A translational continuum of model systems for evaluating treatment strategies in Alzheimer's disease: isradipine as a candidate drug.

A growing body of evidence supports the 'calcium hypothesis' of Alzheimer's disease (AD), which postulates that a variety of insults might disrupt the homeostatic regulation of neuronal calcium (Ca(2+)) in the brain, resulting in the progressive symptoms that typify the disease. However, despite ongoing efforts to develop new methods for testing therapeutic compounds that might be beneficial in AD, no single bioassay permits both rapid screening and in vivo validation of candidate drugs that target specific components of the Ca(2+) regulatory machinery. To address this issue, we have integrated four distinct model systems that provide complementary information about a trial compound: the human neuroblastoma MC65 line, which provides an in vitro model of amyloid toxicity; a transgenic Drosophila model, which develops age-dependent pathologies associated with AD; the 3×TgAD transgenic mouse, which recapitulates many of the neuropathological features that typify AD; and the embryonic nervous system of Manduca, which provides a novel in vivo assay for the acute effects of amyloid peptides on neuronal motility. To demonstrate the value of this 'translational suite' of bioassays, we focused on a set of clinically approved dihydropyridines (DHPs), a class of well-defined inhibitors of L-type calcium channels that have been suggested to be neuroprotective in AD. Among the DHPs tested in this study, we found that isradipine reduced the neurotoxic consequences of ß-amyloid accumulation in all four model systems without inducing deleterious side effects. Our results provide new evidence in support of the Ca(2+) hypothesis of AD, and indicate that isradipine represents a promising drug for translation into clinical trials. In addition, these studies also demonstrate that this continuum of bioassays (representing different levels of complexity) provides an effective means of evaluating other candidate compounds that target specific components of the Ca(2+) regulatory machinery and that therefore might be beneficial in the treatment of AD.

Bone marrow lacking integrin expression facilitates an enhanced susceptibility to EAE in the xenogeneic bone marrow chimeras.

Xenotransplantation of rat bone marrow cells (BMC) into immunodeficient (SCID) mice generates chimeric mice susceptible to paralytic autoimmune CNS inflammation. Herein, we identified a disease relevant subset of transplantable BMC lacking expression of CD11b/c and CD49d. Moreover, disease susceptibility was enhanced in the presence of non-myelin specific T-cells. Only the CD11b/c negative population of BM retained the capability to populate the blood, spleen and spinal cord of recipients and matured after transplant to express CD11b/c. These results indicate non-myelin T cells in combination with integrin negative BM represent pre-pathogenic determinants of an enhanced disease susceptibility to myelin reactive T cells.

Prevention of peroxynitrite-induced apoptosis of motor neurons and PC12 cells by tyrosine-containing peptides.

Although peroxynitrite stimulates apoptosis in many cell types, whether peroxynitrite acts directly as an oxidant or the induction of apoptosis is because of the radicals derived from peroxynitrite decomposition remains unknown. Before undergoing apoptosis because of trophic factor deprivation, primary motor neuron cultures become immunoreactive for nitrotyrosine. We show here using tyrosine-containing peptides that free radical processes mediated by peroxynitrite decomposition products were required for triggering apoptosis in primary motor neurons and in PC12 cells cultures. The same concentrations of tyrosine-containing peptides required to prevent the nitration and apoptosis of motor neurons induced by trophic factor deprivation and of PC12 cells induced by peroxynitrite also prevented peroxynitrite-mediated nitration of motor neurons, brain homogenates, and PC12 cells. The heat shock protein 90 chaperone was nitrated in both trophic factor-deprived motor neurons and PC12 cells incubated with peroxynitrite. Tyrosine-containing peptides did not affect the induction of PC12 cell death by hydrogen peroxide. Tyrosine-containing peptides should protect by scavenging peroxynitrite-derived radicals and not by direct reactions with peroxynitrite as they neither increase the rate of peroxynitrite decomposition nor decrease the bimolecular peroxynitrite-mediated oxidation of thiols. These results reveal an important role for free radical-mediated nitration of tyrosine residues, in apoptosis induced by endogenously produced and exogenously added peroxynitrite; moreover, tyrosine-containing peptides may offer a novel strategy to neutralize the toxic effects of peroxynitrite.

Peptide YY reverses TNF-alpha induced transcription factor binding of interferon regulatory factor-1 and p53 in pancreatic acinar cells.

