Daphnetin, a Natural Coumarin Derivative, Provides the Neuroprotection Against Glutamate-Induced Toxicity in HT22 Cells and Ischemic Brain Injury

Daphnetin (DAP), a coumarin derivative, has been reported to have multiple pharmacological actions including analgesia, antimalarial, anti-arthritic, and anti-pyretic properties. It is unclear whether DAP has neuroprotective effects on ischemic brain injury. In this study, we found that DAP treatment (i.c.v.) reduced the infarct volume at 24 h after ischemia/reperfusion injury and improved neurological behaviors in a middle cerebral artery occlusion mouse model. Moreover, we provided evidences that DAP had protective effects on infarct volume in neonate rats even it was administrated at 4 h after cerebral hypoxia/ischemia injury. To explore its neuroprotective mechanisms of DAP, we examined the protection of DAP on glutamate toxicity-induced cell death in hippocampal HT-22 cells. Our results demonstrated that DAP protected against glutamate toxicity in HT-22 cells in a concentration-dependent manner. Further, we found that DAP maintained the cellular levels of glutathione and superoxide dismutase activity, suggesting the anti-oxidatant activity of DAP. Since DAP has been used for the treatment of coagulation disorder and rheumatoid arthritis for long time with a safety profile, DAP will be a promising agent for the treatment of stroke.     

Pyrroloquinoline Quinine Protects Rat Brain Cortex Against Acute Glutamate-Induced Neurotoxicity

To investigate possible protective effects of pyrroloquinoline quinone (PQQ) on the rat cortex with glutamate injection and to understand the mechanisms linking the in vivo neuroprotection of PQQ. Adult Sprague–Dawley rats received glutamate injection into the rat cortex. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay was performed to observe influences of co-treatment with PQQ (simultaneous injection with PQQ and glutamate) on neural cell apoptosis in the rat cortex. The production of reactive oxygen species (ROS) in the rat cortex was detected by flow cytometry using 2′,7′-dichlorofluorescin diacetate labeling, and the activity of superoxide dismutase, glutathione and malondialdehyde was respectively determined. Real time quantitative RT-PCR and Western blot were applied to measure the mRNA and protein expressions of Nrf1, Nrf2, HO-1 and GCLC in the rat cortex. Western blot was used to detect the phosphorylation of Akt and GSK3β in the rat cortex. Co-treatment with PQQ protected neural cells in the rat cortex from glutamate-induced apoptosis. PQQ decreased the ROS production induced by glutamate injection. PQQ increased the mRNA and protein expressions of Nrf2, HO-1 and GCLC and the phosphorylation of Akt and GSK3β in the cortex of glutamate-injected rats. PQQ could produce neuroprotective effects on the rat cortex. The antioxidant properties of PQQ and PQQ-induced activation of Akt/GSK3β signal pathway might be responsible for the in vivo neuroprotection of PQQ.     

Neuroprotection of Atractylenolide III from Atractylodis macrocephalae Against Glutamate-Induced Neuronal Apoptosis Via Inhibiting Caspase Signaling Pathway

Glutamate-induced excitotoxicity appears to play a crucial role in neurological disorders. Neuroprotection against glutamate-induced excitotoxicity has been proposed as a therapeutic strategy for preventing and/or treating these excitotoxicity-mediated diseases. In the present study, atractylenolide III, which exhibited significantly neuroprotective effect against glutamate-induced neuronal apoptosis, was isolated from Atractylodes macrocephala by means of bioactivity-guided fractionation. The inhibitory effect of atractylenolide III on glutamate-induced neuronal apoptosis was in a concentration-dependent manner. The anti-apoptotic property of atractylenolide III might be mediated, in part, via inhibiting caspase signaling pathway. Atractylenolide III may have therapeutic potential in excitotoxicity-mediated neurological diseases.     

Refocusing the Brain: New Approaches in Neuroprotection Against Ischemic Injury

Neurochemical Research - A new era for neuroprotective strategies is emerging in ischemia/reperfusion. This has forced to review the studies existing to date based in neuroprotection against...     

