Soluble epoxide hydrolase promotes astrocyte survival in retinopathy of prematurity.

Polyunsaturated fatty acids such as docosahexaenoic acid (DHA) positively affect the outcome of retinopathy of prematurity (ROP). Given that DHA metabolism by cytochrome P450 and soluble epoxide hydrolase (sEH) enzymes affects retinal angiogenesis and vascular stability, we investigated the role of sEH in a mouse model of ROP. In WT mice, hyperoxia elicited tyrosine nitration and inhibition of sEH and decreased generation of the DHA-derived diol 19,20-dihydroxydocosapentaenoic acid (19,20-DHDP). Correspondingly, in a murine model of ROP, sEH-/- mice developed a larger central avascular zone and peripheral pathological vascular tuft formation than did their WT littermates. Astrocytes were the cells most affected by sEH deletion, and hyperoxia increased astrocyte apoptosis. In rescue experiments, 19,20-DHDP prevented astrocyte loss by targeting the mitochondrial membrane to prevent the hyperoxia-induced dissociation of presenilin-1 and presenilin-1-associated protein to attenuate poly ADP-ribose polymerase activation and mitochondrial DNA damage. Therapeutic intravitreal administration of 19,20-DHDP not only suppressed astrocyte loss, but also reduced pathological vascular tuft formation in sEH-/- mice. Our data indicate that sEH activity is required for mitochondrial integrity and retinal astrocyte survival in ROP. Moreover, 19,20-DHDP may be more effective than DHA as a nutritional supplement for preventing retinopathy in preterm infants.

Glioprotective Effects of Lingonberry Extract Against Altered Cellular Viability, Acetylcholinesterase Activity, and Oxidative Stress in Lipopolysaccharide-Treated Astrocytes.

Altered astrocytic function is a contributing factor to the development of neurological diseases and neurodegeneration. Berry fruits exert neuroprotective effects by modulating pathways involved in inflammation, neurotransmission, and oxidative stress. The aim of this study was to examine the effects of the lingonberry extract on cellular viability and oxidative stress in astrocytes exposed to lipopolysaccharide (LPS). In the reversal protocol, primary astrocytic cultures were first exposed to 1 µg/mL LPS for 3 h and subsequently treated with lingonberry extract (10, 30, 50, and 100 µg/mL) for 24 and 48 h. In the prevention protocol, exposure to the lingonberry extract was performed before treatment with LPS. In both reversal and prevention protocols, the lingonberry extracts, from 10 to 100 µg/mL, attenuated LPS-induced increase in reactive oxygen species (around 55 and 45%, respectively, P < 0.01), nitrite levels (around 50 and 45%, respectively, P < 0.05), and acetylcholinesterase activity (around 45 and 60%, respectively, P < 0.05) in astrocytic cultures at 24 and 48 h. Also, in both reversal and prevention protocols, the lingonberry extract also prevented and reversed the LPS-induced decreased cellular viability (around 45 and 90%, respectively, P < 0.05), thiol content (around 55 and 70%, respectively, P < 0.05), and superoxide dismutase activity (around 50 and 145%, respectively, P < 0.05), in astrocytes at both 24 and 48 h. Our findings suggested that the lingonberry extract exerted a glioprotective effect through an anti-oxidative mechanism against LPS-induced astrocytic damage.

NFATc3 deficiency protects against high fat diet (HFD)-induced hypothalamus inflammation and apoptosis via p38 and JNK suppression.

