Hippocampal Homogenates Research Articles

Dexmedetomidine inhibits inflammatory reaction in the hippocampus of septic rats by suppressing NF-κB pathway.

Dexmedetomidine (DEX) is known to provide neuroprotective effect in the central nervous system. However, the detailed mechanism remains far more elusive. This study was designed to investigate the relevant mechanisms of DEX's neuroprotective effect. Sprague–Dawley (SD) rats were injected with dexmedetomidine and/or Lipopolysaccharide (LPS) intraperitoneally, and inflammatory cytokines in serum and in the hippocampus were measured by enzyme linked immunosorbent assay (ELISA). NF-κB in the brain tissue extracts was analyzed with western-blot. Then, we investigated whether NF-κB inhibitor prevents the elevation of inflammatory cytokines in rats injected with LPS. Our results indicated that compared with the control group, the rats exposed to LPS showed significant cognitive dysfunction. When compared to controls, the levels of TNF-α and IL-6 in the serum and hippocampus homogenate were increased in rats treated with LPS. DEX pretreatment inhibited the rats' TNF-α, IL-6 and NF-κB levels induced by LPS. In response to LPS, PDTC pretreatment restrains the production of proinflammatory cytokines (TNF-α and IL-6). Rats treated with PDTC and DEX alongside LPS exhibited less TNF-α and IL-6 than the LPS treated group. In combination, PDTC and DEX showed addictive effects. Our data suggest that DEX exerts a neuroprotective effect through NF-κB in part after LPS-induced cognitive dysfunction.

Involvement of SNARE complex in the hippocampus and prefrontal cortex of offspring with depression induced by prenatal stress

BackgroundPrenatal stress (PS) exposure can cause depression-like behavior in offspring, and maladaptive responses including physiological and neurobiological changes. Glutamate neurotransmission is implicated in effects of PS and in antidepressant mechanisms; however, the mechanisms underlying its involvement remain unclear. In the synapse, the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is essential for vesicular docking and neurotransmitter release. MethodsTo explore effects of PS on the SNARE complex, pregnant rats were assigned to a control or PS group. Both male and female offspring in each group were used in this study. PS rats were exposed to restraint stress three times daily for 45 min on days 14–20 of pregnancy. ResultsIn the PS offspring, the expression of the SNARE protein SNAP-25, vesicle-associated membrane protein (VAMP)-2, and Syntaxin 1a was significantly increased in the hippocampus and prefrontal cortex. These observations were associated with increased levels of proteins that chaperone SNARE complex formation, including Munc-18, α-synuclein, CSPα, complexin1, and complexin2. Immunoblotting of hippocampal and prefrontal cortex homogenates revealed significantly increased SNARE complex formation. vGluT1 protein expression was also significantly increased in the offspring. Additionally, PS was associated with increased mRNA expression of VAMP1, VAMP2, SNAP25, Syntaxin1a, and Syntaxin1b in the hippocampus and prefrontal cortex. Increased monomeric SNARE proteins, SNARE complex formation, vesicle-associated proteins, and vGluT1 may explain the increase in glutamate and its downstream excitotoxicity. ConclusionsThese results support the hypothesis that glutamate release and vesicular glutamate transporters play a role in PS-induced depression-like behavior of rat offspring.

Histamine-deficient mice do not respond to the antidepressant-like effects of oleoylethanolamide

It has been suggested that the bioactive lipid mediator oleoylethanolamide (OEA), a potent agonist of the peroxisome proliferator-activated receptor-alpha (PPAR-α) possesses anti-depressant-like effects in several preclinical models. We recently demonstrated that several of OEA's behavioural actions require the integrity of the brain histaminergic system, and that an intact histaminergic neurotransmission is specifically required for selective serotonin re-uptake inhibitors to exert their anti-depressant-like effect. The purpose of our study was to test if OEA requires the integrity of the histaminergic neurotransmission to exert its antidepressant-like effects. Immobility time in the tail suspension test was measured to assess OEA's potential (10 mg/kg i.p.) as an antidepressant drug in histidine decarboxylase null (HDC−/-) mice and HDC+/+ littermates, as well as in PPAR-α+/+ and PPAR-α−/− mice. CREB phosphorylation was evaluated using Western blot analysis in hippocampal and cortical homogenates, as pCREB is considered partially responsible for the efficacy of antidepressants. Serotonin release from ventral hippocampi of HDC+/+ and HDC−/- mice was measured with in-vivo microdialysis, following OEA administration. OEA decreased immobility time and increased brain pCREB levels in HDC+/+ mice, whereas it was ineffective in HDC−/- mice. Comparable results were obtained in PPAR-α+/+ and PPAR-α−/− mice. Microdialysis revealed a dysregulation of serotonin release induced by OEA in HDC−/- mice.Our observations corroborate our hypothesis that brain histamine and signals transmitted by OEA interact to elaborate appropriate behaviours and may be the basis for the efficacy of OEA as an antidepressant-like compound.

