Effect of Flupirtine on Bcl-2 and Glutathione Level in Neuronal Cells Treatedin Vitrowith the Prion Protein Fragment (PrP106-126)

Flupirtine, trade name Katadolon, is a centrally acting nonopioid analgesic that has recently been found to display cytoprotective activityin vitroandin vivoon neurons induced to undergo apoptosis. This report shows that the PrP106-126 fragment of the prion protein, which is the likely etiological agent for a series of encephalopathies, is toxic to cortical neuronsin vitro.Simultaneously, PrP106–126 influences the molecular GSH content and the bcl-2 expression in neurons. Significant toxicity (32% reduction in cell viability) was observed at a concentration of 50 μMof the peptide after 9 days of incubation, while at higher concentrations toxicity increased to 70%. Neurotoxicity was greatly reduced following coincubation with 1 to 3 μg/ml flupirtine. Concomitant with PrP106-126-mediated cytotoxicity, glutathione (GSH) content fell by >70% with respect to untreated controls. This decrease in GSH level was strongly blocked by flupirtine under incubation conditions that reduce cell toxicity. In addition to normalizing GSH content, flupirtine induced the expression of the anti-apoptotically acting proto-oncogenebcl-2.Based on thesein vitrodata and on the favorable pharmacokinetic profile of the drug, we strongly suggest that flupirtine may prove useful for treatment of patients with prion disease.     

Excitotoxicity Through NMDA Receptors Mediates Cerebellar Granule Neuron Apoptosis Induced by Prion Protein 90-231 Fragment

Prion diseases recognize, as a unique molecular trait, the misfolding of CNS-enriched prion protein (PrP^C) into an aberrant isoform (PrP^Sc). In this work, we characterize the in vitro toxicity of amino-terminally truncated recombinant PrP fragment (amino acids 90-231, PrP90-231), on rat cerebellar granule neurons (CGN), focusing on glutamatergic receptor activation and Ca²⁺ homeostasis impairment. This recombinant fragment assumes a toxic conformation (PrP90-231^TOX) after controlled thermal denaturation (1 h at 53 °C) acquiring structural characteristics identified in PrP^Sc (enrichment in β-structures, increased hydrophobicity, partial resistance to proteinase K, and aggregation in amyloid fibrils). By annexin-V binding assay, and evaluation of the percentage of fragmented and condensed nuclei, we show that treatment with PrP90-231^TOX, used in pre-fibrillar aggregation state, induces CGN apoptosis. This effect was associated with a delayed, but sustained elevation of [Ca²⁺]_i. Both CGN apoptosis and [Ca²⁺]_i increase were not observed using PrP90-231 in PrP^C-like conformation. PrP90-231^TOX effects were significantly reduced in the presence of ionotropic glutamate receptor antagonists. In particular, CGN apoptosis and [Ca²⁺]_i increase were largely reduced, although not fully abolished, by pre-treatment with the NMDA antagonists APV and memantine, while the AMPA antagonist CNQX produced a lower, although still significant, effect. In conclusion, we report that CGN apoptosis induced by PrP90-231^TOX correlates with a sustained elevation of [Ca²⁺]_i mediated by the activation of NMDA and AMPA receptors.     

A Neurotoxic Prion Protein Fragment Induces Rat Astroglial Proliferation and Hypertrophy

Prion-related encephalopathies are characterized by the accumulation of an abnormal prion protein isoform (PrP^Sc) and the deposition of PrP amyloid in the brain. This process is accompanied by neuronal loss and astrogliosis. We recently showed that a synthetic peptide corresponding to residues 106–126 of human PrP is amyloidogenic and causes neuronal death by apoptosis in vitro. In the present study we investigated the effects of 1- and 14-day exposures of rat astroglial cultures to mtcromolar concentrations of this peptide as well as peptides homologous to other portions of PrP, a peptide corresponding to residues 25–35 of amyloid-β protein, and a scrambled sequence of PrP 106–126. No significant changes were observed after 1-day exposure of cultures to any peptide. Conversely, 14-day treatment with PrP 106–126 (50 μM) resulted in a 5-fold increase in glial fibrillary acidic protein (GFAP) expression, as evaluated by Northern and Western blot analyses, and a 1.5-fold increment in cell number. Light and electron microscopy immunohistochemistry showed an enlargement in size and density of astroglial processes, and an increase in GFAP-immunoreactive intermediate filaments. These changes were not observed after 14-day treatment of cultures with the other peptides, including PrP 106–126 scrambled. The increase in GFAP expression of astroglial cultures exposed to PrP 106–126 was quantitatively similar to that found in scrapie-infected hamster brains. These results suggest that the PrP region corresponding to residues 106–126 is biologically active, and that cerebral accumulation of peptides including this sequence might be responsible for both the neuronal degeneration and the astrogliosis that occur in prion-related encephalopathies.     