Hypothalamic inflammation and apoptosis cause neural injury, playing an important role in metabolic syndrome development. Nuclear Factors of Activated T cells (NFATc3) show many physiological and pathological effects. However, the function of NFATc3 in high fat diet (HFD)-induced hypothalamus injury remains unknown. The wild type (WT) and NFATc3-knockout (KO) mice were subjected to HFD feeding for 16 weeks to examine NFATc3 function in vivo. Astrocytes isolated from WT or KO mice were cultured and exposed to fructose (Fru) in vitro. The liver damage, hypothalamus injury, pro-inflammatory markers, NF-κB (p65), Caspase-3 and mitogen-activated protein kinases (MAPKs) pathways were evaluated. NFATc3 was significantly up-regulated in hypothalamus from mice challenged with HFD, and in astrocytes incubated with Fru. Both in vivo and in vitro studies indicated that NFATc3-deletion attenuated metabolism syndrome, reduced inflammatory regulators expression, inactivated NF-κB (p65), Caspase-3 and p38/JNK signaling pathway. Of note, we identified that promoting p38 or JNK activation could rescue inflammatory response and apoptosis in NFATc3-KO astrocytes stimulated by Fru. Together, these findings revealed an important role of NFATc3 NFATc3 for HFD-induced metabolic syndrome and particularly hypothalamus injury, and understanding of the regulatory molecular mechanism might provide new and effective therapeutic strategies for prevention and treatment of hypothalamic damage associated with dietary obesity-associated neuroinflammation and apoptosis.

GSK-3ß inhibitor TDZD-8 reduces neonatal hypoxic-ischemic brain injury in mice.

AIMS: Glycogen synthase kinase 3ß (GSK-3ß) is activated following hypoxic-ischemic (HI) brain injury. TDZD-8 is a specific GSK-3ß inhibitor. Currently, the impact of inhibiting GSK-3ß in neonatal HI injury is unknown. We aimed to investigate the effect of TDZD-8 following neonatal HI brain injury. METHODS: Unilateral common carotid artery ligation followed by hypoxia was used to induce HI injury in postnatal day 7 mouse pups pretreated with TDZD-8 or vehicle. The infarct volume, whole-brain imaging, Nissl staining, and behavioral tests were used to evaluate the protective effect of TDZD-8 on the neonatal brain and assess functional recovery after injury. Western blot was used to evaluate protein levels of phosphorylated protein kinase B (Akt), GSK-3ß, and cleaved caspase-3. Protein levels of cleaved caspase-3, neuronal marker, and glial fibrillary acidic protein were detected through immunohistochemistry. RESULTS: Pretreatment with TDZD-8 significantly reduced brain damage and improved neurobehavioral outcomes following HI injury. TDZD-8 reversed the reduction of phosphorylated Akt and GSK-3ß, and the activation of caspase-3 induced by hypoxia-ischemia. In addition, TDZD-8 suppressed apoptotic cell death and reduced reactive astrogliosis. CONCLUSION: TDZD-8 has the therapeutic potential for hypoxic-ischemic brain injury in neonates. The neuroprotective effect of TDZD-8 appears to be mediated through its antiapoptotic activity and by reducing astrogliosis.

Caveolin-1 is a checkpoint regulator in hypoxia-induced astrocyte apoptosis via Ras/Raf/ERK pathway.

Astrocytes, the most numerous cells in the human brain, play a central role in the metabolic homeostasis following hypoxic injury. Caveolin-1 (Cav-1), a transmembrane scaffolding protein, has been shown to converge prosurvival signaling in the central nerve system. The present study aimed to investigate the role of Cav-1 in the hypoxia-induced astrocyte injury. We also examined how Cav-1 alleviates apoptotic astrocyte death. To this end, primary astrocytes were exposed to oxygen-glucose deprivation (OGD) for 6 h and a subsequent 24-h reoxygenation to mimic hypoxic injury. OGD significantly reduced Cav-1 expression. Downregulation of Cav-1 using Cav-1 small interfering RNA dramatically worsened astrocyte cell damage and impaired cellular glutamate uptake after OGD, whereas overexpression of Cav-1 with Cav-1 scaffolding domain peptide attenuated OGD-induced cell apoptosis. Mechanistically, the expressions of Ras-GTP, phospho-Raf, and phospho-ERK were sequestered in Cav-1 small interfering RNA-treated astrocytes, yet were stimulated after supplementation with caveolin peptide. MEK/ERK inhibitor U0126 remarkably blocked the Cav-1-induced counteraction against apoptosis following hypoxia, indicating Ras/Raf/ERK pathway is required for the Cav-1's prosurvival role. Together, these findings support Cav-1 as a checkpoint for the in hypoxia-induced astrocyte apoptosis and warrant further studies targeting Cav-1 to treat hypoxic-ischemic brain injury.