Effects of isopentyl ferulate on oxidative stress biomarkers and a possible GABAergic anxiolytic-like trait in Swiss mice

This study aimed to evaluate the anxiolytic-like effect and the possible neuronal mechanism of action of isopentyl ferulate (IF). For this purpose, we used the marble burying test in Swiss albino mice. The biomarkers involved in oxidative stress were measured in the hippocampus homogenate of the test animals. In addition, the toxicity and antioxidant capacities were tested in Artemia salina and rat erythrocytes, respectively. The results suggest that, an acute administration of the IF at doses of 25, 50, 75 and 150 mg/kg (intraperitoneal, i.p.) significantly (p < 0.05) reduced the marble burying behavior of the animals as compared to the vehicle group, which demonstrates a calming effect of this chemical. It was observed that, the pre-treatment with flumazenil (2.5 mg/kg, i.p.), an antagonist of the gamma-amino butyrinc acid (GABAA) receptor, significantly reversed the marble burying behavioral activity in the animals treated with the IF 150 mg/kg dose. Moreover, the reduction in nitrite content and lipid peroxidation levels, while an increased in the reduced glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT) activities were also observed their hippocampus. Although, IF (2.36–14.16 mM) did not show toxicity in A. salina but exhibited a prominent antioxidant capacity in hydrogen peroxide-induced oxidative damage in rat erythrocytes. In conclusion, IF exhibited an anxiety-like effect in mice along with a potent antioxidant capacity, and we suppose it may have neuroprotective effects possibly via GABAergic transmission pathway.

Axon-terminals expressing EAAT2 (GLT-1; Slc1a2) are common in the forebrain and not limited to the hippocampus

The excitatory amino acid transporter type 2 (EAAT2) represents the major mechanism for removal of extracellular glutamate. In the hippocampus, there is some EAAT2 in axon-terminals, whereas most of the protein is found in astroglia. The functional importance of the neuronal EAAT2 is unknown, and it is debated whether EAAT2-expressing nerve terminals are present in other parts of the brain. Here we selectively deleted the EAAT2 gene in neurons (by crossing EAAT2-flox mice with synapsin 1-Cre mice in the C57B6 background). To reduce interference from astroglial EAAT2, we measured glutamate accumulation in crude tissue homogenates. EAAT2 proteins levels were measured by immunoblotting. Although synapsin 1-Cre mediated gene deletion only reduced the forebrain tissue content of EAAT2 protein to 95.5 ± 3.4% of wild-type (littermate) controls, the glutamate accumulation in homogenates of neocortex, hippocampus, striatum and thalamus were nevertheless diminished to, respectively, 54 ± 4, 46 ± 3, 46 ± 2 and 65 ± 7% of controls (average ± SEM, n = 3 pairs of littermates). GABA uptake was unaffected. After injection of U-13C-glucose, lack of neuronal EAAT2 resulted in higher 13C-labeling of glutamine and GABA in the hippocampus suggesting that neuronal EAAT2 is partly short-circuiting the glutamate-glutamine cycle in wild-type mice. Crossing synapsin 1-Cre mice with Ai9 reporter mice revealed that Cre-mediated excision occurred efficiently in hippocampus CA3, but less efficiently in other regions and hardly at all in the cerebellum. Conclusions: (1) EAAT2 is expressed in nerve terminals in multiple brain regions. (2) The uptake catalyzed by neuronal EAAT2 plays a role in glutamate metabolism, at least in the hippocampus. (3) Synapsin 1-Cre does not delete floxed genes in all neurons, and the contribution of neuronal EAAT2 is therefore likely to be larger than revealed in the present study.

