Enhanced expression of activity-regulated cytoskeleton-associated protein in the medial prefrontal cortex is involved in working memory performance.

Working memory (WM) is a cognitive function important for guiding the on-going or upcoming behavior. A memory-related protein Arc (activity-regulated cytoskeleton-associated protein) is implicated in long-term memory consolidation. Recent evidence further suggests the involvement of hippocampal Arc in spatial WM. The medial prefrontal cortex (mPFC) is a key brain region mediating WM. However, the role of mPFC Arc in WM is still uncertain. To investigate whether mPFC Arc protein is involved in WM performance, delayed non-match to sample (DNMS) T-maze task was performed in rats with or without blocking new synthesis of mPFC Arc. In DNMS task, a 10-s or 30-s delay between the sample run and the choice run was given to evaluate WM performance. To block new Arc protein synthesis during the DNMS task, Arc antisense oligodeoxynucleotides (ODNs) were injected to the bilateral mPFC. The results show that, in rats without surgery for cannula implantation and subsequent intracerebral injection of ODNs, WM was functioning well during the DNMS task with a delay of 10 s but not 30 s, which was accompanied with a significantly increased level of mPFC Arc protein, indicating a possible link between enhanced Arc protein expression and the performance of WM. After preventing the enhancement of mPFC Arc protein expression with Arc antisense ODNs, rat's WM performance was impaired. These findings support enhanced mPFC Arc protein expression playing a role during WM performance.

Chondrocyte Thrombomodulin Protects against Osteoarthritis.

Osteoarthritis (OA) is a prevalent form of arthritis that affects over 32.5 million adults worldwide, causing significant cartilage damage and disability. Unfortunately, there are currently no effective treatments for OA, highlighting the need for novel therapeutic approaches. Thrombomodulin (TM), a glycoprotein expressed by chondrocytes and other cell types, has an unknown role in OA. Here, we investigated the function of TM in chondrocytes and OA using various methods, including recombinant TM (rTM), transgenic mice lacking the TM lectin-like domain (TMLeD/LeD), and a microRNA (miRNA) antagomir that increased TM expression. Results showed that chondrocyte-expressed TM and soluble TM [sTM, like recombinant TM domain 1 to 3 (rTMD123)] enhanced cell growth and migration, blocked interleukin-1ß (IL-1ß)-mediated signaling and protected against knee function and bone integrity loss in an anterior cruciate ligament transection (ACLT)-induced mouse model of OA. Conversely, TMLeD/LeD mice exhibited accelerated knee function loss, while treatment with rTMD123 protected against cartilage loss even one-week post-surgery. The administration of an miRNA antagomir (miR-up-TM) also increased TM expression and protected against cartilage damage in the OA model. These findings suggested that chondrocyte TM plays a crucial role in counteracting OA, and miR-up-TM may represent a promising therapeutic approach to protect against cartilage-related disorders.

High-fat diet reduces novelty-induced expression of activity-regulated cytoskeleton-associated protein.

Chronic high-fat-diet (HFD) consumption can lead to the development of brain insulin resistance, which then exerts deleterious effects on learning and memory. Activity-regulated cytoskeleton-associated protein (Arc) is a memory-related protein, and its expression can be induced by insulin stimulation. In HFD-fed animals, their basal Arc protein levels in cerebral cortex and hippocampus are reduced. However, the effects of HFD on novelty-induced Arc protein expression that is important for cognitive function is still unknown. In the present study, after feeding HFD (60% kcal from fat) for 5 weeks, mice developed brain insulin resistance and had a significant reduction in the novelty-induced but not the basal Arc protein levels in their hippocampi. Further experiments were performed in primary rat hippocampal neurons. The results show that, under the condition of neuronal insulin resistance, acute insulin stimulation induced less activation of the phosphatidylinositol 3-kinase/protein kinase B/p70 ribosomal S6 kinase (PI3K/Akt/p70S6K) pathway, resulting in reduced induction of Arc protein expression. Accordingly, it is suggested that following HFD feeding, the reduction in novelty-induced Arc protein expression in animal's hippocampus is probably related to a suppressed activation of the PI3K/Akt/p70S6K pathway due to the existence of brain insulin resistance.