Pyk2 is essential for astrocytes mobility following brain lesion.

Proline-rich tyrosine kinase 2 (Pyk2) is a calcium-dependent, non-receptor protein-tyrosine kinase of the focal adhesion kinase (FAK) family. Pyk2 is enriched in the brain, especially the forebrain. Pyk2 is highly expressed in neurons but is also present in astrocytes, where its role is not known. We used Pyk2 knockout mice (Pyk2(-/-) ) developed in our laboratory to investigate the function of Pyk2 in astrocytes. Morphology and basic properties of astrocytes in vivo and in culture were not altered in the absence of Pyk2. However, following stab lesions in the motor cortex, astrocytes-mediated wound filling was slower in Pyk2(-/-) than in wild-type littermates. In an in vitro wound healing model, Pyk2(-/-) astrocytes migrated slower than Pyk2(+/+) astrocytes. The role of Pyk2 in actin dynamics was investigated by treating astrocytic cultures with the actin-depolymerizing drug latrunculin B. Actin filaments re-polymerization after latrunculin B treatment was delayed in Pyk2(-/-) astrocytes as compared with wild-type astrocytes. We mimicked wound-induced activation by treating astrocytes in culture with tumor-necrosis factor alpha (TNFα), which increased Pyk2 phosphorylation at Tyr402. TNFα increased PKC activity, and Rac1 phosphorylation at Ser71 similarly in wild-type and Pyk2-deficient astrocytes. Conversely, we found that gelsolin, an actin-capping protein known to interact with Pyk2 in other cell types, was less enriched at the leading edge of migrating Pyk2(-/-) astrocytes, suggesting that its lack of recruitment mediated in part the effects of the mutation. This work shows the critical role of Pyk2 in astrocytes migration during wound healing.

Dorsomedial hindbrain catecholamine regulation of hypothalamic astrocyte glycogen metabolic enzyme protein expression: Impact of estradiol.

The brain astrocyte glycogen reservoir is a vital energy reserve and, in the cerebral cortex, subject among other factors to noradrenergic control. The ovarian steroid estradiol potently stimulates nerve cell aerobic respiration, but its role in glial glycogen metabolism during energy homeostasis or mismatched substrate supply/demand is unclear. This study examined the premise that estradiol regulates hypothalamic astrocyte glycogen metabolic enzyme protein expression during normo- and hypoglycemia in vivo through dorsomedial hindbrain catecholamine (CA)-dependent mechanisms. Individual astrocytes identified in situ by glial fibrillary acidic protein immunolabeling were laser-microdissected from the ventromedial hypothalamic (VMH), arcuate hypothalamic (ARH), and paraventricular hypothalamic (PVH) nuclei and the lateral hypothalamic area (LHA) of estradiol (E)- or oil (O)-implanted ovariectomized (OVX) rats after insulin or vehicle injection, and pooled within each site. Stimulation [VMH, LHA] or suppression [PVH, ARH] of basal glycogen synthase (GS) protein expression by E was reversed in the former three sites by caudal fourth ventricular pretreatment with the CA neurotoxin 6-hydroxydopamine (6-OHDA). E diminished glycogen phosphorylase (GP) protein profiles by CA-dependent [VMH, PVH] or -independent mechanisms [LHA]. Insulin-induced hypoglycemia (IIH) increased GS expression in the PVH in OVX+E, but reduced this protein in the PVH, ARH, and LHA in OVX+O. Moreover, IIH augmented GP expression in the VMH, LHA, and ARH in OVX+E and in the ARH in OVX+O, responses that normalized by 6-OHDA. Results demonstrate site-specific effects of E on astrocyte glycogen metabolic enzyme expression in the female rat hypothalamus, and identify locations where dorsomedial hindbrain CA input is required for such action. Evidence that E correspondingly increases and reduces basal GS and GP in the VMH and LHA, but augments the latter protein during IIH suggests that E regulates glycogen content and turnover in these structures during glucose sufficiency and shortage.