Amelioration by nitric oxide (NO) mimetics on neurobehavioral and biochemical changes in experimental model of Alzheimer’s disease in rats

The present study evaluated the effects of s-nitrosoglutathione (GSNO), a nitrosothiol and sustained NO releaser, on experimental model of sporadic Alzheimer`s disease (sAD) in rats. Levels of Aβ40, Aβ42 and BDNF were assessed in brain hippocampal homogenates for correlative purposes. Intracerebroventricular-Streptozotocin (icv-STZ) induced increased escape latencies (acquisition) and reduced time in target quadrant (probe trial) in Morris Water Maze (MWM) test at 3 months post icv-STZ administration. These behavioural changes were associated with increased Aβ depositions and lowered BDNF levels in brain hippocampal homogenates. Pre-treatment with GSNO (50 μg/kg/day), reduced the icv-STZ induced cognitive deficits in acquisition and probe trials in the MWM. The icv-STZ induced elevations in Aβ40 and Aβ42 and reduced levels of BDNF in hippocampal homogenates were also attenuated after GSNO treatment in these rats. The NO-precursor, l-arginine (100 mg/kg) induced similar effects on behavioural and biochemical parameters tested but was marginally less consistent as compared to those seen with GSNO. The results suggest that GSNO ameliorates the cognitive deficits and associated brain biochemical changes in this experimental model of sporadic AD, and NO-BDNF interactions could play crucial role in these effects.

Hypericum perforatum (ethanolic extract) ameliorates simulated hypobaric hypoxia induced oxidative stress and neuronal damage in brains of Balb/c mice

Background: Hypobaric hypoxia refers to lower oxygen availability at high altitudes and is the cause of high altitude illness. Drugs such as acetazolamide and dexamethasone provide symptomatic relief and are associated with undesired side effects. Plant extracts such as Hypericum perforatum, which are documented to have neuromodulatory role can be more beneficial in ameliorating high altitude illness. Aims and Objective: Progressive cognitive decline is the hallmark characteristic of hypobaric hypoxia induced neuropathology attributed to ensuing oxidative stress and subsequent hippocampal damage. We have explored the efficacy of ethanolic extracts of Hypericum perforatum in amelioration of hypobaric hypoxia induced oxidative stress and associated behavioral deficits in mice.Material and Methods: Male Balb/c mice were exposed to simulated altitude of 25,000 ft. for 7 days (6 hr. per day) in a specially designed chamber. Ethanolic extract of Hypericum perforatum (HPE)(25mg/kg of body weight) was given orally prior to hypoxia exposure and effects were compared to hypoxia and control groups.Results: Animals exposed to hypobaric hypoxia showed sign of cognitive deterioration at day 3 and day 7 in the Elevated Plus Maze and Passive Avoidance Step through behavioral paradigms as compare to normoxic animals. Administration of HPE was able to alleviate the amnesic effect in treatment group, indicated by reduction in transfer latencies at day 3(IR-3 = -0.66±0.07) and day 7 IR-7 = -0.81±0.06) in elevated plus maze task and increased passive avoidance step through latency at day 3, (IR-3 = 3.23±0.67),as compared to ±hypoxic mice. Hypoxia group of animals suffered significant oxidative stress compared to normoxic mice as indicated by up-regulated malondialdehyde and total nitrite levels in hippocampal homogenates. The plasma lactate dehydrogenase activity was also increased following hypoxia indicating tissue damage. Co-treatment with HPE in simulated hypobaric hypoxia insult for seven days resulted in significant reduction in malondialdehyde, total nitrites and plasma LDH levels in animals.Conclusion: Hypericum perforatum extract improves cognitive performance in hypobaric hypoxia exposed mice with a concomitant reduction in oxidative stress burden suggesting its plausible use for preventing high altitude illness.Asian Journal of Medical Sciences Vol.9(2) 2018 1-8

Hypothyroidism increases cyclooxygenase-2 levels and pro-inflammatory response and decreases cell proliferation and neuroblast differentiation in the hippocampus.

The present study investigated the effects of hypothyroidism on cyclooxygenase-2 (COX-2) and pro-inflammatory cytokines in the dentate gyrus to elucidate the roles of COX-2 in the hypothyroid hippocampus. Hypothyroidism was induced in rats by treating with 0.03% 2-mercapto-1-methyl-imidazole dissolved in drinking water for 5 weeks. The animals were sacrificed at 12 weeks of age. Hypothyroidism rats exhibited decreased triiodothyronine and thyroxine levels in the serum, while the levels of thyroid-stimulating hormone and the weight of thyroid glands were significantly higher in the hypothyroid rats compared with those in the vehicle-treated group. COX-2 immunoreactivity was significantly increased in the hippocampal CA2/3 region and the dentate gyrus compared with the vehicle-treated group. Levels of pro-inflammatory cytokines including interleukin (IL)-1β, IL-6 and tumor necrosis factor-α were significantly higher in the hippocampal homogenates of hypothyroid rats. Cell proliferation and neuroblast differentiation based on Ki67 and doublecortin immunohistochemistry were decreased in the dentate gyrus of hypothyroid rats compared with those in the vehicle-treated group. These results suggested that hypothyroidism-mediated COX-2 expression affected hippocampal plasticity by upregulating the levels of pro-inflammatory cytokines in the hippocampus. Therefore, COX-2 may be suggested as a candidate molecule for preventing hypothyroidism-induced neurological side effects.

