Social Interaction Test in Home Cage as a Novel and Ethological Measure of Social Behavior in Mice.

Sociability is the disposition to interact with one another. Rodents have a rich repertoire of social behaviors and demonstrate strong sociability. Various methods have been established to measure the sociability of rodents in simple and direct ways, which includes reciprocal social interaction, juvenile social play, and three-chamber social tests. There are possible confounding factors while performing some of these tasks, such as aggression, avoidance of interaction by the stimulus mouse, exposure to a new environment, and lengthy procedures. The present study devised a method to complement these shortcomings and measure sociability as a group in the home cage setting, which prevents group-housed mice from isolation or exposure to a new environment. The home cage social test can allow high-throughput screening of social behaviors in a short amount of time. We developed two types of home cage setup: a home cage social target interaction test that measures sociability by putting the wire cage in the center area of the cage and a home cage two-choice sociability and social preference test that measures both sociability or social preference by putting cage racks at opposite sides of the cage. Interestingly, our results showed that the two types of home cage setup that we used in this study can extract abnormal social behaviors in various animal models, similar to the three-chamber assay. Thus, this study establishes a new and effective method to measure sociability or social preference that could be a complementary assay to evaluate the social behavior of mice in various setup conditions.

Comparative Behavioral Correlation of High and Low-Performing Mice in the Forced Swim Test.

Behavioral analysis in mice provided important contributions in helping understand and treat numerous neurobehavioral and neuropsychiatric disorders. The behavioral performance of animals and humans is widely different among individuals but the neurobehavioral mechanism of the innate difference is seldom investigated. Many neurologic conditions share comorbid symptoms that may have common pathophysiology and therapeutic strategy. The forced swim test (FST) has been commonly used to evaluate the "antidepressant" properties of drugs yet the individual difference analysis of this test was left scantly investigated along with the possible connection among other behavioral domains. This study conducted an FST-screening in outbred CD-1 male mice and segregated them into three groups: high performers (HP) or the active swimmers, middle performers (MP), and low performers (LP) or floaters. After which, a series of behavioral experiments were performed to measure their behavioral responses in the open field, elevated plus maze, Y maze, three-chamber social assay, novel object recognition, delay discounting task, and cliff avoidance reaction. The behavioral tests battery revealed that the three groups displayed seemingly correlated differences in locomotor activity and novel object recognition but not in other behaviors. This study suggests that the HP group in FST has higher locomotor activity and novelty-seeking tendencies compared to the other groups. These results may have important implications in creating behavior database in animal models that could be used for predicting interconnections of various behavioral domains, which eventually helps to understand the neurobiological mechanism controlling the behaviors in individual subjects.

Tenovin-1 Induces Senescence and Decreases Wound-Healing Activity in Cultured Rat Primary Astrocytes.

Brain aging induces neuropsychological changes, such as decreased memory capacity, language ability, and attention; and is also associated with neurodegenerative diseases. However, most of the studies on brain aging are focused on neurons, while senescence in astrocytes has received less attention. Astrocytes constitute the majority of cell types in the brain and perform various functions in the brain such as supporting brain structures, regulating blood-brain barrier permeability, transmitter uptake and regulation, and immunity modulation. Recent studies have shown that SIRT1 and SIRT2 play certain roles in cellular senescence in peripheral systems. Both SIRT1 and SIRT2 inhibitors delay tumor growth in vivo without significant general toxicity. In this study, we investigated the role of tenovin-1, an inhibitor of SIRT1 and SIRT2, on rat primary astrocytes where we observed senescence and other functional changes. Cellular senescence usually is characterized by irreversible cell cycle arrest and induces senescence- associated ß-galactosidase (SA-ß-gal) activity. Tenovin-1-treated astrocytes showed increased SA-ß-gal-positive cell number, senescence-associated secretory phenotypes, including IL-6 and IL-1ß, and cell cycle-related proteins like phospho-histone H3 and CDK2. Along with the molecular changes, tenovin-1 impaired the wound-healing activity of cultured primary astrocytes. These data suggest that tenovin-1 can induce cellular senescence in astrocytes possibly by inhibiting SIRT1 and SIRT2, which may play particular roles in brain aging and neurodegenerative conditions.

Effects of Several Cosmetic Preservatives on ROS-Dependent Apoptosis of Rat Neural Progenitor Cells.