Nonlethal dose of silver nanoparticles attenuates TNF-α-induced hepatic epithelial cell death through HSP70 overexpression.

Silver nanoparticles (Ag-nps) have been widely used in various biomedical products. Compared with its hazardous effects extensively being studied, rare attention has been paid to the potential protective effect of Ag-nps to human health. The present study was designed to evaluate the protective effects of Ag-nps and heat shock treatment on tumor necrosis factor-α (TNF-α)-induced cell damage in Clone 9 cells. Clone 9 cells were pretreated with nonlethal concentration of Ag-nps (1 µg/ml) or heat shock, and then cell damages were induced by TNF-α (1 ng/ml). Protective effects of Ag-nps administration or heat shock treatment were determined by examining the TNF-α-induced changes in cell viabilities. The results showed that the intensity of cytotoxicity produced by TNF-α was alleviated upon treatment with nonlethal concentration of Ag-nps (1 µg/ml). Similar protective effects were also found upon heat shock treatment. These data demonstrate that Ag-nps and heat shock treatment were equally capable of inducing heat shock protein 70 (HSP70) protein expression in Clone 9 cells. The results suggest that clinically Ag-nps administration is a viable strategy to induce endogenous HSP70 expression instead of applying heat shock. In conclusion, our study for the first time provides evidence that Ag-nps may act as a viable alternative for HSP70 induction clinically.

Baicalein inhibits HMGB1 release and MMP-2/-9 expression in lipopolysaccharide-induced cardiac hypertrophy.

Myocardial dysfunction, a common complication after sepsis, significantly contributes to the death of patients with septic shock. In the search for potentially effective drugs to decrease mortality from sepsis, we investigated the cardioprotective effects of baicalein, a flavonoid present in the root of Scutellaria baicalensis, on lipopolysaccharide (LPS)-induced pro-inflammatory cytokine production and matrix metalloproteinase-2 and -9 (MMP-2/-9) expression. We found that baicalein significantly attenuated LPS-induced cardiac hypertrophy and counteracted reactive oxygen species (ROS) generation in neonatal rat cardiomyocytes. In addition, pretreatment with baicalein inhibited LPS-induced early (e.g., tumor necrosis factor-α (TNF-α) and interleukin-6) and late (e.g., high mobility group box 1 (HMGB1) pro-inflammatory cytokine release, inducible nitric oxide synthase (iNOS) expression and NO production. Finally, baicalein also significantly down-regulated the expression of MMP-2/-9 and attenuated HMGB1 translocation from the nucleus to the cytoplasm. These results suggest that baicalein can protect cardiomyocytes from LPS-induced cardiac injury via the inhibition of ROS and inflammatory cytokine production. These cardioprotective effects are possibly mediated through the inhibition of the HMGB1 and MMP-2/-9 signaling pathways.

Insulin can induce the expression of a memory-related synaptic protein through facilitating AMPA receptor endocytosis in rat cortical neurons.

Learning and memory depend on long-term synaptic plasticity including long-term potentiation (LTP) and depression (LTD). Activity-regulated cytoskeleton-associated protein (Arc) plays versatile roles in synaptic plasticity mainly through inducing F-actin formation, underlying consolidation of LTP, and promoting AMPA receptor (AMPAR) endocytosis, underlying LTD. Insulin can also induce LTD by facilitating the internalization of AMPARs. In neuroblastoma cells, insulin induced a dramatic increase in Arc mRNA and Arc protein levels, which may underlie the memory-enhancing action of insulin. Thus, a hypothesis was made that, in response to insulin, increased AMPAR endocytosis leads to enhanced Arc expression, and vice versa. Primary cultures of neonatal Sprague-Dawley rat cortical neurons were used. Using Western-blot analysis and immunofluorescent staining, our results reveal that inhibiting AMPAR-mediated responses with AMPAR antagonists significantly enhanced whereas blocking AMPAR endocytosis with various reagents significantly prevented insulin (200 nM, 2 h)-induced Arc expression. Furthermore, via surface biotinylation assay, we demonstrate that acute blockade of new Arc synthesis after insulin stimulation using Arc antisense oligodeoxynucleotide prevented insulin-stimulated AMPAR endocytosis. These findings suggest for the first time that an interaction exists between insulin-stimulated AMPAR endocytosis and insulin-induced Arc expression.