MicroRNA-365 Knockdown Prevents Ischemic Neuronal Injury by Activating Oxidation Resistance 1-Mediated Antioxidant Signals

Micro RNA-365(mi R-365) is upregulated in the ischemic brain and is involved in oxidative damage in the diabetic rat. However, it is unclear whether mi R-365 regulates oxidative stress(OS)-mediated neuronal damage after ischemia. Here, we used a transient middle cerebral artery occlusion model in rats and the hydrogen peroxide-induced OS model in primary cultured neurons to assess the roles of mi R-365 in neuronal damage. We found that mi R-365 exacerbated ischemic brain injury and OS-induced neuronal damage and was associated with a reduced expression of OXR1(Oxidation Resistance 1). In contrast, mi R-365 antagomir alleviated both the brain injury and OXR1 reduction. Luciferase assays indicated that mi R-365 inhibited OXR1 expression by directly targeting the 30-untranslated region of Oxr1. Furthermore, knockdown of OXR1 abolished the neuroprotective and antioxidant effects of the mi R-365 antagomir. Our results suggest that mi R-365 upregulationincreases oxidative injury by inhibiting OXR1 expression,while its downregulation protects neurons from oxidative death by enhancing OXR1-mediated antioxidant signals.     

A Prion Protein Fragment Primes Type 1 Astrocytes to Proliferation Signals from Microglia

Gliosis is a hallmark of prion disease. A neurotoxic prion peptide (PrP106-126) induces astrocyte proliferation in the presence of microglia. This peptide also directly enhances microglial proliferation in culture. We have investigated this further to understand the method by which factors released by microglia and PrP106-126 work together to enhance astrocyte proliferation. PrP106-126 in the presence of microglia specifically enhanced type 1 astrocyte proliferation but not Type 2. Astrocytes that do not express the prion protein were more sensitive to oxidative stress and the toxicity of cytosine arabinoside. In the presence of cytosine arabinoside, PrP106-126 was toxic to pure astrocyte cultures. Using conditioned medium from microglia we have shown that PrP^c-expressing astrocytes proliferate in response to factors released by microglia stimulated by granulocyte/macrophage colony-stimulating factor. This response is enhanced in the presence of PrP106-126. PrP^c-deficient astrocytes do not show this response. These results suggest that astrocytes are primed by PrP106-126 to respond more to factors released by proliferating microglia. Astrocytes may proliferate in this system to escape entering the cell suicide pathway.     

炎症反应在脑动脉微栓子导致的大脑神经元损伤机制中的作用

目的:探讨炎症反应在脑动脉微栓子导致的大脑神经元损伤机制中的作用.方法:48只SD大鼠随机分为微栓子3 d组、微栓子7 d组、假手术3 d组和假手术7 d组 (均n=12).微栓子组将25～50 mm全血凝块微栓子注入SD大鼠左侧颈内动脉,建立脑梗死阈值下微栓子导致的脑损伤模型.损伤后3 d和7 d分批处死大鼠,CD1...     