Estradiol regulation of hypothalamic astrocyte adenosine 5'-monophosphate-activated protein kinase activity: role of hindbrain catecholamine signaling.

Recent work challenges the conventional notion that metabolic monitoring in the brain is the exclusive function of neurons. This study investigated the hypothesis that hypothalamic astrocytes express the ultra-sensitive energy gauge adenosine 5'-monophosphate-activated protein kinase (AMPK), and that the ovarian hormone estradiol (E) controls activation of this sensor by insulin-induced hypoglycemia (IIH). E- or oil (O)-implanted ovariectomized (OVX) rats were pretreated by caudal fourth ventricular administration of the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA) prior to sc insulin or vehicle injection. Individual astrocytes identified in situ by glial fibrillary acidic protein immunolabeling were laser-microdissected from the ventromedial (VMH), arcuate (ARH), and paraventricular (PVH) nuclei and the lateral hypothalamic area (LHA), and pooled within each site for Western blot analysis of AMPK and phosphoAMPK (pAMPK) protein expression. In the VMH, baseline astrocyte AMPK and pAMPK levels were respectively increased or decreased in OVX+E versus OVX+O; these profiles did not differ between E and O rats in other hypothalamic loci. In E animals, astrocyte AMPK protein was reduced [VMH] or augmented [PVH; LHA] in response to either 6-OHDA or IIH. IIH increased astrocyte pAMPK expression in each structure in vehicle-, but not 6-OHDA-pretreated E rats. Results provide novel evidence for hypothalamic astrocyte AMPK expression and hindbrain catecholamine-dependent activation of this cell-specific sensor by hypoglycemia in the presence of estrogen. Further research is needed to determine the role of astrocyte AMPK in reactivity of these glia to metabolic imbalance and contribution to restoration of neuro-metabolic stability.

A chemically standardized extract of Ziziphus jujuba fruit (Jujube) stimulates expressions of neurotrophic factors and anti-oxidant enzymes in cultured astrocytes.

The fruit of Ziziphus jujuba Mill., known as jujube or Chinese date, is commonly consumed as a health supplement worldwide. To study the role of jujube in brain benefits, the expression of neurotrophic factors and anti-oxidant enzymes in the jujube-treated cultured astrocytes was determined. Application of a chemical standardized water extract of jujube in cultured astrocytes for 24 h stimulated the expressions of nerve growth factor, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor in a concentration-dependent manner. The pre-treatment with H89, a protein kinase A inhibitor, attenuated the jujube-induced expression of neurotrophic factors. In parallel, the treatment of jujube water extract induced the transcriptional expressions of the enzymes responsible for anti-oxidation, i.e. NAD(P)H: quinine oxidoreductase 1, glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit and glutathione S-transferase, in a concentration-dependent manner. These results proposed the benefits of jujube in regulating expressions of neurotrophic factors and anti-oxidant enzymes in cultured astrocytes.

[Effects of electroacupuncture on the expressions of neuroal nitric oxide synthase and astrocyte in dentate gyrus of rats with Parkinson's disease].

