AIM: To explore the possibility that expressions of different heat shock proteins (HSPs) are specifically involved in the protection of thermal preconditioning against apoptosis of cerebellar granule neurons (CGNs) induced by repolarization. METHODS: Western blotting was used to detect expressions of HSP27, HSP70, and HSP90 induced by thermal preconditioning (TP) in CGNs; reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression level of HSP70 mRNA; apoptosis of CGNs was induced by switching culture medium containing KCl 25 mmol/L to one containing KCl 5 mmol/L. RESULTS: No expression of HSP27 in cerebellar granule neurons was observed with TP at 44 ºC. Expression of HSP90 was obvious in CGNs both without and with TP at 44 ºC for different periods. Expression of HSP70 protein in CGNs was lower with TP at 44 ºC for 5 min, but it increased gradually after the period was prolonged to 30, 60, or 90 min. HSP70 mRNA was detected after TP 44 ºC for 30, 60, and 90 min and increased gradually with time. HSP70 antisense oligodeoxynucleotides 10 μmol/L for 72 h inhibited the protective effects of TP at 44 ºC on apoptosis of CGNs induced by repolarization. CONCLUSION: HSP70 is involved in protective effects of thermal preconditioning on apoptosis in cerebellar granule neurons induced by repolarization.

The b-amyloid peptide Ab.is deposited in neuritic plaques which are characteristic features of Alzheimer's disease AD.. Prominent neurodegeneration and glial activation occurs around these plaques leading to the hypothesis that Ab may play a causative role in the neuronal loss and the inflammatory response associated with AD. Here we show that Ab-induced toxicity of cultured fetal rat cortical neurons is associated with internucleosomal DNA fragmentation beginning just 6 h after neurons are exposed to Ab. Additionally, constitutive NF-k B activity readily measured in fetal rat cortical neurons decreases in a concentration-and time-dependent fashion following exposure to Ab, but there is no corresponding decrease in NF-k B mRNA or protein p65.. An upregulation of both Ik Ba protein and mRNA which occurs in cortical neurons exposed to Ab may be responsible for retaining NF-k B in the cytoplasm accounting for the observed decrease in activated NF-k B. The latter is supported by the observation that pretreatment of cortical cultures with an antisense oligonucleotide to Ik Ba mRNA is neuroprotective. In contrast to cortical neurons, exposure of rat primary astroglial cultures to Ab results in a concentration-and time-dependent activation of NF-k B with subsequent upregulation of IL-1b and IL-6. Our data suggest that Ab-induced neurotoxicity as well as astrocyte activation may be medicated by the NF-k BrRel family of proteins, and thus alterations in NF-k B-directed gene expression may contribute to both the neurodegeneration and inflammatory response which occur in AD.

We have recently reported that mastoparan, a peptide toxin isolated from wasp venom, induces apoptosis in cultured cerebellar granule neurons that can be blocked by cholera toxin, an activator of Gs. Measurements of intracelhilar free calcium concentration ([Ca2+]i) reveal that mastoparan induces a dramatic elevation of [Ca2+]i that is frequently followed by enhanced leakage of fura-2 out of the neurons, suggesting that this rise in [Ca2+]i may be due to a more generalized change in membrane permeability. However, the mastoparan-induced initial elevation of [Ca2+]i is maintained in the absence of extracellular Ca2+, suggesting that the rise of [Ca2+]i is from intracellular stores. This conclusion is supported by the observation that depletion of [Ca2+]i stores by pretreatment with either caffeine or thapsigargin attenuates both the rise in [Ca2+]i and cell death induced by mastoparan. Phospholipase C (PLC) inhibitors, neomycin and U73122 block mastoparan-induced increases of [Ca2+]i and protect against neuronal death. Pretreatment with cholera toxin, but not pertussis toxin, reduced the mastoparan-induced rise in [Ca2+]i. Taken together, our data suggest that mastoparan initiates cell death in cerebellar granule neurons by inducing Ca2+ release from intracellular stores, probably via activation of PLC and IP3. A secondary or parallel process results in disruption of plasma membrane integrity and may be ultimately responsible for the death of these neurons by mastoparan.

AIM: To study the effect of caffeine on apoptosis induced by inhibition of I-phosphaddylinositol 3-kinase in cerebellur granule neurons. METHODS: Cerebellar granule neurons culture, agar gel eletrophoresis, and tess-activated protein kinase (SAPK)/c-Jun N-tennial, protein kimtse (JNK) assay kit to mesure SAPK/JNK activity. RESULTS: LY294002 evoked apoptosis concentmtion-dependemly in cerebellar granule neurons. But death resulting from LY2940002 was prevented by caffeine in a concentratlon-dependent manner. The survival effect of caffeine was not affected by inbitors of ryanodine-sensitive Ca2+ release, nor was it inhibited by L-type channel blockers and N-methyi-D-aspartate (NMDA) receptor blocker. In addition, RP-cAMP, H89, and KN62 were not able to inhibit the protective effect of caffeine. Phosphorylation of c-Jun was necessary for the induction of apoptosis induced by LY294002 in cerebellar granule neurons. But caffeine lirectly inhibited the activation of JNK and decreased phospho-c-Jun in granule neurons. CONCLUSION: Caffeine inhibited the activation of JNK and decremsed the phosphorylabon of c-Jun to protect granule neurons from LY294002-ieduced apoptosis.

AIM: To investigate possible intracellular signal molecules involved in diphenylhydantoin (DPH)-mediated apoptosis of cerebellar granule neurons (CGN) and explore possible molecular mechanisms of neurotoxicity of DPH. METHODS: Fluorescein diacetate (FDA) stain, hochest 33258 stain, and agar gel electrophoresis were used to test morphological and biological characters of primary CGN and cortical neurons (CN) in the presence or absence of 100 μmol/L DPH; Western blot and RT-PCR were employed to further investigate apoptotic/survival signal moleculars involved in the neuronal apoptotic signal transdution. RESULTS: DPH 100 μmol/L induced a typical apoptosis of CGN but had no toxicity on CN. Cerebellar granule neural apoptosis induced by 100 μmol/L DPH was significantly inhibited by pre-treatment with SB203580 (10 μmol/L) or CEP-11004 (1 μmol/L) for 1 h. DPH markedly upregulated the levels of phospho-c-Jun (active c-Jun), total c-Jun protein and c-jun mRNA in CGN. The levels of phosphoc-Jun dramatically elevated by DPH at 8 h were significantly inhibited by SB203580 (10 μmol/L) or CEP-11004 (1 μmol/L). Moreover, the activities of p44/42 (ERK1/ERK2), other members of MAP kinases and generally believed to be important survival effetors in CGN, were markedly suppressed. However, the activities of both JNK and p38 were little affected in the process of apoptosis of CGN induced by 100 μmol/L DPH. CONCLUSION: The selective toxicity of DPH on CGN is likely due to its ability to induce apoptosis of CGN, it is a process involved activation of c-Jun and suppression of the activity of p44/42.