Benzalkonium chloride, diazolidinyl urea, and imidazolidinyl urea are commonly used preservatives in cosmetics. Recent reports suggested that these compounds may have cellular and systemic toxicity in high concentration. In addition, diazolidinyl urea and imidazolidinyl urea are known formaldehyde (FA) releasers, raising concerns for these cosmetic preservatives. In this study, we investigated the effects of benzalkonium chloride, diazolidinyl urea, and imidazolidinyl urea on ROS-dependent apoptosis of rat neural progenitor cells (NPCs) in vitro. Cells were isolated and cultured from embryonic day 14 rat cortices. Cultured cells were treated with 1-1,000 nM benzalkonium chloride, and 1-50 µM diazolidinyl urea or imidazolidinyl urea at various time points to measure the reactive oxygen species (ROS). PI staining, MTT assay, and live-cell imaging were used for cell viability measurements. Western blot was carried out for cleaved caspase-3 and cleaved caspase-8 as apoptotic protein markers. In rat NPCs, ROS production and cleaved caspase-8 expression were increased while the cell viability was decreased in high concentrations of these substances. These results suggest that several cosmetic preservatives at high concentrations can induce neural toxicity in rat brains through ROS induction and apoptosis.

Activation of Glucagon-Like Peptide-1 Receptor Promotes Neuroprotection in Experimental Autoimmune Encephalomyelitis by Reducing Neuroinflammatory Responses.

The signaling axis of glucagon-like peptide-1 (GLP-1)/GLP-1 receptor (GLP-1R) has been an important component in overcoming diabetes, and recent reports have uncovered novel beneficial roles of this signaling axis in central nervous system (CNS) disorders, such as Alzheimer's disease, Parkinson's disease, and cerebral ischemia, accelerating processes for exendin-4 repositioning. Here, we studied whether multiple sclerosis (MS) could be a complement to the CNS disorders that are associated with the GLP-1/GLP-1R signaling axis. Both components of the signaling axis, GLP-1 and GLP-1R proteins, are expressed in neurons, astrocytes, and microglia in the spinal cord of normal mice. In particular, they are abundant in Iba1-positive microglia. Upon challenge by experimental autoimmune encephalomyelitis (EAE), an animal model of MS, the mRNA expression of both GLP-1 and GLP-1R was markedly downregulated in EAE-symptomatic spinal cords, indicating attenuated activity of GLP-1/GLP-1R signaling in EAE. Such a downregulation obviously occurred in LPS-stimulated rat primary microglia, a main cell type to express both GLP-1 and GLP-1R, further indicating attenuated activity of GLP-1/GLP-1R signaling in activated microglia. To investigate whether increased activity of GLP-1R has a therapeutic benefit, exendin-4 (5 µg/kg, i.p.), a GLP-1R agonist, was administered daily to EAE-symptomatic mice. Exendin-4 administration to symptomatic EAE mice significantly improved the clinical signs of the disease, along with the reversal of histopathological sequelae such as cell accumulation, demyelination, astrogliosis, microglial activation, and morphological transformation of activated microglia in the injured spinal cord. Such an improvement by exendin-4 was comparable to that by FTY720 (3 mg/kg, i.p.), a drug for MS. The neuroprotective effects of exendin-4 against EAE were also associated with decreased mRNA expression of proinflammatory cytokines, such as interleukin (IL)-17, IL-1ß, IL-6, and tumor necrosis factor (TNF)-α, all of which are usually upregulated in injured sites of the EAE spinal cord. Interestingly, exendin-4 exposure similarly reduced mRNA levels of IL-1ß and TNF-α in LPS-stimulated microglia. Furthermore, exendin-4 administration significantly attenuated activation of NF-κB signaling in EAE spinal cord and LPS-stimulated microglia. Collectively, the current study demonstrates the therapeutic potential of exendin-4 for MS by reducing immune responses in the CNS, highlighting the importance of the GLP-1/GLP-1R signaling axis in the development of a novel therapeutic strategy for MS.

Sex Differences in Autism-Like Behavioral Phenotypes and Postsynaptic Receptors Expression in the Prefrontal Cortex of TERT Transgenic Mice.