Lipid Rafts in Neurodegeneration and Neuroprotection

The collective properties of the lipids that form biological membranes give rise to a very high level of lateral organization within the membranes. Lipid-driven membrane organization allows the segregation of membrane-associated components into specific lipid rafts, which function as dynamic platforms for signal transduction, protein processing, and membrane turnover. A number of events essential for the functional integrity of the nervous system occur in lipid rafts and depend on lipid raft organization. Alterations of lipid composition that lead to abnormal lipid raft organization and consequent deregulation of lipid raft-dependent signaling are often associated with neurodegenerative diseases. The amyloidogenic processing of proteins involved in the pathogenesis of major nervous system diseases, including Alzheimer’s disease and Parkinson’s disease, requires lipid raft-dependent compartmentalization at the membrane level. Improved understanding of the forces that control lipid raft organization will facilitate the development of novel strategies for the effective prevention and treatment of neurodegenerative and age-related brain diseases.     

HDAC6 at the Intersection of Neuroprotection and Neurodegeneration

Histone deacetylase 6 (HDAC6) catalyzes multiple reactions. We summarize the current knowledge on HDAC6, its targets and functions. Among others, HDAC6 recognizes damaged proteins and assures that these proteins are destroyed by autophagy. On the other hand, HDAC6 also modifies the tracks used by the clearance mechanism so that axonal transport becomes less efficient. We hypothesize that a disturbance in the equilibrium between the different functions of HDAC6 could play an important role in neurodegeneration.     

Neuroprotection of Ro25-6981 Against Ischemia/Reperfusion-Induced Brain Injury via Inhibition of Autophagy

In this study, we investigated the neuroprotective effect of Ro25-6981 against cerebral ischemia/reperfusion injury. Ro25-6981 alone or in combination with rapamycin was intracerebroventricularly administered to rats which suffered transient forebrain ischemia inducing by 4-vessel occlusion and reperfusion. Nissl staining was used to determine the survival of CA1 pyramidal cells of the hippocampus, while immunohistochemistry was performed to measure neuron-specific enolase (NSE) expression. The expression of autophagy-related proteins, such as microtubule-associated protein l light chain 3 (LC3), Beclin 1, and sequestosome 1 (p62), was assessed by immunoblotting. Nissl staining showed that neuronal damage was reduced in the hippocampal CA1 pyramidal layer in rats that received Ro25-6981. The protective effect of Ro25-6981 was dose-dependent, with a significant effect in the middle-dose range. The expression of NSE increased after Ro25-6981 treatment. Ro25-6981 significantly decreased LC3II (which is membrane bound) and Beclin 1, and increased p62. In addition, Ro25-6981 decreased rapamycin-induced neuronal damage and excessive activation of autophagy after I/R. Taken together, the results suggest that Ro25-6981 could suppress ischemic brain injury by regulating autophagy-related proteins during ischemia/reperfusion.     

Moderate Ethanol Preconditioning of Rat Brain Cultures Engenders Neuroprotection Against Dementia-Inducing Neuroinflammatory Proteins: Possible Signaling Mechanisms

There is no question that chronic alcohol (ethanol) abuse, a leading worldwide problem, causes neuronal dysfunction and brain damage. However, various epidemiologic studies in recent years have indicated that in comparisons with abstainers or never-drinkers, light/moderate alcohol consumers have lower risks of age-dependent cognitive decline and/or dementia, including Alzheimer’s disease (AD). Such reduced risks have been variously attributed to favorable circulatory and/or cerebrovascular effects of moderate ethanol intake, but they could also involve ethanol “preconditioning” phenomena in brain glia and neurons. Here we summarize our experimental studies showing that moderate ethanol preconditioning (MEP; 20–30 mM ethanol) of rat brain cultures prevents neurodegeneration due to β-amyloid, an important protein implicated in AD, and to other neuroinflammatory proteins such as gp120, the human immunodeficiency virus 1 envelope protein linked to AIDS dementia. The MEP neuroprotection is associated with suppression of neurotoxic protein-evoked initial increases in [Ca⁺²]_i and proinflammatory mediators—e.g., superoxide anion, arachidonic acid, and glutamate. Applying a sensor → transducer → effector model to MEP, we find that onset of neuroprotection correlates temporally with elevations in “effector” heat shock proteins (HSP70, HSP27, and phospho-HSP27). The effector status of HSPs is supported by the fact that inhibiting HSP elevations due to MEP largely restores gp120-induced superoxide potentiation and subsequent neurotoxicity. As upstream mediators, synaptic N-methyl-d-aspartate receptors may be initial prosurvival sensors of ethanol, and protein kinase C epsilon and focal adhesion kinase are likely transducers during MEP that are essential for protective HSP elevations. Regarding human consumption, we speculate that moderate ethanol intake might counter incipient cognitive deterioration during advanced aging or AD by exerting preconditioning-like suppression of ongoing neuroinflammation related to amyloidogenic protein accumulation.     