OBJECTIVE: To observe the changes in the expression of neuroal nitric oxide synthase (nNOS) and glial fibrillary acidic protein (GFAP) in dentate gyrus (DG) of rats with Parkinson's disease (PD) and effects of electroacupuncture (EA). METHODS: On the 7th day of stereotactic injection with 6-hydroxydopamine into right medial forebrain bundle, apomorphine-induced rotation was carried out to select the PD rats. The selected 12 rats were randomly divided into a model group (n = 6) and an EA group (n = 6). Moreover a normal group was established (n = 6). Then, the rats in EA group were treated with EA at "Hegu" (LI 4) and "Taichong" (LR 3), once a day for 21 days, and the other groups without any treatment. After EA treatment, the expressions of nNOS and GFAP in right DG were examined by immunohistochemistry method. RESULTS: The expression of nNOS in the right DG was weak in normal group, and the expression of nNOS in model group was significantly higher than that in normal group (P < 0.01), while in EA group, it was significantly lower than that in model (P < 0.01), with no difference between EA group and normal group (P > 0.05). The expression level of GFAP in model group was significantly higher than that in normal group (P < 0.01), while there was no difference in the number of GFAP positive cells between the above two groups (P > 0.05), and the number of GFAP positive cells in EA group was significantly increased compared with that in model group (P < 0.01), while with no significant difference in the expression level of GFAP between EA group and model group (P > 0.05). CONCLUSION: EA can reverse the increase of nNOS expression and promote the activation of astrocyte in DG on the injured side in rats with PD.

Mutant copper-zinc superoxide dismutase (SOD1) induces protein secretion pathway alterations and exosome release in astrocytes: implications for disease spreading and motor neuron pathology in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is the most common motor neuron disease and is still incurable. The mechanisms leading to the selective motor neuron vulnerability are still not known. The interplay between motor neurons and astrocytes is crucial in the outcome of the disease. We show that mutant copper-zinc superoxide dismutase (SOD1) overexpression in primary astrocyte cultures is associated with decreased levels of proteins involved in secretory pathways. This is linked to a general reduction of total secreted proteins, except for specific enrichment in a number of proteins in the media, such as mutant SOD1 and valosin-containing protein (VCP)/p97. Because there was also an increase in exosome release, we can deduce that astrocytes expressing mutant SOD1 activate unconventional secretory pathways, possibly as a protective mechanism. This may help limit the formation of intracellular aggregates and overcome mutant SOD1 toxicity. We also found that astrocyte-derived exosomes efficiently transfer mutant SOD1 to spinal neurons and induce selective motor neuron death. We conclude that the expression of mutant SOD1 has a substantial impact on astrocyte protein secretion pathways, contributing to motor neuron pathology and disease spread.

Coordination of hypothalamic and pituitary T3 production regulates TSH expression.

Type II deiodinase (D2) activates thyroid hormone by converting thyroxine (T4) to 3,5,3'-triiodothyronine (T3). This allows plasma T4 to signal a negative feedback loop that inhibits production of thyrotropin-releasing hormone (TRH) in the mediobasal hypothalamus (MBH) and thyroid-stimulating hormone (TSH) in the pituitary. To determine the relative contributions of these D2 pathways in the feedback loop, we developed 2 mouse strains with pituitary- and astrocyte-specific D2 knockdown (pit-D2 KO and astro-D2 KO mice, respectively). The pit-D2 KO mice had normal serum T3 and were systemically euthyroid, but exhibited an approximately 3-fold elevation in serum TSH levels and a 40% reduction in biological activity. This was the result of elevated serum T4 that increased D2-mediated T3 production in the MBH, thus decreasing Trh mRNA. That tanycytes, not astrocytes, are the cells within the MBH that mediate T4-to-T3 conversion was defined by studies using the astro-D2 KO mice. Despite near-complete loss of brain D2, tanycyte D2 was preserved in astro-D2 KO mice at levels that were sufficient to maintain both the T4-dependent negative feedback loop and thyroid economy. Taken together, these data demonstrated that the hypothalamic-thyroid axis is wired to maintain normal plasma T3 levels, which is achieved through coordination of T4-to-T3 conversion between thyrotrophs and tanycytes.

Regulation of enhanced cerebrovascular expression of proinflammatory mediators in experimental subarachnoid hemorrhage via the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway.