Autism spectrum disorder (ASD) remains unexplained and untreated despite the high attention of research in recent years. Aside from its various characteristics is the baffling male preponderance over the female population. Using a validated animal model of ASD which is the telomerase reverse transcriptase overexpressing mice (TERT-tg), we conducted ASD-related behavioral assessments and protein expression experiments to mark the difference between male and females of this animal model. After statistically analyzing the results, we found significant effects of TERT overexpression in sociability, social novelty preference, anxiety, nest building, and electroseizure threshold in the males but not their female littermates. Along these differences are the male-specific increased expressions of postsynaptic proteins which are the NMDA and AMPA receptors in the prefrontal cortex. The vGluT1 presynaptic proteins, but not GAD, were upregulated in both sexes of TERT-tg mice, although it is more significantly pronounced in the male group. Here, we confirmed that the behavioral effect of TERT overexpression in mice was male-specific, suggesting that the aberration of this gene and its downstream pathways preferentially affect the functional development of the male brain, consistent with the male preponderance in ASD.

Eupatilin exerts neuroprotective effects in mice with transient focal cerebral ischemia by reducing microglial activation.

Microglial activation and its-driven neuroinflammation are characteristic pathogenetic features of neurodiseases, including focal cerebral ischemia. The Artemisia asiatica (Asteraceae) extract and its active component, eupatilin, are well-known to reduce inflammatory responses. But the therapeutic potential of eupatilin against focal cerebral ischemia is not known, along with its anti-inflammatory activities on activated microglia. In this study, we investigated the neuroprotective effect of eupatilin on focal cerebral ischemia through its anti-inflammation, particularly on activated microglia, employing a transient middle cerebral artery occlusion/reperfusion (tMCAO), combined with lipopolysaccharide-stimulated BV2 microglia. Eupatilin exerted anti-inflammatory responses in activated BV2 microglia, in which it reduced secretion of well-known inflammatory markers, including nitrite, IL-6, TNF-α, and PGE2, in a concentration-dependent manner. These observed in vitro effects of eupatilin led to in vivo neuroprotection against focal cerebral ischemia. Oral administration of eupatilin (10 mg/kg) in a therapeutic paradigm significantly reduced brain infarction and improved neurological functions in tMCAO-challenged mice. The same benefit was also observed when eupatilin was given even within 5 hours after MCAO induction. In addition, the neuroprotective effects of a single administration of eupatilin (10 mg/kg) immediately after tMCAO challenge persisted up to 3 days after tMCAO. Eupatilin administration reduced the number of Iba1-immunopositive cells across ischemic brain and induced their morphological changes from amoeboid into ramified in the ischemic core, which was accompanied with reduced microglial proliferation in ischemic brain. Eupatilin suppressed NF-κB signaling activities in ischemic brain by reducing IKKα/ß phosphorylation, IκBα phosphorylation, and IκBα degradation. Overall, these data indicate that eupatilin is a neuroprotective agent against focal cerebral ischemia through the reduction of microglial activation.

Supplementation of Korean Red Ginseng improves behavior deviations in animal models of autism.

BACKGROUND: Autism spectrum disorder (ASD) is heterogeneous neurodevelopmental disorders that primarily display social and communication impairments and restricted/repetitive behaviors. ASD prevalence has increased in recent years, yet very limited therapeutic targets and treatments are available to counteract the incapacitating disorder. Korean Red Ginseng (KRG) is a popular herbal plant in South Korea known for its wide range of therapeutic effects and nutritional benefits and has recently been gaining great scientific attention, particularly for its positive effects in the central nervous system. OBJECTIVES: Thus, in this study, we investigated the therapeutic potential of KRG in alleviating the neurobehavioral deficits found in the valproic acid (VPA)-exposed mice models of ASD. DESIGN: Starting at 21 days old (P21), VPA-exposed mice were given daily oral administrations of KRG solution (100 or 200 mg/kg) until the termination of all experiments. From P28, mice behaviors were assessed in terms of social interaction capacity (P28-29), locomotor activity (P30), repetitive behaviors (P32), short-term spatial working memory (P34), motor coordination (P36), and seizure susceptibility (P38). RESULTS: VPA-exposed mice showed sociability and social novelty preference deficits, hyperactivity, increased repetitive behavior, impaired spatial working memory, slightly affected motor coordination, and high seizure susceptibility. Remarkably, long-term KRG treatment in both dosages normalized all the ASD-related behaviors in VPA-exposed mice, except motor coordination ability. CONCLUSION: As a food and herbal supplement with various known benefits, KRG demonstrated its therapeutic potential in rescuing abnormal behaviors related to autism caused by prenatal environmental exposure to VPA.