Caffeine Prevents Memory Impairment Induced by Hyperhomocysteinemia

Mesenchymal stem cell (MSC) therapy is a promising prospect for the treatment of Alzheimer’s disease (AD); however, the underlying mechanisms by which MSCs mediate positive effects are still unclear. We speculated that MSCs mediate microglial autophagy and enhance the clearance of Aβ. To test this hypothesis, we cultured BV2 microglial cells with umbilical cord mesenchymal stem cells conditioned medium (ucMSCs-CM) in the presence or absence of Aβ25–35 oligomers. We investigated BV2 cell proliferation, cell death, and Aβ25–35 phagocytosis as well as protein expression levels of LC3, Beclin-1, p62, insulin-degrading enzyme (IDE), and neprilysin (Nep) with western blotting. The results showed that ucMSCs-CM inhibited the proliferation and decreased cell death of BV2 cells induced by Aβ25–35. ucMSCs-CM also promoted the phagocytosis of Aβ25–35 by BV2 cells and changed the expression of autophagy-related proteins LC3, Beclin-1, and p62. Treatment also upregulated the expression of Aβ-degrading enzymes IDE and Nep. Furthermore, the culture medium in BV2 cells with Aβ25–35 and ucMSCs-CM prevented neuronal cell SH-SY5Y from cell death compared to control medium without ucMSCs-CM. Altogether, these data suggested that ucMSCs-CM protect microglial and neuronal cells from Aβ25–35-induced cell death and promote Aβ phagocytosis by modulating autophagy and enhancing the expression of Aβ-degrading enzymes in microglia.     

β淀粉样蛋白致神经细胞凋亡机制的研究进展

阿尔茨海默病（AD）是老年期痴呆最主要类型。β淀粉样蛋白（Aβ）可诱导神经细胞凋亡，是AD发生发展的关键因素。近年来，Aβ的神经毒性及其产生机制一直是对AD研究的热点问题，Aβ导致体外培养的神经细胞凋亡的可能机制包括氧化应激和钙稳态失衡，NF-κB基因调控，糖原合酶激酶3β和细胞周期依赖性激酶的活动等，这些机制的阐明对揭示AD发病原因及探索有效防治措施具有重要意义。     

Effect of Microglia Activation on Dopaminergic Neuronal Injury Induced by Manganese, and Its Possible Mechanism

Manganese (Mn) is an essential trace element. It is known to have various functions, such as participating in enzymatic synthesis, and promoting hematopoiesis. On the other hand, it can cause toxic injury upon excess intake. However, toxic effects and its mechanism on glial cells are unclear. In the present study, we demonstrated that MnCl₂ can activate microglia, and that this can cause dopaminergic neuronal injury. Investigation of the underlying mechanisms showed that inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) was induced and highly expressed following Mn treatment. Moreover, pretreatment with S-methylisothiourea (SMT. iNOS inhibitor), Mn-induced iNOS expression and dopaminergic neuronal injury were partly reverse. Pretreatment with minocycline (microglia activation inhibitor), Mn-induced activation of microglia and dopaminergic neuronal injury was partly reverse. Taken together, our results showed that Mn can cause microglia activation, which can up-regulate the level of IL-1β, TNF-α and iNOS, and these inflammatory factors can cause dopaminergic neuronal injury. SMT and minocycline prevent Mn-induced dopaminergic neuronal injury.     

Efficacy of Novel Acridine Derivatives in the Inhibition of hPrP90-231 Prion Protein Fragment Toxicity

Quinacrine is one of the few molecules tested to treat patients affected by prion diseases, although the clinical outcome is largely unsatisfactory. To identify novel derivatives with higher neuroprotective activity, we evaluated the effects of a small library of acridine derivatives. The 6-chloro-2-methoxyacridine derivatives bearing on position 9 a quinolizidin-1-ylamino (Q1, Q2) or a quinolizidin-1-ylalkylamino residue (Q3, Q4, Q6, Q7), the thio-bioisoster of Q3 (Q5), the 9-(N-lupinylthiopropyl)amino derivative (Q8) and simple acridines (Q9 and Q10) were considered. We compared the effects of quinacrine and these novel analogues in the inhibition of the cytotoxic activity and protease K (PK) resistance of the human prion protein fragment 90-231 (hPrP90-231). We demonstrate that quinacrine caused a significant reduction of hPrP90-231 toxicity due to its binding to the fragment and the prevention of its conversion in a toxic isoform. All acridine derivatives analyzed showed high affinity binding for hPrP90-231, but only Q3 and Q10, caused a significant reduction of hPrP90-231 cytotoxicity, with higher efficacy than quinacrine. We attempted to correlate the cytoprotective effects of the new compounds with some biochemical parameters (binding affinity to hPrP90-231, intrinsic fluorescence quenching, hydrophobic amino acid exposure), but a direct relationship occurred only with the reduction of PK resistance, likely due to the prevention of the acquisition of the β-sheet-rich toxic conformation. These data represent interesting leads for further modifications of the basic side chain and the substituent pattern of the acridine nucleus to develop novel compounds with improved antiprion activity to be tested in in vivo experimental setting.     