BACKGROUND: Subarachnoid hemorrhage (SAH) is associated with high morbidity and mortality. It is suggested that the associated inflammation is mediated through activation of the mitogen-activated protein kinase (MAPK) pathway which plays a crucial role in the pathogenesis of delayed cerebral ischemia after SAH. The aim of this study was first to investigate the timecourse of altered expression of proinflammatory cytokines and matrix metalloproteinase in the cerebral arteries walls following SAH. Secondly, we investigated whether administration of a specific mitogen-activated protein kinase kinase (MEK)1/2 inhibitor, U0126, given at 6 h after SAH prevents activation of the MEK/extracellular signal-regulated kinase 1/2 pathway and the upregulation of cerebrovascular inflammatory mediators and improves neurological function. METHODS: SAH was induced in rats by injection of 250 µl of autologous blood into basal cisterns. U0126 was given intracisternally using two treatment regimens: (A) treatments at 6, 12, 24 and 36 h after SAH and experiments terminated at 48 h after SAH, or (B) treatments at 6, 12, and 24 h after SAH and terminated at 72 h after SAH. Cerebral arteries were harvested and interleukin (IL)-6, IL-1ß, tumor necrosis factor α (TNF)α, matrix metalloproteinase (MMP)-9 and phosphorylated ERK1/2 (pERK1/2) levels investigated by immunohistochemistry. Early activation of pERK1/2 was measured by western blot. Functional neurological outcome after SAH was also analyzed. RESULTS: Expression levels of IL-1ß, IL-6, MMP-9 and pERK1/2 proteins were elevated over time with an early increase at around 6 h and a late peak at 48 to 72 h post-SAH in cerebral arteries. Enhanced expression of TNFα in cerebral arteries started at 24 h and increased until 96 h. In addition, SAH induced sensorimotor and spontaneous behavior deficits in the animals. Treatment with U0126 starting at 6 h after SAH prevented activation of MEK-ERK1/2 signaling. Further, U0126 significantly decreased the upregulation of inflammation proteins at 48 and 72 h following SAH and improved neurological function. We found no differences between treatment regimens A and B. CONCLUSIONS: These results show that SAH induces early activation of the MEK-ERK1/2 pathway in cerebral artery walls, which is associated with upregulation of proinflammatory cytokines and MMP-9. Inhibition of the MEK-ERK1/2 pathway by U0126 starting at 6 h post-SAH prevented upregulation of cytokines and MMP-9 in cerebral vessels, and improved neurological outcome.

Chronic h1-antihistamine treatment increases seizure susceptibility after withdrawal by impairing glutamine synthetase.

AIM: To investigate the effect of chronic H1-antihistamine treatment on seizure susceptibility after drug withdrawal in nonepileptic rats and to further study its relation to glutamine synthetase (GS), which is the key enzyme for glutamate metabolism and gamma aminobutyric acid (GABA) synthesis. METHODS: After drug withdrawal from a 2-week treatment with diphenhydramine or pyrilamine, seizure susceptibility was determined by amygdaloid kindling or pentylenetetrazol model; meanwhile, the GS expression or activity was analyzed. The glutamine, glutamate, and GABA contents were measured by high-performance liquid chromatography. RESULTS: Seizure susceptibility significantly increased in amygdaloid kindling and pentylenetetrazol model 10 days after drug withdrawal from a 2-week treatment with H1-antihistamines. Meanwhile, GS activity and expression in the cortex or hippocampus decreased simultaneously with a marked decline of glutamine and GABA content. Comparable inhibition of GS activity by methionine sulfoximine was also sufficient to increase the susceptibility, while supplementation with glutamine reversed the high susceptibility 10 days after diphenhydramine withdrawal. Moreover, the seizure susceptibility increased 10 days after diphenhydramine withdrawal in wild-type mice but not in histidine decarboxylase knockout mice, which lack histamine. CONCLUSIONS: Chronic H1-antihistamine treatment produces long-lasting increase in seizure susceptibility in nonepileptic rodents after drug withdrawal and its mechanism involves impairment of GS through blocking the action of histamine.

Structure-dependent inhibition of gelatinases by dietary antioxidants in rat astrocytes and sera of multiple sclerosis patients.