Exogenous Glutamate-Induced Modulation of Neurosecretory Process in Nerve Terminals Obtained from the Rat Brain

We studied intracellular processes in nerve terminals of neurons of the rat brain in response to application of exogenous glutamate. Using a рН-sensitive fluorescence probe, acridine orange (AO), and labeled gammaaminobutyric acid ([³Н]GABA), we estimated the effect of application of glutamate on the level of acidification of synaptic vesicles and also on the release of GABA from nerve terminals (synaptosomes) obtained from hippocampal tissue. Our experiments showed that glutamate in a dose-dependent manner stimulated the [³Н]GABA release from nerve terminals, and then we observed re-uptake of this neurotransmitter. A selective blocker of GABA transporters, NO-711, completely blocked the uptake of neurotransmitter but did not influence its release; this observation indicates that the glutamate-induced GABA release was from the vesicular, not cytosolic, pool. We confirmed that glutamate stimulates the process of exocytosis in experiments using AO, where we obtained data indicating that this process is two-phase. The first phase, which reflects probably calcium-induced exocytosis, looked like a “burst” of fluorescent signal typical of the response of synaptosomes to the action of KCl applied in depolarization concentration. Both phases of the response were completely blocked or significantly suppressed in calcium-free medium or in the presence of 25 μM Cd²⁺. The second (slow) phase of the response developed after a certain lag period and was characterized by a gradual increase in the intensity of fluorescent signal. This phase was completely dependent on the presence of sodium in the extracellular medium and completely blocked when sodium was replaced by choline or N-methyl-D-glucamine. We hypothesize that the second phase of the response can reflect either spontaneous unstimulated exocytosis or dissipation of the proton gradient in synaptic vesicles induced by the entry of Na⁺ into the nerve terminal.     

Drebrin Regulates Neuroblast Migration in the Postnatal Mammalian Brain

After birth, stem cells in the subventricular zone (SVZ) generate neuroblasts that migrate along the rostral migratory stream (RMS) to become interneurons in the olfactory bulb (OB). This migration is crucial for the proper integration of newborn neurons in a pre-existing synaptic network and is believed to play a key role in infant human brain development. Many regulators of neuroblast migration have been identified; however, still very little is known about the intracellular molecular mechanisms controlling this process. Here, we have investigated the function of drebrin, an actin-binding protein highly expressed in the RMS of the postnatal mammalian brain. Neuroblast migration was monitored both in culture and in brain slices obtained from electroporated mice by time-lapse spinning disk confocal microscopy. Depletion of drebrin using distinct RNAi approaches in early postnatal mice affects neuroblast morphology and impairs neuroblast migration and orientation in vitro and in vivo. Overexpression of drebrin also impairs migration along the RMS and affects the distribution of neuroblasts at their final destination, the OB. Drebrin phosphorylation on Ser142 by Cyclin-dependent kinase 5 (Cdk5) has been recently shown to regulate F-actin-microtubule coupling in neuronal growth cones. We also investigated the functional significance of this phosphorylation in RMS neuroblasts using in vivo postnatal electroporation of phosphomimetic (S142D) or non-phosphorylatable (S142A) drebrin in the SVZ of mouse pups. Preventing or mimicking phosphorylation of S142 in vivo caused similar effects on neuroblast dynamics, leading to aberrant neuroblast branching. We conclude that drebrin is necessary for efficient migration of SVZ-derived neuroblasts and propose that regulated phosphorylation of drebrin on S142 maintains leading process stability for polarized migration along the RMS, thus ensuring proper neurogenesis.     