Ginkgo biloba Extract (EGb 761®) Inhibits Glutamate-induced Up-regulation of Tissue Plasminogen Activator Through Inhibition of c-Fos Translocation in Rat Primary Cortical Neurons.

EGb 761(®) , a standardized extract of Ginkgo biloba leaves, has antioxidant and antiinflammatory properties in experimental models of neurodegenerative disorders such as stroke and Alzheimer's disease. Tissue plasminogen activator (tPA) acts a neuromodulator and plays a crucial role in the manifestation of neurotoxicity leading to exaggerated neuronal cell death in neurological insult conditions. In this study, we investigated the effects of EGb 761 on the basal and glutamate-induced activity and expression of tPA in rat primary cortical neurons. Under basal condition, EGb 761 inhibited both secreted and cellular tPA activities, without altering tPA mRNA level, as modulated by the activation of p38. Compared with basal condition, EGb 761 inhibited the glutamate-induced up-regulation of tPA mRNA resulting in the normalization of overt tPA activity and expression. c-Fos is a component of AP-1, which plays a critical role in the modulation of tPA expression. Interestingly, EGb 761 inhibited c-Fos nuclear translocation without affecting c-Fos expression in glutamate-induced rat primary cortical neurons. These results demonstrated that EGb 761 can modulate tPA activity under basal and glutamate-stimulated conditions by both translational and transcriptional mechanisms. Thus, EGb 761 could be a potential and effective therapeutic strategy in tPA-excessive neurotoxic conditions.

High sucrose consumption during pregnancy induced ADHD-like behavioral phenotypes in mice offspring.

In recent years, the average consumption of sugar in humans from all ages has remarkably increased, exceeding the recommended limit. Pregnancy is a critical time for the global development of offsprings who are vulnerable to the deleterious effects of environmental factors. In this study, we investigated whether high sucrose consumption during pregnancy could affect the attention-deficit hyperactivity disorder (ADHD)-like neurobehavioral outcomes in offspring mice. Pregnant mice were randomly grouped and orally administered with either water as control (Con) or 30% wt/vol sucrose diluted in water at 6 (Suc6) or 9 (Suc9) g/kg dosage per day from gestational days 6 to 15. After the weaning period, offspring mice underwent a series of behavioral testing for locomotor activity, attention, and impulsivity. Although there is no obvious difference in gross development of offspring mice such as weight gain, high sucrose-exposed offspring mice showed a significantly increased locomotor activity. Moreover, these mice exhibited a dose-dependent decrease in attention and increase in impulsivity. In the striatum, a significantly increased dopamine transporter (DAT) mRNA expression was found in the Suc9 group along with dose-dependent decreases in the Drd1, Drd2 and Drd4 dopamine receptor subtypes. Furthermore, synaptosomal DAT protein expression was increased about twofold in the Suc9 group. Prenatal fructose exposure also induced hyperactive behavior in offspring mice suggesting the essential role of fructose in the dysregulated neurobehavioral development. These findings suggest prenatal sucrose consumption as a new risk factor for ADHD, which may need further attention and investigation in humans.

The Epigenetic Reader BRD2 as a Specific Modulator of PAI-1 Expression in Lipopolysaccharide-Stimulated Mouse Primary Astrocytes.

The post translational modification of lysine acetylation is a key mechanism that regulates chromatin structure. Epigenetic readers, such as the BET domains, are responsible for reading histone lysine acetylation which is a hallmark of open chromatin structure, further providing a scaffold that can be accessed by RNA polymerases as well as transcription factors. Recently, several reports have assessed and highlighted the roles of epigenetic readers in various cellular contexts. However, little is known about their role in the regulation of inflammatory genes, which is critical in exquisitely tuning inflammatory responses to a variety of immune stimuli. In this study, we investigated the role of epigenetic readers BRD2 and BRD4 in the lipopolysaccharide (LPS)-induced immune responses in mouse primary astrocytes. Inflammatory stimulation by LPS showed that the levels of Brd2 mRNA and protein were increased, while Brd4 mRNA levels did not change. Knocking down of Brd2 mRNA using specific small interfering RNA (siRNA) in cultured mouse primary astrocytes inhibited LPS-induced mRNA expression and secretion of plasminogen activator inhibitor-1 (PAI-1). However, no other pro-inflammatory cytokines, such as Il-6, Il-1ß and Tnf-α, were affected. Indeed, treatment with bromodomain-containing protein inhibitor, JQ1, blocked Pai-1 mRNA expression through the inhibition of direct BRD2 protein-binding and active histone modification on Pai-1 promoter. Taken together, our data suggest that BRD2 is involved in the modulation of neuroinflammatory responses through PAI-1 and via the regulation of epigenetic reader BET protein, further providing a potential novel therapeutic strategy in neuroinflammatory diseases.

