ABSTRACT
Pharmacological modulation of the GABA(A) receptor has gained increasing attention as a potential treatment for central processes affected in Alzheimer disease (AD), including neuronal survival and cognition. The proteolytic cleavage of the amyloid precursor protein (APP) through the alpha-secretase pathway decreases in AD, concurrent with cognitive impairment. This APP cleavage occurs within the beta-amyloid peptide (Abeta) sequence, precluding formation of amyloidogenic peptides and leading to the release of the soluble N-terminal APP fragment (sAPPalpha) which is neurotrophic and procognitive. In this study, we show that at nanomolar-low micromolar concentrations, etazolate, a selective GABA(A) receptor modulator, stimulates sAPPalpha production in rat cortical neurons and in guinea pig brains. Etazolate (20 nM-2 microM) dose-dependently protected rat cortical neurons against Abeta-induced toxicity. The neuroprotective effects of etazolate were fully blocked by GABA(A) receptor antagonists indicating that this neuroprotection was due to GABA(A) receptor signalling. Baclofen, a GABA(B) receptor agonist failed to inhibit the Abeta-induced neuronal death. Furthermore, both pharmacological alpha-secretase pathway inhibition and sAPPalpha immunoneutralization approaches prevented etazolate neuroprotection against Abeta, indicating that etazolate exerts its neuroprotective effect via sAPPalpha induction. Our findings therefore indicate a relationship between GABA(A) receptor signalling, the alpha-secretase pathway and neuroprotection, documenting a new therapeutic approach for AD treatment.
Subject(s)
Amyloid beta-Protein Precursor/metabolism , Cerebral Cortex/metabolism , Etazolate/pharmacology , Neurons/metabolism , Neuroprotective Agents/pharmacology , Receptors, GABA-A/metabolism , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Alzheimer Disease/physiopathology , Amyloid Precursor Protein Secretases/drug effects , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/antagonists & inhibitors , Amyloid beta-Peptides/biosynthesis , Amyloid beta-Peptides/toxicity , Animals , Cells, Cultured , Cerebral Cortex/drug effects , Dose-Response Relationship, Drug , GABA Agents/pharmacology , GABA Agonists/pharmacology , GABA Antagonists/pharmacology , Guinea Pigs , Male , Neurons/drug effects , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/biosynthesis , Peptide Fragments/toxicity , Phosphodiesterase Inhibitors/pharmacology , Protein Structure, Tertiary/physiology , Rats , Rats, Wistar , Receptors, GABA-A/drug effects , Signal Transduction/drug effects , Signal Transduction/physiologyABSTRACT
beta-Amyloid peptides (Abeta) that form the senile plaques of Alzheimer disease consist mainly of 40- and 42-amino acid (Abeta 40 and Abeta 42) peptides generated from the cleavage of the amyloid precursor protein (APP). Generation of Abeta involves beta-secretase and gamma-secretase activities and is regulated by membrane trafficking of the proteins involved in Abeta production. Here we describe a new small molecule, EHT 1864, which blocks the Rac1 signaling pathways. In vitro, EHT 1864 blocks Abeta 40 and Abeta 42 production but does not impact sAPPalpha levels and does not inhibit beta-secretase. Rather, EHT 1864 modulates APP processing at the level of gamma-secretase to prevent Abeta 40 and Abeta 42 generation. This effect does not result from a direct inhibition of the gamma-secretase activity and is specific for APP cleavage, since EHT 1864 does not affect Notch cleavage. In vivo, EHT 1864 significantly reduces Abeta 40 and Abeta 42 levels in guinea pig brains at a threshold that is compatible with delaying plaque accumulation and/or clearing the existing plaque in brain. EHT 1864 is the first derivative of a new chemical series that consists of candidates for inhibiting Abeta formation in the brain of AD patients. Our findings represent the first pharmacological validation of Rac1 signaling as a target for developing novel therapies for Alzheimer disease.
Subject(s)
Amyloid beta-Peptides/metabolism , Endopeptidases/metabolism , Enzyme Inhibitors/pharmacology , Protein Processing, Post-Translational/drug effects , Pyrones/pharmacology , Quinolines/pharmacology , rac1 GTP-Binding Protein/antagonists & inhibitors , Aminoquinolines/pharmacology , Amyloid Precursor Protein Secretases , Amyloid beta-Peptides/biosynthesis , Animals , Aspartic Acid Endopeptidases , Cell Line , Cyclin D1/metabolism , Dose-Response Relationship, Drug , Guinea Pigs , Humans , Male , Molecular Structure , NF-kappa B/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Pyrimidines/pharmacology , Pyrones/chemistry , Quinazolines/chemistry , Quinazolines/pharmacology , Quinolines/chemistry , rac1 GTP-Binding Protein/metabolismABSTRACT
BACKGROUND: We have previously reported activating mutations of the gene coding for the fibroblast growth factor receptor 3 (FGFR3) in invasive cervical carcinoma. To further analyze the role of FGFR3 in cervical tumor progression, we extended our study to screen a total of 75 invasive tumors and 80 cervical intraepithelial neoplasias (40 low-grade and 40 high-grade lesions). RESULTS: Using single strand conformation polymorphism (SSCP) followed by DNA sequencing, we found FGFR3 mutation (S249C in all cases) in 5% of invasive cervical carcinomas and no mutation in intraepithelial lesions. These results suggest that, unlike in bladder carcinoma, FGFR3 mutation does not or rarely occur in non invasive lesions. Compared to patients with wildtype FGFR3 tumor, patients with S249C FGFR3 mutated tumors were older (mean age 64 vs. 49.4 years, P = 0.02), and were more likely to be associated with a non-16/18 HPV type in their tumor. Gene expression analysis demonstrated that FGFR3 mutated tumors were associated with higher FGFR3b mRNA expression levels compared to wildtype FGFR3 tumors. Supervised analysis of Affymetrix expression data identified a significant number of genes specifically differentially expressed in tumors with respect to FGFR3 mutation status. CONCLUSION: This study suggest that tumors with FGFR3 mutation appear to have distinctive clinical and biological characteristics that may help in defining a population of patients for FGFR3 mutation screening.