Neuroprotective Effects of Etidronate and 2,3,3-Trisphosphonate Against Glutamate-Induced Toxicity in PC12 Cells

Neurochemical Research - Etidronate is one of the best known bisphosphonates (BP) derivatives. It is often used as a reference drug in research related to hypercalcaemia and other common bone...     

Geldanamycin Provides Posttreatment Protection Against Glutamate-Induced Oxidative Toxicity in a Mouse Hippocampal Cell Line

Abstract : The benzoquinoid ansamycin geldanamycin interferes with many cell signaling pathways and is currently being evaluated as an anticancer agent. The main intracellular target of geldanamycin is the 90-kDa heat shock protein, hsp90. In this report we demonstrate that geldanamycin is effective at preventing glutamate-induced oxidative toxicity in the HT22 mouse hippocampal cell line, even if given 4 h after glutamate treatment. Geldanamycin prevents glutamate-induced internucleosomal DNA cleavage in the HT22 cells but does not reverse the depletion of glutathione levels brought about by glutamate treatment. Both anabolic and catabolic effects are generated by geldanamycin treatment of HT22 cells, as evidenced by the induction of hsp70 expression and degradation of c-Raf-1 protein, respectively. Thus, geldanamycin may provide an effective strategy for manipulating signaling pathways in neuronal cells that use hsp90 as they proceed through a programmed cell death pathway in response to oxidative stress.     

Tau Phosphorylation and Cleavage in Ethanol-Induced Neurodegeneration in the Developing Mouse Brain

Previous studies indicated that ethanol-induced neurodegeneration in postnatal day 7 (P7) mice, widely used as a model for the fetal alcohol spectrum disorders, was accompanied by glycogen synthase kinase-3β (GSK-3β) and caspase-3 activation. Presently, we examined whether tau, a microtubule associated protein, is modified by GSK-3β and caspase-3 in ethanol-treated P7 mouse forebrains. We found that ethanol increased phosphorylated tau recognized by the paired helical filament (PHF)-1 antibody and by the antibody against tau phosphorylated at Ser199. Ethanol also generated tau fragments recognized by an antibody against caspase-cleaved tau (C-tau). C-tau was localized in neurons bearing activated caspase-3 and fragmented nuclei. Over time, cell debris and degenerated projections containing C-tau appeared to be engulfed by activated microglia. A caspase-3 inhibitor partially blocked C-tau formation. Lithium, a GSK-3β inhibitor, blocked ethanol-induced caspase-3 activation, phosphorylated tau elevation, C-tau formation, and microglial activation. These results indicate that tau is phosphorylated by GSK-3β and cleaved by caspase-3 during ethanol-induced neurodegeneration in the developing brain.     

Glutamate-Induced Excitotoxicity in Retina: Neuroprotection with Receptor Antagonist,Dextromethorphan, but Not with Calcium Channel Blockers

The purpose of our studies was to evaluate different strategies for possible neuroprotection in glutamate-induced neurotoxicity in the retina. In a first set of experiments we attempted to determine if dextrorphan antagonism of glutamate action on NMDA receptors would protect against excitotoxic injury associated with secondary damage seen after surgical laser treatment in retina. In a second set of experiments, the effects of different calcium channel blockers in an in-vitro model of N-methyl-D-aspartate (NMDA)-induced retinal ganglion cell excitotoxicity that utilized rabbit retinal explants were evaluated. Dextrorphan infusion prior to laser treatment of rabbit retina produced a significant decrease in the area of neural retinal damage. We attribute the apparent dextrorphan protection to attenuation of glutamate mediated excitotoxicity secondary to laser induced cell death. Preincubation of rabbit retinal explants with verapamil, nimodipine or -conotoxin MVIIA did not cause a significant change in NMDA induced cell death in the ganglion cell layer.     

