptors improves stroke injury via anti-inflammatory and anti-apoptotic mechanisms.

Although deletion of EP3 receptors is known to ameliorate stroke injury in experimental stroke models, the underlying mechanisms and the effects of EP3-specific antagonists remain poorly understood. Here we demonstrate the protective effect of postischemic treatment with an EP3 antagonist, ONO-AE3-240, through anti-inflammatory and anti-apoptotic effects. In transient focal ischemia models, peritoneal injection of an EP3 antagonist after occlusion-reperfusion reduced infarction, edema and neurological dysfunctions to almost the same levels of those in EP3 knockout (KO) mice. Furthermore, neuronal apoptosis in the ischemic cortex investigated by terminal dUTP nick-end labeling (TUNEL) and caspase-3 immunostaining were ameliorated in EP3 antagonist-treated mice or EP3 KO mice as compared with vehicle-treated mice or wild-type (WT) mice, respectively. There were no significant differences between ONO-AE3-240-injected or EP3 KO mice and vehicle-injected or WT mice, respectively, in mean arterial blood pressure, cerebral blood flow or body temperature. The double-immunostaining showed that EP3 receptor-positive cells were also positive for CD-11b and partially for Neu-N, the marker for microglia and neurons. Deletion of EP3 receptors also reduced damage of the blood-brain barrier, activation of microglia and infiltration of neutrophils into the ischemic cortex. These results suggest that EP3 receptors are involved in stroke injury through the enhancement of inflammatory and apoptotic reactions in the ischemic cortex. Thus, EP3 antagonists may be valuable for the treatment of human stroke.

MARCKS regulates lamellipodia formation induced by IGF-I via association with PIP2 and beta-actin at membrane microdomains.

Myristoylated alanine-rich C kinase substrate (MARCKS) is considered to participate in formation of F-actin-based lamellipodia, which represents the first stage of neurite formation. However, the mechanism of how MARCKS is involved in lamellipodia formation is not precisely unknown. Using SH-SY5Y cells, we demonstrated here that MARCKS was translocated from cytosol to detergent-resistant membrane microdomains, known as lipid rafts, within 30 min after insulin-like growth factor-I (IGF-I) stimulation, which was accompanied by MARCKS dephosphorylation, beta-actin accumulation in lipid rafts, and lamellipodia formation. The protein kinase C inhibitor, Ro-31-8220, and Rho-kinase inhibitors, HA1077 and Y27632, themselves decreased basal phosphorylation levels of MARCKS and coincidently elicited translocation of MARCKS to lipid rafts. On the other hand, the phosphoinositide 3-kinase inhibitor, LY294002, abolished IGF-I-induced dephosphorylation, translocation of MARCKS to lipid rafts, and lamellipodia formation. Treatment of cells with neomycin, a PIP2-masking reagent, attenuated the translocation of MARCKS to lipid rafts and the lamellipodia formation induced by IGF-I, although dephosphorylation of MARCKS was not affected. Immunocytochemical and immunoprecipitation analysis indicated that IGF-I stimulation induced the translocation of MARCKS to lipid rafts in the edge of lamellipodia and formation of the complex with PIP2. Moreover, we demonstrated that knockdown of endogenous MARCKS resulted in significant attenuation of IGF-I-induced beta-actin accumulation in the lipid rafts and lamellipodia formation. These results suggest a novel role for MARCKS in lamellipodia formation induced by IGF-I via the translocation of MARCKS, association with PIP2, and accumulation of beta-actin in the membrane microdomains.

Microsomal prostaglandin E synthase-1 is a critical factor of stroke-reperfusion injury.

Although augmented prostaglandin E(2) (PGE(2)) synthesis and accumulation have been demonstrated in the lesion sites of rodent transient focal ischemia models, the role of PGE(2) in neuronal survival has been controversial, showing both protective and toxic effects. Here we demonstrate the induction of microsomal PGE synthase 1 (mPGES-1), an inducible terminal enzyme for PGE(2) synthesis, in neurons, microglia, and endothelial cells in the cerebral cortex after transient focal ischemia. In mPGES-1 knockout (KO) mice, in which the postischemic PGE(2) production in the cortex was completely absent, the infarction, edema, apoptotic cell death, and caspase-3 activation in the cortex after ischemia were all reduced compared with those in wild-type (WT) mice. Furthermore, the behavioral neurological dysfunctions observed after ischemia in WT mice were significantly ameliorated in KO mice. The ameliorated symptoms observed in KO mice after ischemia were reversed to almost the same severity as WT mice by intracerebroventricular injection of PGE(2) into KO mice. Our observations suggest that mPGES-1 may be a critical determinant of postischemic neurological dysfunctions and a valuable therapeutic target for treatment of human stroke.

Microglia-specific expression of microsomal prostaglandin E2 synthase-1 contributes to lipopolysaccharide-induced prostaglandin E2 production.

Microsomal prostaglandin E2 synthase (mPGES)-1 is an inducible protein recently shown to be an important enzyme in inflammatory prostaglandin E2 (PGE2) production in some peripheral inflammatory lesions. However, in inflammatory sites in the brain, the induction of mPGES-1 is poorly understood. In this study, we demonstrated the expression of mPGES-1 in the brain parenchyma in a lipopolysaccharide (LPS)-induced inflammation model. A local injection of LPS into the rat substantia nigra led to the induction of mPGES-1 in activated microglia. In neuron-glial mixed cultures, mPGES-1 was co-induced with cyclooxygenase-2 (COX-2) specifically in microglia, but not in astrocytes, oligodendrocytes or neurons. In microglia-enriched cultures, the induction of mPGES-1, the activity of PGES and the production of PGE2 were preceded by the induction of mPGES-1 mRNA and almost completely inhibited by the synthetic glucocorticoid dexamethasone. The induction of mPGES-1 and production of PGE2 were also either attenuated or absent in microglia treated with mPGES-1 antisense oligonucleotide or microglia from mPGES-1 knockout (KO) mice, respectively, suggesting the necessity of mPGES-1 for microglial PGE2 production. These results suggest that the activation of microglia contributes to PGE2 production through the concerted de novo synthesis of mPGES-1 and COX-2 at sites of inflammation of the brain parenchyma.