Activation of Trim17 by PPARγ is involved in di(2-ethylhexyl) phthalate (DEHP)-induced apoptosis on Neuro-2a cells.

Di-(2-ethylhexyl) phthalate (DEHP) is widely used as a plasticizer in plastics. Its reproductive toxicity and teratogenic effects are well known. DEHP can cause liver damage and peroxisome proliferation, as well as carcinogenesis. Animal study has shown that DEHP causes neurodegeneration in rat brain. Prenatal exposure to DEHP disrupts brain development and decreases brain weight in rats. But its mechanism of action in the brain is not clear. This study used a neuroblastoma cell line, Neuro-2a cells, to investigate the toxic effect of DEHP. The results revealed that DEHP inhibits cell proliferation, activate caspase-3, induce apoptosis in a dose and time dependent manner, and activate expression of the PPARγ and Trim17 protein. Administration of the PPARγ agonist (troglitazone) enhanced DEHP-induced Trim17 protein expression and this enhancement could be reversed by the PPARγ antagonist (GW9662). These results suggest that DEHP activates the Trim17 protein via PPARγ leading to cleavage pro-caspase-3 and apoptosis. This finding may account for the central nervous system toxicity of DEHP and implies DEHP can impair fetal brain development.

Nonlethal aluminum maltolate can reduce brain-derived neurotrophic factor-induced Arc expression through interrupting the ERK signaling in SH-SY5Y neuroblastoma cells.

Although many studies have demonstrated that aluminum (Al) exposure impairs learning and memory, its underlying mechanism is still uncertain. Long-lasting forms of synaptic plasticity that underlie memory are dependent on new protein synthesis. In particular, activity-regulated cytoskeleton-associated protein (Arc) has a versatile role in synaptic plasticity, and its synthesis can be induced by brain-derived neurotrophic factor (BDNF). BDNF-induced Arc expression has been suggested to play a fundamental role in the stabilization of synaptic plasticity. In the present study, the pretreatment of Al(malt)3 at nonlethal level (200 µM, 24 h) significantly reduced BDNF (10 ng/ml, 1h)-induced Arc expression in SH-SY5Y human neuroblastoma cells. BDNF-induced activation of ERK but not PI3K signaling pathway was interfered with the Al(malt)3 pretreatment, resulting in the subsequent reduction of BDNF-induced phosphorylation of 4EBP1, p70S6K, and eIF4E. Reduced phospho-4EBP1 and phospho-eIF4E hindered the initiation step of translation, which may lead to a reduction in BDNF-induced Arc expression. However, reduced phospho-p70S6K did not influence the phosphorylation of eEF2K and eEF2, indicating no significant effect on BDNF-enhanced translation elongation. Therefore, even at nonlethal level, Al(malt)3 pretreatment reduced BDNF-induced Arc expression, which was caused by interrupting the ERK signaling pathway as well as the subsequent translation initiation.

The protective effect of Rho-associated kinase inhibitor on aluminum-induced neurotoxicity in rat cortical neurons.

Aluminum (Al) is a neurotoxicant and is implicated in several neurodegenerative diseases, including Alzheimer's disease (AD). In AD brains, one of the pathological hallmarks is the extracellular deposition of senile plaques, which are mainly composed of aggregated amyloid-beta (Abeta). Endoproteolysis of the amyloid-beta precursor protein (AbetaPP) by the beta-secretase and the gamma-secretase generates Abeta. AbetaPP can also be cleaved by the alpha-secretase within the Abeta region, which releases a soluble fragment sAPPalpha and precludes the formation of Abeta. Al has been reported to increase the level of Abeta, promote Abeta aggregation, and increase Abeta neurotoxicity. In contrast, small G protein Rho and its effector, Rho-associated kinase (ROCK), are known to negatively regulate the amount of Abeta. Inhibition of the Rho-ROCK pathway may underlie the ability of nonsteroidal anti-inflammatory drugs and statins to reduce Abeta production. Whether the Rho-ROCK pathway is involved in Al-induced elevation and aggregation of Abeta is unknown. In the present study, cultured rat cortical neurons were treated with Al(malt)(3) in the absence or presence of ROCK inhibitor Y-27632. After the treatment of Al(malt)(3), the cell viability and the level of sAPPalpha were reduced, whereas the amyloid fibrils in the conditioned media were increased. Treatment with Y-27632 prevented these adverse effects of Al(malt)(3) and thus maintained neuronal survival. These results reveal that the activation of the Rho-ROCK signaling pathway was involved in Al-induced effects in terms of the cell viability, the production of sAPPalpha, and the formation of amyloid fibril, which provides a novel mechanism underlying Al-induced neurotoxicity.