Energy Substrates Protect Hippocampus Against Endogenous Glutamate-Mediated Neurodegeneration in Awake Rats

Excitotoxicity due to excessive glutamatergic neurotransmission is a well-studied phenomenon that has been related to the mechanisms of neuronal death occurring in some disorders of the CNS. We have previously shown that the intrahippocampal perfusion by microdialysis of 4-aminopyridine (4-AP) in rats stimulates endogenous glutamate release from nerve endings and this results in excitotoxic effects such as immediate seizures and delayed neuronal death, due to the overactivation of N-methyl-d-aspartate (NMDA) receptors. To study whether mitochondrial energy dysfunction and oxidative stress could be involved in this 4-AP-induced excitotoxicity, we evaluated in awake rats the protective effect of several energy substrates and antioxidant compounds, using microdialysis, electroencephalographic (EEG) recording and histological analysis. The 4-AP-induced behavioral and EEG seizures, which progressed to status epilepticus in about 30 min, were prevented by the NMDA receptor antagonist MK-801, whereas acetoacetate, dl- and l-β-hydroxybutyrate did not protect against seizures but increased the latency to the onset of status epilepticus; pyruvate, α-ketoglutarate and glutathione ethyl ester did not show any protective effect. 4-AP also produced nearly complete loss of pyramidal neurons in CA1 and CA3 regions of the ipsilateral hippocampus 24 h after the experiment. MK-801 totally prevented this neuronal death and the energy substrates tested protected by about 50 %, whereas the antioxidants showed only a weak protection. We conclude that ketone bodies possess weak anticonvulsant effects and that energy metabolism impairment plays a more important role than oxidative stress in the delayed hippocampal neurodegeneration resulting from the excitotoxic action of 4-AP mediated by endogenous glutamate.     

Neuroprotective Effects of Biochanin A Against Glutamate-Induced Cytotoxicity in PC12 Cells Via Apoptosis Inhibition

l-Glutamate plays a crucial role in neuronal cell death, which is known to be associated with various neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, and Huntington’s diseases. In this study, we investigated the protective effects of biochanin A, a phytoestrogen compound found mainly in Trifolium pratense, against l-glutamate-induced cytotoxicity in a PC12 cell line. Exposure of the cells to 10 mM l-glutamate was found to significantly increase cell viability loss and apoptosis, whereas pretreatment with various concentrations of biochanin A attenuated the cytotoxic effects of l-glutamate. Specifically, the pretreatment led to not only decreases in the release of lactate dehydrogenase, the number of apoptotic cells, and the activity of caspase-3 but also an increase in the total glutathione level in the l-glutamate-treated PC12 cells. These results indicate that biochanin A may be able to exert neuroprotective effects against l-glutamate-induced cytotoxicity. Furthermore, our findings also imply that biochanin A may act as an antiapoptotic agent in order to perform its protective function.     

Postnatal Development of Thiamine Metabolism in Rat Brain

The activities of thiamine diphosphatase (TDPase), thiamine triphosphatase (TTPase), and thiamine pyrophosphokinase and the contents of thiamine and its phosphate esters were determined in rat brain cortex, cerebellum, and liver from birth to adulthood. Microsomal TTPase activity in the cerebral cortex and cerebellum increased from birth to 3 weeks, whereas that in the liver did not change during postnatal development. Microsomal TDPase activity in the cerebral cortex showed a transient increase at 1-2 weeks, but that in the cerebellum did not change during development. In contrast to the activity of the brain enzyme, that of liver microsomal TDPase increased stepwise after birth. Thiamine pyrophosphokinase activity in the cerebellum increased from birth to 3 weeks and then decreased, whereas that in the cerebral cortex and liver showed less change during development. TDP and thiamine monophosphate (TMP) levels increased after birth and plateaued at 3 weeks whereas TTP and thiamine levels showed little change during development in the cerebral cortex and cerebellum. The contents of thiamine and its phosphate esters in the liver showed more complicated changes during development. It is concluded that thiamine metabolism in the brain changes during postnatal development in a different way from that in the liver and that the development of thiamine metabolism differs among brain regions.