Interleukin-10 and PD150606 modulate expression of AMPA receptor GluA1 and GluA2 subunits under hypoxic conditions.

The goal of this study was to evaluate the effects of anti-inflammatory cytokine, interleukin-10 (IL-10), and calpain inhibitor, PD150606, on the expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits in rat hippocampal slices exposed to repeated brief hypoxic episodes. We studied both individual and combinatory effects of PD150606 and IL-10 on the expression of AMPA receptor subunits under hypoxic conditions for GluA1 and GluA2 as well as their phosphorylated forms - pSer831-GluA1 and pSer880-GluA2. Additionally, we studied whether brief hypoxic episodes and IL-10 may affect mRNA expression of transcriptional factors such as hypoxia-inducible factor-1α and nuclear factor κB (NF-κB). Western blotting analysis of hippocampal slice homogenates revealed that IL-10 and PD150606, both individually and in combination, ameliorate hypoxia-induced decrease in the expression of GluA1 and pSer831-GluA1, with different level of efficiency measured at 10, 50, and 90 min after hypoxia induction. Interestingly, brief hypoxic episodes did not induce any changes in the expression of GluA2 and pSer880-GluA2 subunits, whereas PD150606 showed biphasic effect, decreasing the expression of GluA2 and pSer880-GluA2 at 10 min and potentiating it at 90 min after hypoxia induction. IL-10 alone did not show any effect but was able to reverse PD150606 action on the expression of pSer880-GluA2 at 10 min and further potentiated it for GluA2 at 90 min after hypoxia. Finally, PCR analysis revealed that modulation of GluA1 and GluA2 expressions by hypoxia, and IL-10 was not associated with changes in the expression of hypoxia-inducible factor-1α and nuclear factor-κB (NF-κB) transcriptional factors.

Decrease in glucose transporter 1 levels and translocation of glucose transporter 3 in the dentate gyrus of C57BL/6 mice and gerbils with aging

In the present study, we compared the cell-specific expression and changes protein levels in the glucose transporters (GLUTs) 1 and 3, the major GLUTs in the mouse and gerbil brains using immuno-histochemistry and Western blot analysis. In both mouse and gerbils, GLUT1 immunoreactivity was mainly found in the blood vessels in the dentate gyrus, while GLUT3 immunoreactivity was detected in the subgranular zone and the molecular layer of the dentate gyrus. GLUT1-immunoreactivity in blood vessels and GLUT1 protein levels were significantly decreased with age in the mice and gerbils, respectively. In addition, few GLUT3-immunoreactive cells were found in the subgranular zone in aged mice and gerbils, but GLUT3-immunoreactivity was abundantly found in the polymorphic layer of dentate gyrus in mice and gerbils with a dot-like pattern. Based on the double immunofluorescence study, GLUT3-immunoreactive structures in gerbils were localized in the glial fibrillary acidic protein-immunoreactive astrocytes in the dentate gyrus. Western blot analysis showed that GLUT3 expression in the hippocampal homogenates was slightly, although not significantly, decreased with age in mice and gerbils, respectively. These results indicate that the reduction in GLUT1 in the blood vessels of dentate gyrus and GLUT3 in the subgranular zone of dentate gyrus may be associated with the decrease in uptake of glucose into brain and neuroblasts in the dentate gyrus. In addition, the expression of GLUT3 in the astrocytes in polymorphic layer of dentate gyrus may be associated with metabolic changes in glucose in aged hippocampus.