Amyloid-beta interrupts the PI3K-Akt-mTOR signaling pathway that could be involved in brain-derived neurotrophic factor-induced Arc expression in rat cortical neurons.

The deposition of amyloid-beta (Abeta) contributes to the pathogenesis of Alzheimer's disease. Even at low levels, Abeta may interfere with various signaling cascades critical for the synaptic plasticity that underlies learning and memory. Brain-derived neurotrophic factor (BDNF) is well known to be capable of inducing the synthesis of activity-regulated cytoskeleton-associated protein (Arc), which plays a fundamental role in modulating synaptic plasticity. Our recent study has demonstrated that treatment of fibrillar Abeta at a nonlethal level was sufficient to impair BDNF-induced Arc expression in cultured rat cortical neurons. In this study, BDNF treatment alone induced the activation of the phosphatidylinositol 3-kinase-Akt-mammlian target of rapamycin (PI3K-Akt-mTOR) signaling pathway, the phosphorylation of eukaryotic initiation factor 4E binding protein (4EBP1) and p70 ribosomal S6 kinase (p70S6K), the dephosphorylation of eukaryotic elongation factor 2 (eEF2), and the expression of Arc. Interrupting the PI3K-Akt-mTOR signaling pathway by inhibitors prevented the effects of BDNF, indicating the involvement of this pathway in BDNF-induced 4EBP1 phosphorylation, p70S6K phosphorylation, eEF2 dephosphorylation, and Arc expression. Nonlethal Abeta pretreatment partially blocked these effects of BDNF. Double- immunofluorescent staining in rat cortical neurons further confirmed the coexistence of eEF2 dephosphorylation and Arc expression following BDNF treatment regardless of the presence of Abeta. These results reveal that, in cultured rat cortical neurons, Abeta interrupts the PI3K-Akt-mTOR signaling pathway that could be involved in BDNF-induced Arc expression. Moreover, this study also provides the first evidence that there is a close correlation between BDNF-induced eEF2 dephosphorylation and BDNF-induced Arc expression. (c) 2009 Wiley-Liss, Inc.

Functional changes on ascending auditory pathway in rats caused by germanium dioxide exposure: an electrophysiological study.

The semiconductor element, germanium (Ge), is essential for the manufacture of modern integrated circuits. Because of its anti-tumor and immunomodulative effects, Ge-containing compounds are also used as health-promoting ingredients in food. However, some histological studies have shown the toxic effects of Ge-containing compounds on various organs, including the central nervous system. Even now, the effect of germanium on auditory system function is not completely clear. To clarify this question, brainstem auditory evoked potentials (BAEPs) were applied to examine the effect of germanium dioxide (GeO(2)) on the ascending auditory pathway. Since the voltage-gated sodium channel is important to neuron activation and nerve conduction, the effect of GeO(2) on voltage-gated sodium channels was also examined. The result revealed GeO(2) elevated the BAEPs threshold dose-dependently. GeO(2) also prolonged latencies and interpeak latencies (IPLs) of BAEPs, but the amplitudes of suprathreshold intensities (90dB) did not show any obvious change. In addition, the results of whole cell patch clamp studies indicated GeO(2) reduced inward sodium current. These results suggest the toxic effect of GeO(2) on the conduction of the auditory system, and that inhibitory effect of GeO(2) on the voltage-gated sodium channels might play a role in GeO(2)-induced abnormal hearing loss.

Germanium dioxide induces mitochondria-mediated apoptosis in Neuro-2A cells.