An in Vivo Quantifiable Model of Cochlear Neuronal Degeneration Induced by Central Process Injury

In the available in vivo experimental models for cochlear neuronal degeneration, the peripheral (hair cell side) process of the cochlear nerve has been injured in order to induce neuronal degeneration. However, there has been no dependable experimental model in which cochlear neuronal degeneration begins from the central (brain stem side) process. This lack of a central process injury model has probably been due to the experimental difficulties that had to be overcome in order to reproducibly and selectively injure the central process of the cochlear neurons while maintaining the patency of the internal auditory artery in small experimental animals such as rats. Using rats, we first developed a central process injury model in which the reduction of the spiral ganglion cells due to retrograde degeneration of cochlear neurons can be quantitatively evaluated. In our experimental model, the cochlear nerve was compressed and injured by a compression-recording (CR) electrode placed at the internal auditory meatus. First, the cochlear nerve was compressed until the compound action potentials of the cochlear nerve became flat, and then the CR electrode was advanced by various compression speeds (5, 10, or 200 μm/s) to reach the same depth (400μm). In our model, therefore, the reduction of the spiral ganglion cells was caused compression speed dependently. This method made it possible to produce compression injury to the cochlear nerve without evidence of damage to the blood supply to the cochlea via the internal auditory artery. This model gives us the means to obtain knowledge that was previously impossible to derive from the peripheral process injury models.     

7-Chlorokynurenate Ameliorates Neuronal Injury Mediated by HIV Envelope Protein gp120 in Rodent Retinal Cultures

Prior studies with in vitro model systems have suggested that at least part of the neurological manifestations of AIDS may stem from neuronal injury involving the HIV-1 coat protein gp120. This form of neuronal damage is most probably mediated indirectly by a complex set of cellular interactions among macrophages, astrocytes, and neurons, resulting in a final common pathway of overstimulation of N -methyl- d -aspartate (NMDA) receptors. We studied the neuroprotective effect from gp120-induced neuronal injury of an antagonist of the glycine site of the NMDA receptor, 7-chlorokynurenate. In identified rat retinal ganglion cells in culture, we found that 50 μM 7-chlorokynurenate significantly abrogated the injury engendered by 20 pM gp120. Addition of 300 μM exogenous glycine prevented this protective effect of 50 μM 7-chlorokynurenate. These data suggest that glycine site antagonists of the NMDA receptor may have therapeutic potential for ameliorating neuronal damage associated with gp120.     

Ammonia Prevents Activation of NMDA Receptors by Glutamate in Rat Cerebellar Neuronal Cultures

Acute ammonia toxicity is mediated by activation of NMDA receptors and is prevented by chronic moderate hyperammonaemia. The aim of this work was to assess whether the protective effect of chronic hyperammonaemia is due to impaired activation of the NMDA receptor. It is shown that chronic hyperammonaemia in rats decreases the binding of [³H]MK-801 to synaptosomal membranes from the hippocampus but not the amount of NMDAR1 receptor protein as determined by immunoblotting. In primary cultures of cerebellar neurons, long-term treatment with 1 mM ammonia also decreased significantly the binding of [³H]MK-801. These results suggest that ammonia impairs NMDA receptor activation. To confirm this possibility we tested the effect of long-term treatment of the cultured neurons with 1 mM ammonia on three well known events evoked by activation of the NMDA receptor: neuronal death induced by glutamate, increase in aspartate aminotransferase activity and increase in free intracellular [Ca²⁺]. Long-term treatment with ammonia prevented noticeably the effects of glutamate or NMDA on all these parameters. These results indicate that long-term treatment of neurons with 1 mM ammonia leads to impaired function of the NMDA receptor, which cannot be activated by glutamate or NMDA. Activation of protein kinase C by a phorbol ester restored the ability of the NMDA receptor to be activated in neurons treated with ammonia. This suggests that ammonia impairs NMDA receptor function by decreasing protein kinase C-dependent phosphorylation.     