We investigated whether polyphenols modulate the expression and activity of the enzymes gelatinases A (MMP-2) and B (MMP-9), involved in the pathogenesis of multiple sclerosis (MS). LPS-activated primary rat astrocytes were treated with the flavonoids quercetin (QRC) and cathechins [green tea extract (GTE)] and the non-flavonoids resveratrol (RSV) and tyrosol/hydroxytyrosol (Oliplus). As assessed by zymography and RT-PCR, RSV and Oliplus, but not QRC and GTE, dose-dependently inhibited the LPS-induced levels and mRNA expression of MMP-2 and MMP-9. By contrast, in cell-free systems direct inhibition of gelatinase activity in MS sera was determined by QRC and GTE, but not by RSV. Oliplus was only partially effective. Our results indicate that the flavonoids and non-flavonoids tested exert their inhibitory effect on MMPs, displaying different mechanisms of action, possibly related to their structure. Therefore, their combined use may represent a powerful tool for the down-regulation of MMPs in the course of MS.

Effect of berberine and Berberis aetnensis C. Presl. alkaloid extract on glutamate-evoked tissue transglutaminase up-regulation in astroglial cell cultures.

Berberis aetnensis C. Presl. is a bushy-spiny shrub common on Mount Etna (Sicily, Italy), containing various alkaloids with several pharmacological properties. This study assessed the effect of berberine and of the alkaloid extract of B. aetnensis roots on the glutamate-evoked tissue transglutaminase (TG2) up-regulation in rat astrocyte primary cultures, used as an in vitro model of excitotoxicity. The findings show that the alkaloid extract of B. aetnensis roots consists mainly of berberine. Furthermore, berberine and the alkaloid extract of B. aetnensis roots were able to restore the oxidative status modified by glutamate and the levels of TG2 to control values. It was found that berberine or the alkaloid extract of B. aetnensis roots are able to ameliorate the excessive production of glutamate, protein misfolding and aggregation, mitochondrial fragmentation, and neurodegeneration. Thus, it is suggested that berberine and the alkaloid extract of B. aetnensis roots, may represent a natural therapeutic strategy in the neuropathological conditions associated with excitotoxicity.

Complex contribution of cyclophilin D to Ca2+-induced permeability transition in brain mitochondria, with relation to the bioenergetic state.

Cyclophilin D (cypD)-deficient mice exhibit resistance to focal cerebral ischemia and to necrotic but not apoptotic stimuli. To address this disparity, we investigated isolated brain and in situ neuronal and astrocytic mitochondria from cypD-deficient and wild-type mice. Isolated mitochondria were challenged by high Ca(2+), and the effects of substrates and respiratory chain inhibitors were evaluated on permeability transition pore opening by light scatter. In situ neuronal and astrocytic mitochondria were visualized by mito-DsRed2 targeting and challenged by calcimycin, and the effects of glucose, NaCN, and an uncoupler were evaluated by measuring mitochondrial volume. In isolated mitochondria, Ca(2+) caused a large cypD-dependent change in light scatter in the absence of substrates that was insensitive to Ruthenium red or Ru360. Uniporter inhibitors only partially affected the entry of free Ca(2+) in the matrix. Inhibition of complex III/IV negated the effect of substrates, but inhibition of complex I was protective. Mitochondria within neurons and astrocytes exhibited cypD-independent swelling that was dramatically hastened when NaCN and 2-deoxyglucose were present in a glucose-free medium during calcimycin treatment. In the presence of an uncoupler, cypD-deficient astrocytic mitochondria performed better than wild-type mitochondria, whereas the opposite was observed in neurons. Neuronal mitochondria were examined further during glutamate-induced delayed Ca(2+) deregulation. CypD-knock-out mitochondria exhibited an absence or a delay in the onset of mitochondrial swelling after glutamate application. Apparently, some conditions involving deenergization render cypD an important modulator of PTP in the brain. These findings could explain why absence of cypD protects against necrotic (deenergized mitochondria), but not apoptotic (energized mitochondria) stimuli.