Germanium (Ge) is commonly used in the semiconductor industry as well as health-promoting and medical field. Biologically, germanium possesses erythropoietic, anti-microbial, anti-tumor, anti-amyloidosis, and immunomodulative effects. However, toxic effects of Ge-containing compounds on kidney, muscle, neuronal cells, and nerves have been reported. Mitochondrial dysfunction was found to be involved in the pathogenesis of GeO(2)-induced nephropathy and myopathy. Since it is well known that mitochondria play a major role in apoptosis triggered by many stimuli, an effort was made to examine whether the Ge-induced neurotoxicity occurs through mitochondria-mediated apoptosis. A mouse neuroblastoma cell line, Neuro-2A, was used in the present study. After incubating with 0.1-800microM of GeO(2) for 0-72h, the cell viability of Neuro-2A cells was inhibited in a dose- and time-dependent manner. Further analysis showed that aside from the changes in the nuclear morphology responsible for apoptosis, the release of cytochrome c, the loss of mitochondrial membrane potential, the translocation of Bax, and the reduction of Bcl-2 expression were also observed in Neuro-2A cells after GeO(2) treatment. These results indicate that the mitochondria-mediated apoptosis is involved in this in vitro model of GeO(2)-induced neurotoxicity.

Implication of nNOS in the enlargement of AChR aggregates but not the initial aggregate formation in a novel coculture model.

The aggregation of nicotinic acetylcholine receptors (AChRs) is an early hallmark of the formation of neuromuscular junction (NMJ), and nitric oxide is recently known to play an important role. In many NMJ studies, nerve-muscle coculture model was used, and NG108-15 cells, a neuroblastoma x glioma hybrid cell line, were the most frequently used nerve cells. However, possible contributions from glial cells could not be excluded. In this study, Neuro-2a neuroblastoma cells were used instead of [corrected] coculture with myotubes, and the relationship between AChR aggregation and spatiotemporal expression and activation of nNOS (neuronal nitric oxide synthase) was examined. Upon coculture, AChR aggregates were observed by FITC-conjugated alpha-bungarotoxin, and double labeling of AChRs and neurofilament showed that the neurites of a Neuro-2a cell innervated several myotubes. After treating the cocultures with single dose of L-NAME at the end of 1-day [corrected] coculturing, only slight effect on AChR aggregation could be found indicating that nNOS is not related to the initial formation of AChR aggregates. In contrast, when L-NAME treatment was given at the end of a 3-day coculturing, the day just before reaching the maximum extent of AChR aggregation, new AChR aggregates were hardly formed and the preformed AChR aggregates were even dispersed indicating that the enlargement of AChR aggregates is highly dependent on the nNOS activity. Double-labeling study of nNOS and AChR further showed that the coupling of membranous nNOS to regions nearby the AChR aggregates was essential for the enlargement of AChR aggregates. These results not only revealed the spatiotemporal relationship between AChR aggregation and nNOS activity but also verified the feasibility and usefulness of using Neuro-2a cells in a coculture model.

Lead-induced attenuation in the aggregation of acetylcholine receptors during the neuromuscular junction formation.

Lead (Pb2+) toxicity is more common in children and is associated with cognitive deficits, which may reflect lead-induced changes in central synaptic development and function. Aside from neurotoxicity, lead exposure may also impact mature neuromuscular junction (NMJ) and cause muscle weakness. NMJ is known as a peripheral cholinergic synapse and its signaling cascades responsible for development are similar to those for the central synapses. However, the effect of lead exposure on the formation of NMJ in mammals is unclear. In the present study, a NG108-15/C2C12 coculture model was used to measure the acetylcholine receptor (AChR) aggregates formed on the myotubes which was an early hallmark for the NMJ formation. AChR aggregates were identified by alpha-bungarotoxin under fluorescent microscope. Single dose of lead acetate with final concentrations at 10(-3), 10(-1), or 10 microM was applied to dishes at the beginning of coculturing. Following 3-day exposure, although NG108-15 cells could extend long neurites to nearby myotubes, obvious dose-dependent attenuation in AChR aggregation was shown. The averaged area of an AChR aggregate, the averaged number of AChR aggregates per myotube, and the total area of AChR aggregates per myotube were all significantly decreased. In addition, the distribution percentages of various sizes of AChR aggregates showed that almost half of the AChR aggregates were formed with a size of 2-5 microm2 regardless of lead exposure. After treating 10 microM of lead acetate, significantly more AChR aggregates ranged from 2 to 20 microm2 were formed and significantly less AChR aggregates larger than 20 microm2 were formed. These results indicated that lead exposure reduced the extent of AChR aggregation concerning both the size and number of AChR aggregates and large AChR aggregates could hardly be formed after acute high-level lead exposure. No significant change was found in the total amount of AChRs on the myotubes after lead exposure, which indicated that the attenuation of AChR aggregation was not caused by reducing the synthesis of AChRs but by remaining dispersed pattern of AChRs on the myotubes. These data suggest that lead exposure exerts detrimental effects on the formation of NMJ.