Proteomic Analyses of Exothermic Processes in Rat Brain Homogenate

Alzheimer’s disease (AD) is the most widespread neurodegenerative disorder which can be induced by scopolamine, but the underlying molecular mechanism is poorly understood. Recently, differential scanning calorimetry (DSC) has been used to study healthy and scopolamine-treated mice. A well-expressed exothermic transition minimum in the range of 35 - 45°C was determined in the DSC profiles of healthy mice supernatants. To explain this process, using two-dimensional gel electrophoresis (2D-PAGE) coupled with MALDI-TOF-TOF, poorly soluble membrane proteins in hippocampal proteome of rat brain tissue were identified. The different behavior of the hippocampal proteome from the healthy rats before and after heating to 45oC was identified. Due to the demonstrated change in protein level of tau protein and tubulin in the rat hippocampus after heating to 45°C, it was suggested that the observed exothermic process at 35-45°C in rat may be due to the partial unfolding of tau protein, which leads to the release of tubulin. Both proteins together are involved in protein fibrillation and aggregation. Another important result is the discovery of different profiles for the proteome of hippocampal rat homogenates with scopolamine-induced neurodegenerative disorder and its characteristics of healthy rats. The reported results from this study can help clarify the molecular mechanisms of scopolamine-induced dementia and neurodegenerative processes in general.

Probing the putative α7 nAChR/NMDAR complex in human and murine cortex and hippocampus: Different degrees of complex formation in healthy and Alzheimer brain tissue.

α7 nicotinic acetylcholine receptors (nAChRs) and N-methyl-D-aspartate receptors (NMDARs) are key mediators of central cholinergic and glutamatergic neurotransmission, respectively. In addition to numerous well-established functional interactions between α7 nAChRs and NMDARs, the two receptors have been proposed to form a multimeric complex, and in the present study we have investigated this putative α7 nAChR/NMDAR assembly in human and murine brain tissues. By α-bungarotoxin (BGT) affinity purification, α7 and NMDAR subunits were co-purified from human and murine cortical and hippocampal homogenates, substantiating the notion that the receptors are parts of a multimeric complex in the human and rodent brain. Interestingly, the ratios between GluN1 and α7 levels in BGT pull-downs from cortical homogenates from Alzheimer’s disease (AD) brains were significantly lower than those in pull-downs from non-AD controls, indicating a reduced degree of α7 nAChR/NMDAR complex formation in the diseased tissue. A similar difference in GluN1/α7 ratios was observed between pull-downs from cortical homogenates from adult 3xTg-AD and age-matched wild type (WT) mice, whereas the GluN1/α7 ratios determined in pull-downs from young 3xTg-AD and age-matched WT mice did not differ significantly. The observation that pretreatment with oligomeric amyloid-β1–42 reduced GluN1/α7 ratios in BGT pull-downs from human cortical homogenate in a concentration-dependent manner provided a plausible molecular mechanism for this observed reduction. In conclusion, while it will be important to further challenge the existence of the putative α7 nAChR/NMDAR complex in future studies applying other methodologies than biochemical assays and to investigate the functional implications of this complex for cholinergic and glutamatergic neurotransmission, this work supports the formation of the complex and presents new insights into its regulation in healthy and diseased brain tissue.

The effects of vitamin D administration on brain inflammatory markers in high fat diet induced obese rats

BackgroundObesity induced brain inflammation is associated with cognitive disorders. We aimed to investigate the influence of vitamin D on hypothalamus and hippocampus inflammatory response in high-fat diet induced obese rats.MethodsIn the beginning of the study, 40 rats were divided into two groups: control diet and high fat diet (HFD) for 16 weeks; then each group subdivided into two groups including: N, ND + vitamin D, HFD and HFD + vitamin D. Vitamin D supplementation was done for 5 weeks at 500 IU/kg dosage. IL-6, IL-1β, NF-Kβ and acetylcholine (ACH) and brain derived neurotropic factor (BDNF) concentrations in hippocampus and hypothalamus homogenate samples were measured by commercial ELISA kits.ResultsVitamin D administration, reduced food intake and weight gain in studied groups (P < 0.001). Vitamin D reduced hippocampus acetylcholine concentrations in ND + vitamin D group (P < 0.001). High fat diet increased hippocampus IL-6 concentrations significantly (P < 0.05) compared with normal diet receiving groups. Vitamin D could not have significant effects on IL-6 concentrations. Vitamin D administrations reduced IL-1β, NF-Kβ and acetylcholine concentration and BDNF concentrations in ND + vitamin D compared with ND group. These reductions were not significant in HFD + vitamin D versus HFD group.ConclusionAccording to our results, vitamin D reduced food intake and weight gain and modulated the HFD induced inflammatory response in hippocampus and hypothalamus of high fat diet induced obesity. Therefore, this neurosteroid, can be suggested as a supplemental therapeutic tool in prevention of obesity related cognitive and neurodegenerative problems.

Chlorogenic Acid Prevents Alcohol-induced Brain Damage in Neonatal Rat.