Astrocytic poly(ADP-ribose) polymerase-1 activation leads to bioenergetic depletion and inhibition of glutamate uptake capacity.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a ubiquitous nuclear enzyme involved in genomic stability. Excessive oxidative DNA strand breaks lead to PARP-1-induced depletion of cellular NAD(+), glycolytic rate, ATP levels, and eventual cell death. Glutamate neurotransmission is tightly controlled by ATP-dependent astrocytic glutamate transporters, and thus we hypothesized that astrocytic PARP-1 activation by DNA damage leads to bioenergetic depletion and compromised glutamate uptake. PARP-1 activation by the DNA alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), caused a significant reduction of cultured cortical astrocyte survival (EC(50) = 78.2 +/- 2.7 microM). HPLC revealed MNNG-induced time-dependent reductions in NAD(+) (98%, 4 h), ATP (71%, 4 h), ADP (63%, 4 h), and AMP (66%, 4 h). The maximal [(3)H]glutamate uptake rate (V(max)) also declined in a manner that corresponded temporally with ATP depletion, falling from 19.3 +/- 2.8 in control cells to 2.1 +/- 0.8 nmol/min/mg protein 4 h post-MNNG. Both bioenergetic depletion and loss of glutamate uptake capacity were attenuated by genetic deletion of PARP-1, directly indicating PARP-1 involvement, and by adding exogenous NAD(+) (10 mM). In mixed neurons/astrocyte cultures, MNNG neurotoxicity was partially mediated by extracellular glutamate and was reduced by co-culture with PARP-1(-/-) astrocytes, suggesting that impairment of astrocytic glutamate uptake by PARP-1 can raise glutamate levels sufficiently to have receptor-mediated effects at neighboring neurons. Taken together, these experiments showed that PARP-1 activation leads to depletion of the total adenine nucleotide pool in astrocytes and severe reduction in neuroprotective glutamate uptake capacity.

Involvement of oxidative pathways in cytokine-induced secretory phospholipase A2-IIA in astrocytes.

Recent studies have suggested the involvement of secretory phospholipase A2-IIA (sPLA2-IIA) in neuroinflammatory diseases. Although sPLA2-IIA is transcriptionally induced through the NF-kappaB pathway by pro-inflammatory cytokines, whether this induction pathway is affected by other intracellular signaling pathways has not been investigated in detail. In this study, we demonstrated the induction of sPLA2-IIA mRNA and protein expression in astrocytes by cytokines and detected the protein in the culture medium after stimulation. We further investigated the effects of oxidative pathways and botanical antioxidants on the induction pathway and observed that IL-1beta-induced sPLA2-IIA mRNA expression in astrocytes is dependent on ERK1/2 and PI-3 kinase, but not p38 MAPK. In addition to apocynin, a known NADPH oxidase inhibitor, botanical antioxidants, such as resveratrol and epigallocatechin gallate, also inhibited IL-1beta-induced sPLA2-IIA mRNA expression. These compounds also suppressed IL-1beta-induced ERK1/2 activation and translocation of the NADPH oxidase subunit p67 phox from cytosol to membrane fraction. Taken together, these results support the involvement of reactive oxygen species from NADPH oxidase in cytokine induction of sPLA2-IIA in astrocytes and promote the use of botanical antioxidants as protective agents for inhibition of inflammatory responses in these cells.

Effects of maslinic acid, a natural triterpene, on glycogen metabolism in cultured cortical astrocytes.

Maslinic acid (2- alpha,3- beta-dihydroxyolean-12-en-28-oic acid) is a triterpenoid compound present in plants of Olea europaea. In the present study, we investigated the effect of maslinic acid on astrocytic glycogen metabolism. Glycogen phosphorylase (GP) activity in homogenates of cultured astrocytes was analyzed, and maslinic acid exhibited GP inhibition with an IC (50) value of 5.7 microM. Moreover, the influence of maslinic acid on glycogen synthesis and glycogenolysis was also investigated. Pre-incubation with maslinic acid dose-dependently increased cellular glycogen content and prevented the excessive glycogenolysis induced by norepinephrine. In conclusion, maslinic acid is suggested to be a potent inhibitor of astrocytic glycogen phosphorylase.