f-Amyloid protein (A,B) has been implicated in the pathogenesis of Alzheimer disease (AD) by the facts that mutations in its precursor protein lead to the overproduction of A13 (1-5) and that A,B is directly toxic to some types of cells (6)(7)(8). In addition to AD there are numerous other human diseases that are characterized by the deposition of amyloid in various tissues (9). Although these amyloids contain different proteins, all are characterized by the antiparallel }3-sheet conformation of their major protein component (9). As in AD, the tissues surrounding these amyloid deposits frequently show a toxic response. Since the protein components share a secondary structure but have no obvious sequence homology, it is possible that they have a common mechanism for cellular toxicity that is nonspecificially linked to their unique state of peptide conformation or assembly. The alternative is that the responsive cells all have unique receptors for the respective peptides.The mechanism by which AP3 causes cell death is the best understood of the human amyloidoses. Although the molecular details are not completely known, A,3 causes increased H202 accumulation in cells, resulting in free radical-induced lipid peroxidation and, ultimately, cell death (10). The source of H202 is thought to be from the activation of an NADPHlinked oxidase similar to that found in neutrophils, for A,3 toxicity is blocked by relatively specific inhibitors of this enzyme (10). The following experiments address the issue of whether there is a common mechanism of toxicity that is shared by other peptides associated with human amyloidoses and examine how these mechanisms relate to their secondary and tertiary structure. MATERIALS AND METHODSCell Lines. B12 cells are from a collection of cell lines made from nitrosoethylurea-induced rat brain tumors (11). A suspension-growing variant of B12 was selected by growth in Petri dishes over a 6-month period and subsequent cloning. The attached and suspension cell lines have indistinguishable cytotoxic properties versus A,3 (10). Rat primary cultures were made from embryonic day 18 cerebral cortex and cultured on poly(L-lysine)-coated dishes in 50% Dulbecco's modified Eagle medium (DMEM)/50% Ham's F12 medium containing N2 supplements (12). No mitotic inhibitors were required in these minimal conditions, and >95% of the cells were neuronal as defined by staining with neuron-specific enolase and the absence of glial fibrillary acidic protein-positive cells. Dialyzed fetal calf and horse sera were from GIBCO/BRL. The peptides were obtained from Bachem except for the Leu-Lys peptides, which were synthesized according to Brack and Caille (13) and have an average molecular mass ranging from 5000 to 12,000 Da. All peptides-were initially dissolved in water and diluted into culture medium immediately before use. With the exception of poly(Leu), they were soluble in water but formed insoluble aggregates of various sizes in culture medium. The fluorescent dyes were from Molecular Probes, diphenylene iodonium...
It is frequently argued that both amyloid beta (Abeta) and oxidative stress are involved in the pathogenesis of Alzheimer's disease (AD). We show here that clonal nerve cell lines and primary cortical neurons that are resistant to Abeta toxicity have an enhanced flux of glucose through both the glycolytic pathway and the hexose monophosphate shunt. AD brain also has increased enzymatic activities in both pathways relative to age-matched controls. The Abeta-induced changes in glucose metabolism are due to the activation of the transcription factor hypoxia inducible factor 1 (HIF-1). As a result of Abeta-induced changes in glucose metabolism, Abeta-resistant cells are more readily killed by glucose starvation and by classes of antipsychotic drugs that inhibit glucose uptake.
Hydrogen sulfide (H2S) is a neuromodulator in the brain and a relaxant for smooth muscle. H2S protects primary cortical neurons from oxidative stress by increasing the intracellular concentrations of glutathione, the major antioxidant in cells. However, changes in glutathione alone are not sufficient to account for full protection in all types of nerve cells. H2S is here shown to protect an immortalized mouse hippocampal cell line from oxidative glutamate toxicity by activating ATP-dependent K+ (KATP) and Cl- channels, in addition to increasing the levels of glutathione. The present study therefore identifies a novel pathway for H2S protection from oxidative stress.
Huntington's disease (HD) is an inherited, progressive and ultimately fatal neurodegenerative disorder that is characterized by psychiatric, cognitive and motor symptoms. Among the pathways implicated in HD are those involving mitogen-activated protein kinase signaling and particularly the Ras-extracellular signal-regulated kinase (ERK) cascade. Studies in both cells and animal models suggest that ERK activation might provide a novel therapeutic target for the treatment of HD but compounds that specifically activate ERK are few. To test the hypothesis that pharmaceutical activation of ERK might be protective for HD, a polyphenol, fisetin, which was previously shown to activate the Ras-ERK cascade, was tested in three different models of HD: PC12 cells expressing mutant Httex1 under the control of an inducible promoter, Drosophila expressing mutant Httex1 and the R6/2 mouse model of HD. The results indicate that fisetin can reduce the impact of mutant huntingtin in each of these disease models. Prompted by this observation, we determined that the related polyphenol, resveratrol, also activates ERK and is protective in HD models. Notably, although more than a dozen small molecule inhibitors of ERK activation are in clinical trials, very few small molecule activators of ERK signaling are reported. Thus, fisetin, resveratrol and related compounds might be useful for the treatment of HD by virtue of their unique ability to activate ERK.
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