The present investigation evaluates the neuroprotective effect of chlorogenic acid (CA) in alcohol-induced brain damage in neonatal rats. Ethanol (12 % v/v, 5 g/kg) was administered orally in the wistar rat pups on postnatal days (PD) 7-9. Chlorogenic acid (100 and 200 mg/kg, p.o.) was administered continuously from PD 6 to 28. Cognitive function was estimated by Morris water maze (MWM) test. However, activity of acetylcholinesterase, inflammatory mediators, parameters of oxidative stress and activity of caspase-3 enzyme was estimated in the tissue homogenate of cerebral cortex and hippocampus of ethanol-exposed pups. It has been observed that treatment with CA attenuates the altered cognitive function in ethanol-exposed pups. There was a significant decrease in the activity of acetylcholinesterase in the CA treated group compared to the negative control group. However, treatment with CA significantly ameliorates the increased oxidative stress and concentration of inflammatory mediators in the brain tissues of ethanol-exposed pups. Activity of caspase-3 enzyme was also found significantly decreased in the CA treated group compared to the negative control group. The present study concludes that CA attenuates the neuronal damage induced in alcohol exposed neonatal rat by decreasing the apoptosis of neuronal cells.

Alterations in the Interplay between Neurons, Astrocytes and Microglia in the Rat Dentate Gyrus in Experimental Models of Neurodegeneration.

The hippocampus is negatively affected by aging and neurodegenerative diseases leading to impaired learning and memory abilities. A diverse series of progressive modifications in the intercellular communication among neurons, astrocytes and microglia occur in the hippocampus during aging or inflammation. A detailed understanding of the neurobiological modifications that contribute to hippocampal dysfunction may reveal new targets for therapeutic intervention. The current study focussed on the interplay between neurons and astroglia in the Granule Layer (GL) and the Polymorphic Layer (PL) of the Dentate Gyrus (DG) of adult, aged and LPS-treated rats. In GL and PL of aged and LPS-treated rats, astrocytes were less numerous than in adult rats. In GL of LPS-treated rats, astrocytes acquired morphological features of reactive astrocytes, such as longer branches than was observed in adult rats. Total and activated microglia increased in the aged and LPS-treated rats, as compared to adult rats. In the GL of aged and LPS-treated rats many neurons were apoptotic. Neurons decreased significantly in GL and PL of aged but not in rats treated with LPS. In PL of aged and LPS-treated rats many damaged neurons were embraced by microglia cells and were infiltrated by branches of astrocyte, which appeared to be bisecting the cell body, forming triads. Reactive microglia had a scavenging activity of dying neurons, as shown by the presence of neuronal debris within their cytoplasm. The levels of the chemokine fractalkine (CX3CL1) increased in hippocampal homogenates of aged rats and rats treated with LPS, and CX3CL1 immunoreactivity colocalized with activated microglia cells. Here we demonstrated that in the DG of aged and LPS-treated rats, astrocytes and microglia cooperate and participate in phagocytosis/phagoptosis of apoptotic granular neurons. The differential expression/activation of astroglia and the alteration of their intercommunication may be responsible for the different susceptibility of the DG in comparison to the CA1 and CA3 hippocampal areas to neurodegeneration during aging and inflammation.

Oxidative stress with tau hyperphosphorylation in memory impaired 1,2-diacetylbenzene-treated mice

Long-term exposure to organic solvent may be related to the incidence of neuronal diseases, such as, Alzheimer’s disease, depression, multiple sclerosis, dementia, Parkinson’s disease. Previously, the authors reported 1,2-diacetylbenzene (DAB; a neurotoxic metabolite of 1,2-diethylbenzene) causes central and peripheral neuropathies that lead to motor neuronal deficits. Furthermore, it is known DAB increases oxidative stress and protein adduct levels and impairs hippocampal neurogenesis in mice. The authors examined the relevance of oxidative stress and tau hyperphosphorylation in the hippocampus. Five-week-old male C57BL/6 mice were treated with 1 or 5mg/kg/day DAB for 2weeks. Neither overall body weight increases nor behavioral differences were observed after treatment, but kidney and liver weights decreased. Increased ROS production, activated glycogen synthase kinase-3β (GSK-3β) and tau hyperphosphorylation were observed in hippocampal homogenates. To assess memory impairment, the Morris Water Maze was used. Animals in the DAB-treated groups took longer to reach the platform. Movement patterns of DAB treated mice were more complicated and their swimming speeds were lower than those of controls. When SHSY5Y neuroblastoma cells were pretreated with NAC (an antioxidant) or a GSK-3β inhibitor, the expression of active GSK-3β and tau hyperphosphorylation were reduced. These results suggest ROS produced by DAB causes tau hyperphosphorylation via GSK-3β phosphorylation and it might be related to impaired memory deficit.