Proteinase 3 Induces Neuronal Cell Death Through Microglial Activation.

Proteinase 3 (PR3) is released from neutrophil granules and is involved in the inflammatory process. PR3 is implicated in antimicrobial defense and cell death, but the exact role of PR3 in the brain is less defined. Microglia is the major immune effector cells in the CNS and is activated by brain injury. In the present study, the effect of PR3 on glial activation was investigated. Microglial activation was assessed by the intracellular level of reactive oxygen species and expression of inflammatory cytokines. The conditioned media from activated microglia by PR3 was used for measuring the neurotoxic effects of PR3-stimulated microglia. The effects of PR3 in vivo were measured by microinjecting PR3 into the rat brain. Herein we show that PR3 increased the inflammatory responses including the intracellular ROS and pro-inflammatory cytokine production in rat primary microglia. Conditioned media from PR3-treated microglia induced neuronal cell death in a concentration dependent manner. Furthermore, microinjected PR3 into the striatum of the rat brain induced microglial activation and neuronal cell death. Interestingly treatment with anti-PR3 monoclonal antibody and protease inhibitors ameliorated microglial activation induced by PR3 in primary microglia and striatum, which also prevented neuronal cell death in both conditions. The data presented here suggest that PR3 is a direct modulator of microglial activation and causes neuronal death through the augmentation of inflammatory responses. We suggest that PR3 could be a new modulator of neuroinflammation, and blocking PR3 would be a promising novel therapeutic target for neuroinflammatory disease such as stroke and Alzheimer's disease.

Ginkgo biloba extract (Egb761) attenuates zinc-induced tau phosphorylation at Ser262 by regulating GSK3ß activity in rat primary cortical neurons.

In the brain, an excessive amount of zinc promotes the deposition of ß-amyloid proteins and the intraneuronal accumulation of neurofibrillary tangles composed of hyperphosphorylated tau proteins. These consequences are key neuropathological traits that reflect Alzheimer's disease. Egb761, a standardized Ginkgo biloba extract, is a powerful antioxidant known to exhibit neuroprotective actions. In this study, we investigated whether Egb761 can counteract the zinc-induced tau phosphorylation in rat primary cortical neurons. To determine the modification of tau phosphorylation by Egb761 treatment, we conducted Western blot analyses, MTT assay, ROS measurements and immunocytochemistry. We found that zinc-induced tau phosphorylation occurred at Ser262 in a time- and dose-dependent manner while other tau sites were not phosphorylated. Tau phosphorylation at Ser262 was increased 30 min after zinc treatment and peaked 3 h after zinc treatment (control: 100 ± 1.2%, 30 min: 253 ± 2.24%, 3 h: 373 ± 1.3%). Interestingly, Egb761 treatment attenuated the zinc-induced tau hyperphosphorylation at Ser262 in a concentration-dependent manner while the antioxidant N-acetylcysteine showed a similar effect. Furthermore, Egb761 prevented the zinc-induced activation of p38 MAPK and GSK3ß, as well as the zinc-induced increase in ROS production and neuronal cell death. Lithium chloride also inhibited the zinc-induced tau phosphorylation but did not affect ROS levels. These results suggest the potential of Egb761 for inhibiting the zinc-induced tau phosphorylation at Ser262 through its anti-oxidative actions involving the regulation of GSK3ß. Therefore, Egb761 may be a candidate for the treatment of tauopathy present in neurological disorders such as Alzheimer's disease.

Effects of donepezil, an acetylcholinesterase inhibitor, on neurogenesis in a rat model of vascular dementia.