BACKGROUND: Cytokine activation in the pancreatitis induces local and systemic cellular damage. Transcription factors interferon regulatory factor-1 (IRF-1) and the tumor suppressor gene p53 collaborate to enhance p21 related cell cycle regulation during pathological disease progression. However, little is known about their role in the pancreas after cytokine challenge. Our laboratory has previously shown that TNF-alpha induces the binding of many transcription factors, including NF-kappa B, and treatment with the gut hormone, Peptide YY (PYY), ameliorates the effects. We hypothesized that TNF-alpha would induce IRF-1 and p53 protein binding in pancreatic acinar cells and that PYY would attenuate the effect. MATERIALS AND METHODS: Rat pancreatic acinar AR42J cells were treated with rat recombinant TNF-alpha (200 ng/ml). To verify that our model was inducing pancreatitis, alpha-amylase activity was measured in the cell culture supernatant by fluorescence spectroscopy. PYY [3-36] was added at 500 pM 30 min post-TNF treatment; cells were harvested at 2 h for extraction of nuclear protein. Transcription factor binding of IRF-1 and p53 were determined by protein/DNA array analysis using chemiluminescence detection, and relative spot densities were measured by densitometry. A two-fold increase or decrease in density was considered significant. RESULTS: Amylase enzyme activity was significantly (P < 0.05) elevated in the TNF-alpha-treated cells by 2 h. Protein/DNA array analysis revealed significant up-regulation of both IRF-1 and p53 protein in nuclear extracts. Induction by TNF-alpha increased IRF-1 protein binding 3.5-fold, while binding levels of p53 protein increased six-fold. The addition of PYY to TNF-treated cells reduced IRF-1 and p53 binding to control levels. CONCLUSIONS: We have shown for the first time that short-term exposure to TNF-alpha induces the binding activity of transcription factors IRF-1 and p53 in rat pancreatic acinar cells, and that addition of PYY reduces it. Regulation of transcription factor activity by PYY may have therapeutic potential in altering the progression of pancreatitis.

Peptide YY attenuates STAT1 and STAT3 activation induced by TNF-alpha in acinar cell line AR42J.

BACKGROUND: STAT1 and STAT3, members of the cytoplasmic family of signal transducers and activators of transcription factors (STAT), have been associated with numerous inflammatory pathologies, including inflammatory bowel disease, hepatitis, and acute lung injury. But little is known about their role in the pancreas. Peptide YY (PYY), an inhibitory gastrointestinal hormone, ameliorates pancreatitis in vivo and in vitro. In addition, we have shown that PYY attenuates transcription factors, such as nuclear transcription factor (NF)-kappaB and Smad3/4, which mediate inflammation. We hypothesized that tumor necrosis factor (TNF)-alpha would induce STAT1 and STAT3, and PYY would attenuate their transcription factor binding. STUDY DESIGN: Rat pancreatic acinar cells were treated with recombinant TNF-alpha (200 ng/mL); PYY (3-36; 500 pM) was added 30 minutes post-TNF-alpha treatment. Cells were harvested at 2 hours, and nuclear protein and conditioned media were extracted. Levels of amylase secretion and cytokine production were measured using commercially available kits. STAT transcription factor binding was determined by protein/DNA array analysis and densitometry; results were verified again by electrophoretic mobility shift assay (EMSA) and ELISA-based assay. RESULTS: Amylase production was considerably increased (p < 0.05) as early as 5 minutes after addition of exogenous TNF-alpha and remained elevated for 24 hours. PYY decreased amylase production to control levels. A notable increase (p < 0.05) in the production of cytokines interleukin (IL)-1beta, IL-4, IL-6, IL-10, and TNF-alpha was observed with TNF-alpha treatment; production was reduced with PYY. TNF-alpha substantially upregulated STAT1 and STAT3 (two-fold or greater); PYY downregulated their binding activity to control levels. Results from both the electrophoretic mobility shift assay- and the ELISA-based assays verified STAT1 and STAT3 responses to TNF-alpha and PYY. CONCLUSIONS: In pancreatic acinar cells, TNF-alpha activated STAT1 and STAT3, known mediators of inflammatory cytokines. Interestingly, PYY attenuated their protein/DNA binding, which may have an impact on development of the disease. Additional investigation of STAT proteins and PYY could provide new therapeutic strategies for pancreatitis.