Inhibition of 12/15 LOX ameliorates cognitive and cholinergic dysfunction in mouse model of hypobaric hypoxia via. attenuation of oxidative/nitrosative stress

12/15 Lipoxygenase has recently been described as potent propagator of oxidative stress and is closely associated with cognitive decline in neurodegenerative diseases. The mechanism/s behind 12/15 LOX involvement in cognitive deficits remain obscure. The current study has been designed to investigate the underlying role of 12/15LOX and effect of 12/15 LOX inhibition on hypobaric hypoxia-induced memory impairment and cholinergic deficits. Male Balb/c mice subjected to simulated hypobaric hypoxia/reoxygenation condition for 3days showed marked working memory impairment concomitant with hippocampal neuronal damage and malondialdehyde production which were significantly attenuated by baicalein, a specific inhibitor of 12/15LOX. Hypobaric hypoxia-exposed mice had consistently increased expression of 12/15LOX and elevated 12(S) HETE levels in the hippocampus as well as plasma which were significantly mitigated following baicalein treatment. 12/15LOX inhibition also reduced hypobaric hypoxia-mediated upregulation of hippocampal HIF-1α protein expression along with reduction in expression of inflammatory genes. The inhibition of 12/15 LOX resulted in a significant decrease in NO levels in the hippocampal homogenate associated with downregulated iNOS, nNOS transcription but not eNOS speculating that 12/15 LOX is critically involved in HIF-1α, mediated by nitric oxide-induced neurotoxicity. We also observed a similar effect of 12/15 LOX inhibition on hippocampal COX2 expression. 12/15LOX inhibition could effectively modulate central cholinergic indices during hypobaric hypoxia by restoring mAChR-1, α7NAChR expression and AChE, ChAT activity in the hippocampus comparable to normal mice. We report here the mechanistic involvement of 12/15LOX in orchestrating hypoxia-associated neuronal damage and HIF-1α-dependent neuroinflammation resulting in cognitive decline.

Elevated sterol regulatory elementary binding protein 1 and GluA2 levels in the hippocampal nuclear fraction of Genetic Absence Epilepsy Rats from Strasbourg

Studies in animal models and human tissues show that nuclear translocation of sterol regulatory element binding protein 1 (SREBP1) and glutamate A2 subunit (GluA2) of cell-surface AMPA receptor (AMPAR) trigger neuronal excitotoxicity-induced apoptosis in stroke. However, it is not known whether a similar type of underlying pathophysiology occurs in absence epilepsy. To explore this issue, we examined the levels of mature SREBP1, GluA2, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), p53, and activated to total caspase 3 ratio in nuclear fractions (NF) of hippocampal homogenate from 8 to 10 week old male Genetic Absence Epilepsy Rats from Strasbourg (GAERS) and non-epileptic control (NEC) strains. Mature SREBP1 and GluA2 levels were elevated approximately two-fold in NFs of GAERS hippocampal homogenates compared to NEC animals. Significant increases in GAPDH (∼15-fold) and total caspase 3 (∼10-fold) levels were also found in NFs of GAERS hippocampal homogenates in comparison to the non-epileptic strain. Data from the current study suggest that absence epilepsy in GAERS is associated with nuclear translocation of mature SREBP1, GluA2 subunit of AMPARs, and recruitment of pro-cell death signaling proteins such as GAPDH and caspase 3. These changes may contribute to hippocampal neuronal/glial cell death in GAERS. Therefore, inhibiting the nuclear accumulation of mature SREBP1 and GluA2 translocation may reduce the pathophysiology of absence epilepsy.