Vascular dementia (VaD) is the second most common form of dementia caused by cerebrovascular disease. Several recent reports demonstrated that cholinergic deficits are implicated in the pathogenesis of VaD and that cholinergic therapies have shown improvement of cognitive function in patients with VaD. However, the precise mechanisms by which donepezil achieves its effects on VaD are not fully understood. Donepezil hydrochloride is an acetylcholinesterase inhibitor (AChEI) currently used for the symptomatic treatment of Alzheimer's disease (AD). Several lines of evidence have demonstrated that AChEIs such as donepezil promote neurogenesis in the central nervous system. We investigated whether donepezil regulated hippocampal neurogenesis after bilateral common carotid artery occlusion (BCCAO) in rats, a commonly used animal model of VaD. To evaluate the effect of donepezil on neurogenesis, we orally treated rats with donepezil (10mg/kg) once a day for 3weeks, and injected BrdU over the same 3-week period to label newborn cells. The doses of donepezil that we used have been reported to activate cholinergic activity in rats. After 3weeks, a water maze task was performed on these rats to test spatial learning, and a subsequent histopathological evaluation was conducted. Donepezil improved memory impairment and increased the number of BrdU-positive cells in the dentate gyrus (DG) of BCCAO animals. These results indicated that donepezil improves cognitive function and enhances the survival of newborn neurons in the DG in our animal model of VaD, possibly by enhancing the expression of choline acetyltransferase and brain-derived neurotropic factor.

Resveratrol down-regulates a glutamate-induced tissue plasminogen activator via Erk and AMPK/mTOR pathways in rat primary cortical neurons.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene, RSV) is a polyphenolic compound present in a variety of plant species (including grapes) that produces a myriad of biological activities including anti-inflammatory, antioxidant and neuroprotective effects. In this study, we investigate the effects of resveratrol on the basal and glutamate-stimulated expression and activity of a tissue plasminogen activator (tPA) that plays neuromodulatory or neurotoxic roles in many different neurological situations. Under basal conditions, resveratrol decreased the tPA expression and activity without affecting the tPA mRNA level in rat primary cortical neurons. RSV induced AMPK phosphorylation and inhibited mTOR phosphorylation. Inhibition of AMPK phosphorylation using compound C prevented resveratrol-induced down-regulation of tPA activity. This suggested that AMPK/mTOR-dependent translational inhibition contributes to the down-regulation of the tPA. Under glutamate-stimulated conditions of rat primary cortical neurons, tPA activity and expression were increased along with increased tPA mRNA expression but afterward treatment of RSV inhibited the glutamate-induced increase in tPA activity and expression and tPA mRNA expression. Glutamate stimulation induced activation of Akt and MAPK pathways as well as mTOR which were inhibited by RSV. Interestingly, the Erk pathway inhibitor U0126, but neither PI3K-Akt inhibitor LY294002 nor p38 inhibitor SB203580, mimicked the inhibitory action of RSV on glutamate-induced tPA up-regulation. This suggested the essential role of Erk in the transcriptional up-regulation of tPA expression, which is targeted by RSV. Glutamate stimulation induced neuronal cell death as determined by PI staining and MTT assay. However, RSV protected the cultured rat primary cortical neurons from glutamate-induced cell death as paralleled with the changes in tPA expression. These results suggested that RSV can modulate tPA activity under basal and stimulated conditions by both translational and transcriptional mechanisms. The regulation of the tPA by RSV provides additional therapeutic targets on top of the growing number of molecular substrates of RSV's action in the brain.

Valproic Acid Regulates α-Synuclein Expression through JNK Pathway in Rat Primary Astrocytes.

Although the role of α-synuclein aggregation on Parkinson's disease is relatively well known, the physiological role and the regulatory mechanism governing the expression of α-synuclein are unclear yet. We recently reported that α-synuclein is expressed and secreted from cultured astrocytes. In this study, we investigated the effect of valproic acid (VPA), which has been suggested to provide neuroprotection by increasing α-synuclein in neuron, on α-synuclein expression in rat primary astrocytes. VPA concentrationdependently increased the protein expression level of α-synuclein in cultured rat primary astrocytes with concomitant increase in mRNA expression level. Likewise, the level of secreted α-synuclein was also increased by VPA. VPA increased the phosphorylation of Erk1/2 and JNK and pretreatment of a JNK inhibitor SP600125 prevented the VPA-induced increase in α-synuclein. Whether the increased α-synuclein in astrocytes is involved in the reported neuroprotective effects of VPA awaits further investigation.

Transcriptional Upregulation of Plasminogen Activator Inhibitor-1 in Rat Primary Astrocytes by a Proteasomal Inhibitor MG132.