Vitamin E Prevents Alzheimer's Amyloid beta-Peptide (1-42)-Induced Neuronal Protein Oxidation and Reactive Oxygen Species Production

Amyloid beta-peptide (Abeta) is a 42-43 amino acid peptide known to accumulate in Alzheimer's disease (AD) brain. We previously reported that the neurotoxicity caused by Abeta is a result of its associated free radicals, which can play an important role in generating oxidative stress. Abeta(25-35)-associated oxidative stress-induced neuronal death in vitro is well established by many laboratories, including ours. However, the oxidative stress-induced by the full-length [Abeta(1-42)] peptide is not well investigated. The protective effect of antioxidant vitamin E in full-length peptide-induced oxidative stress also has not been reported. Here, we report that the increased protein oxidation, reactive oxygen species (ROS) formation, and neurotoxicity induced by Abeta(1-42) in primary rat embryonic hippocampal neuronal culture are prevented by the free radical scavenger and antioxidant vitamin E. To test the hypothesis that vitamin E's protective effect may be due to inhibition of fibril formation, electron microscopy studies were undertaken. Vitamin E does not inhibit Abeta(1-42) fibril formation, suggesting that the neuroprotection afforded by this molecule stems from other processes, most probably through the scavenging of Ab-associated free radicals. These results may have implications on the treatment of Alzheimer's disease.     

A Quantitative and Qualitative Analysis of Prion Protein Immunohistochemical Staining in Creutzfeldt-Jakob Disease Using Four Anti Prion Protein Antibodies

Creutzfeldt-Jakob disease (CJD) is the most common spongiform encephalopathy affecting humans. Prion protein (PrP) immunohistochemistry may be useful for studying the localization of prion protein and assessing its role in CJD, the accumulation of a specific protease resistant PrP isoform being apparently pathognomic to the spongiform encephalopathies. However, a number of factors influence the results of immunostaining, making interpretation and comparisons between the staining of different PrP antisera difficult. This study has examined qualitatively and quantitatively the staining produced by four antisera raised to a variety of prion protein homologues in two cases of CJD and two age-matched controls. Quantitative analysis was provided through the use of custom designed image analysis software. Kuru, granular and multicentric plaques, cellular, perivacuolar and white matter PrP deposits were observed in CJD cases with all four antisera. No significant immunostaining was seen in the control tissue. Some antibody specific staining patterns were observed qualitatively; however, quantitative analysis showed statistically significant correlations between all the antisera on the diseased brain tissue. Prion protein immunohistochemistry is thus useful in interpreting patterns of protein distribution in diseased brain but care may be required in interpreting the results of a single antibody.     

Reversible and Irreversible Neuronal Injury Induced by Intrahippocampal Infusion of the mGluR Agonist 1S,3R-ACPD in the Rat

Metabotropic glutamate receptor (mGluR)-induced neuronal injury in the brain was further investigated in the rat. The highly selective mGluR agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) was infused stereotaxically into the left dorsal hippocampus of adult rats. Control (2 μl saline injected) rats had minimal tissue injury that was confined to the area around the injection site. In contrast, dose of 250 nmol/2μl 1S,3R-ACPD produced a moderate number of swollen and injured cells in polymorphic, pyramidal and molecular layers of the injected hippocampus which was observed at 4 and 8 h post-injection. However, at 24 h few injured or necrotic cells were found. A dose of 1000 nmol/2μl 1S,3R-ACPD produced severe cellular injury in polymorphic, pyramidal and molecular layers of the hippocampus at 4, 8, or 24 h. At 24 h after this higher dose of 1S,3R-ACPD, a number of necrotic cells (i.e. pyramidal neurons of area CA1) were found. Both doses of 1S,3R-ACPD produced seizures in animals that were charcaterized by multiple episodes of wet dog shakes, staring, immobility, facial automatisms, rearing, bilateral forelimb clonus, and loss of postural control. These data support a possible role for excesive mGluR activation in pathological states of convulsions and neurodegeneration.