REVERSAL OF LYSOSOMAL DYSFUNCTION RESCUES BETA-AMYLOID PATHOGENESIS IN ALZHEIMER’S DISEASE

Amyloid beta (Aβ) deposit is a major neuropathological hallmark of Alzheimer's disease (AD). It has been identified that intracellular Aβ (iAβ) accumulation is one of the crucial events in AD pathogenesis. Amount of evidence suggest that insufficient cellular clearance of iAβ results from lysosomal dysfunctions and enhancement of lysosomal function restores AD pathology. Therefore, identifying genes that promote the clearance of iAβ by reinforcing lysosomal activity and discovering the underlying mechanism are vital for AD intervention. For cell-based functional screening, cells were transfected with cDNA encoding lysosome-associated proteins and then further incubated with synthetic oligomeric Aβ1–42. Cytotoxicity was measured by staining with EthD. Hippocampal homogenates from AD patients with Braak stage V or VI and non-AD patients were provided by the Harvard Brain Tissue Resource Center (McLean Hospital, Belmont, Massachusetts, USA). For detection of intracellular Aβ1–42, membrane-associated Aβ1–42were washed with trypsin-EDTA and harvested cells were subjected to immunoblotting using 4G8 and 6E10 antibodies. Activity-defective mutant HYAL D129A/E131A was generated by mutagenesis. For lysosomal activity assay using fusion protein degraded by lysosome, GFP-CINCCKVL was amplified from pEGFP-C1 vector using synthesized primers (EGFP-5′-HindIII, EGFP-CINCCKVL-3′-EcoRI) and subcloned into pcDNA3.0. HYAL was isolated as the most effective gene that suppressed Aβ-induced cell death. Compared to normal controls, HYAL expression was significantly decreased in AD brains. Downregulation of HYAL aggravated Aβ-induced cell death and increased iAβ level. In overexpression assay, HYAL dramatically decreased Aβ-neurotoxicity and reduced iAβ level. An activity-defective mutant altered neither Aβ-induced toxicity nor the iAβ level, indicating that catalytic activity is critical for its protective role. Degradation of iAβ was halted by lysosomal inhibitors but not by proteasomal inhibitor, indicating that iAβ is degraded mostly by lysosomal pathway. In lysosomal activity assay, ectopically expressed HYAL hindered abnormal accumulation of lysosomal substrates due to Aβ1–42 exposure, suggesting that HYAL serve as positive lysosomal regulators in Aβ pathogenesis. These results suggest that HYAL exerts a protective role by facilitating Aβ degradation, possibly through enhancing lysosomal function. Reference: (1) Frank M. et al. Nat. Rev. Neurosci. 2007; 430(7000):499–509. (2) Nixon, R.A., and Yang, D.-S. Neurobiol. Dis. 2011; 43, 38–45.

COMPARISON OF BINDING PROPERTIES OF THK5117, THK5351, PBB3 AND T807 IN AUTOPSIES OF ALZHEIMER DISEASE CASES

Several families of tau PET tracers recently developed have been studied in vivo in patients after having shown good in vitro binding-properties. Some differences in binding properties have been observed between the tau PET tracers in Alzheimer disease and non-AD tauopathies. Recent observations report presence of off-target binding. Here we aimed at characterizing the binding properties as well as possible off-target binding of the tau tracers by comparing two THK compounds, THK5117 and THK5351 with T807 and PBB3, head-to-head in the same human brain tissue, using autoradiography on large frozen section from AD cases and binding assay in brain homogenates. Autoradiographies on large frozen section were done using 3H-THK5351, 3H-THK5117, 11C-THK5351, 11C-PBB3 and 18F-T807. Competition autoradiography using unlabeled T807 were performed for the 11C compound. In vitro binding assay competition using 3H-THK5351 with unlabeled THK5351, THK5117 and T807 were performed in hippocampus homogenates. Competitions binding assay and autoradiography were performed using 3H-THK5117, 3H-L-deprenyl with unlabeled THK5117, R-deprenyl, rasagiline and chlorgyline to characterize the MAO off-target component. 11C-PPB3, 11C-THK5351 and 18F-T807 showed similar regional distribution in autoradiography but with different intensity. Unlabeled THK5351 displaced 50% of 11C-THK5351 binding and less than 10% of 11C-PBB3 binding in all regions. Unlabeled PBB3 displaced 50 % of 11C-PBB3 binding. Unlabeled T807 displaced up to 70 % of 18F-T807 binding in frontal and temporal cortex, 30% of 11C-THK5351 in frontal and temporal cortex and up to 40% in the basal ganglia, and displaced less than 20 %of 11C-PBB3 in all regions. In vitro binding assay using 3H-THK5351 in competition with unlabeled THK5351, THK5117 and T807 showed at least two binding sites. While a 4 times differences in affinity for the binding site in nanomolar range was observed between THK5351 and T807, they shared one binding site in the picomolar range. Tau tracers from three different families showed all different binding properties in AD cases. Greater similarity in binding properties was observed between THK5351 and T807 than with PBB3. These results may have an influence on the outcome of the clinical use in vivo PET study targeting different form of tauopathies.