Plasminogen activator inhibitor-1 (PAI-1) is a member of serine protease inhibitor family, which regulates the activity of tissue plasminogen activator (tPA). In CNS, tPA/PAI-1 activity is involved in the regulation of a variety of cellular processes such as neuronal development, synaptic plasticity and cell survival. To gain a more insights into the regulatory mechanism modulating tPA/PAI-1 activity in brain, we investigated the effects of proteasome inhibitors on tPA/PAI-1 expression and activity in rat primary astrocytes, the major cell type expressing both tPA and PAI-1. We found that submicromolar concentration of MG132, a cell permeable peptide-aldehyde inhibitor of ubiquitin proteasome pathway selectively upregulates PAI-1 expression. Upregulation of PAI-1 mRNA as well as increased PAI-1 promoter reporter activity suggested that MG132 transcriptionally increased PAI-1 expression. The induction of PAI-1 downregulated tPA activity in rat primary astrocytes. Another proteasome inhibitor lactacystin similarly increased the expression of PAI-1 in rat primary astrocytes. MG132 activated MAPK pathways as well as PI3K/Akt pathways. Inhibitors of these signaling pathways reduced MG132-mediated upregulation of PAI-1 in varying degrees and most prominent effects were observed with SB203580, a p38 MAPK pathway inhibitor. The regulation of tPA/PAI-1 activity by proteasome inhibitor in rat primary astrocytes may underlie the observed CNS effects of MG132 such as neuroprotection.

Proteinase 3 induces oxidative stress-mediated neuronal death in rat primary cortical neuron.

The recruitment of neutrophils into the cerebral microcirculation occurs, especially, in acute brain diseases like a focal cerebral ischemia and plays important role in pathological processes. Proteinase 3 is one of the three major proteinases expressed in neutrophils but no reports are available whether proteinase 3 can modulate neuronal survival. In this study, treatment of cultured rat primary cortical neuron with proteinase 3 induced overt reactive oxygen species production and decreased total glutathione contents as well as disruption of mitochondrial transmembrane potential. Proteinase 3 induced neuronal cell death as evidenced by MTT analysis as well as propidium iodide staining, which was prevented by pretreatment with an antioxidant, N-acetyl cysteine. Proteinase 3 increased activation of procaspase-3 and altered expression level of apoptotic regulator proteins, such as Bcl-2, Bax, and Bcl-xL. Similar to in vitro data, a direct microinjection of proteinase 3 into striatum of rat brain induced neuronal death, which was mediated by reactive oxygen species. These results suggest that proteinase 3 is new essential regulator of neuronal cell death pathway in a condition of excess neutrophil encounter in neuroinflammatory conditions.

Glucose deprivation reversibly down-regulates tissue plasminogen activator via proteasomal degradation in rat primary astrocytes.

AIMS: Tissue plasminogen activator (tPA) is an essential neuromodulator whose involvement in multiple functions such as synaptic plasticity, cytokine-like immune function and regulation of cell survival mandates rapid and tight tPA regulation in the brain. We investigated the possibility that a transient metabolic challenge induced by glucose deprivation may affect tPA activity in rat primary astrocytes, the main cell type responsible for metabolic regulation in the CNS. MAIN METHODS: Rat primary astrocytes were incubated in serum-free DMEM without glucose. Casein zymography was used to determine tPA activity, and tPA mRNA was measured by RT-PCR. The signaling pathways regulating tPA activity were identified by Western blotting. KEY FINDINGS: Glucose deprivation rapidly down-regulated the activity of tPA without affecting its mRNA level in rat primary astrocytes; this effect was mimicked by translational inhibitors. The down-regulation of tPA was accompanied by increased tPA degradation, which may be modulated by a proteasome-dependent degradation pathway. Glucose deprivation induced activation of PI3K-Akt-GSK3ß, p38 and AMPK, and inhibition of these pathways using LY294002, SB203580 and compound C significantly inhibited glucose deprivation-induced tPA down-regulation, demonstrating the essential role of these pathways in tPA regulation in glucose-deprived astrocytes. SIGNIFICANCE: Rapid and reversible regulation of tPA activity in rat primary astrocytes during metabolic crisis may minimize energy-requiring neurologic processes in stressed situations. This effect may thereby increase the opportunity to invest cellular resources in cell survival and may allow rapid re-establishment of normal cellular function after the crisis.