Modifier of cell adhesion regulates N-cadherin-mediated cell-cell adhesion and neurite outgrowth.

Modifier of cell adhesion (MOCA) is a member of the dedicator of cytokinesis 180 family of proteins and is highly expressed in CNS neurons. MOCA is associated with Alzheimer's disease tangles and regulates the accumulation of amyloid precursor protein and beta-amyloid. Here, we report that MOCA modulates cell-cell adhesion and morphology. MOCA increases the accumulation of adherens junction proteins, including N-cadherin and beta-catenin, whereas reducing endogenous MOCA expression lowers cell-cell aggregation and N-cadherin expression. MOCA colocalizes with N-cadherin and actin in areas of cell-cell and cell substratum contact and is expressed in neuronal processes. MOCA accumulates during neuronal differentiation, and its expression enhances NGF-induced neurite outgrowth and morphological complexity. We conclude that MOCA regulates N-cadherin-mediated cell-cell adhesion and neurite outgrowth.

Co-induction of growth-associated protein GAP-43 and neuronal nitric oxide synthase in the cochlear nucleus following cochleotomy.

In adult animals, cochlear lesioning leads to a reactive synaptogenesis with a reemergence of growth-associated protein, GAP-43, in the auditory brainstem nuclei. In addition, nitric oxide (NO) is also implicated in synaptogenesis. Three isoforms of nitric oxide synthase (NOS) responsible for generating NO have been identified and, in neurons, the predominant isoform is neuronal NOS (nNOS). Studies in visual or olfactory systems have found that the NOS expression often correlates with periods of axonal outgrowth and synapse formation; whether NO plays a similar role in the auditory brainstem needs to be examined. In the present study, a unilateral cochleotomy was performed in adult mice to examine the relationship between the reemergence of GAP-43 and the expression pattern of nNOS. Following surgery, GAP-43 re-emerged in the ipsilateral anterior ventral cochlear nucleus (AVCN) and the immunoreactivity reached a climax around postoperative day (POD) 8; the same expression pattern as that reported in the previous literature is the indicator of synaptogenesis. As for the nNOS immunoreactivity, a dramatic redistribution from a mostly cytoplasmal to a predominantly membranous localization in the ipsilateral AVCN was found especially at POD 4. A similar redistribution pattern in the ipsilateral AVCN for the N-methyl-D-aspartate (NMDA) receptor was also observed at POD 4, corresponding to the fact that the activation of nNOS is coupled to calcium influx via the NMDA-receptor. Furthermore, the expression of cyclic guanosine monophosphate (cGMP) is an indicator for activity of soluble guanylyl cyclase (sGC), the substrate of NO, which reveals the target area of NO. Therefore, cGMP immunoreactivity was also examined and an obvious increase of cytoplasmal cGMP expression was observed around POD 4. Accordingly, it is suggested that nNOS activity correlates closely with the reactive synaptogenesis following a cochleotomy. Further evidence is shown by the results of fluorescent double staining; nNOS-positive cells were surrounded by GAP-43 labeled regions that appeared to be presynaptic boutons, and the vast majority of nNOS-positive cells also expressed cGMP. The former result indicates that, after surgery, there should be new terminal endings projecting onto the nNOS-positive cells in the AVCN. Furthermore, the latter result suggests a possible role of an autocrine mediator for nNOS in the AVCN.