calpain-inhibitor-iii and Alzheimer-Disease

The key neuropathological features of Alzheimer's disease are abnormal deposition of Aβ plaques and insoluble Aβ peptides in extracellular brain and intracellular neurofibril tangles induced by abnormal tau hyperphosphorylation. μ-Calpain is one of the factors that bridge these Aβ- and hyperphosphorylated tau-mediated pathological pathways. Undecylenic acid (UDA), a naturally occurring unsaturated fatty acid, was discovered as a μ-calpain inhibitor by screening a chemical library using a substrate specific μ-calpain assay method. UDA inhibited Aβ oligomerization and Aβ fibrillation and reversed Aβ-induced neuronal cell death. In addition, UDA scavenged ROS and reversed the levels of proapoptotic proteins induced by ROS in SH-SY5Y cells. UDA inhibited μ-calpain activity with better potency than the known peptide-like μ-calpain inhibitor, MDL28170, in SH-SY5Y and HEK293T cells transfected with the catalytic subunit of μ-calpain. These results suggest that UDA is a novel non-peptide-like μ-calpain inhibitor with good cell permeability and potent neuroprotective effect.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Calpain; Cell Death; Cell Line, Tumor; Cell Survival; Data Interpretation, Statistical; Dipeptides; Drug Discovery; HEK293 Cells; Humans; Microscopy, Atomic Force; Neurons; Neuroprotective Agents; Permeability; Plaque, Amyloid; Reactive Oxygen Species; Ricinus; Small Molecule Libraries; Undecylenic Acids

Cdk5 dysregulation is a major event in the neurodegenerative process of Alzheimer's disease (AD). In vitro studies using differentiated neurons exposed to Abeta exhibit Cdk5-mediated tau hyperphosphorylation, cell cycle re-entry and neuronal loss. In this study we aimed to determine the role of Cdk5 in neuronal injury occurring in an AD mouse model obtained through the intracerebroventricular (icv) injection of the Abeta(1-40) synthetic peptide. In mice icv-injected with Abeta, Cdk5 activator p35 is cleaved by calpains, leading to p25 formation and Cdk5 overactivation. Subsequently, there was an increase in tau hyperphosphorylation, as well as decreased levels of synaptic markers. Cell cycle reactivation and a significant neuronal loss were also observed. These neurotoxic events in Abeta-injected mice were prevented by blocking calpain activation with MDL28170, which was administered intraperitoneally (ip). As MDL prevents p35 cleavage and subsequent Cdk5 overactivation, it is likely that this kinase is involved in tau hyperphosphorylation, cell cycle re-entry, synaptic loss and neuronal death triggered by Abeta. Altogether, these data demonstrate that Cdk5 plays a pivotal role in tau phosphorylation, cell cycle induction, synaptotoxicity, and apoptotic death in postmitotic neurons exposed to Abeta peptides in vivo, acting as a link between diverse neurotoxic pathways of AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Calpain; Cell Division; Cyclin-Dependent Kinase 5; Dipeptides; Disease Models, Animal; Female; G2 Phase; Injections, Intralesional; Injections, Intraperitoneal; Mice; Mice, Inbred C57BL; Nerve Degeneration; Peptide Fragments; Phosphorylation; Protease Inhibitors; tau Proteins

In this report, a novel positive-negative epitope tagging approach was developed to study the cellular processing of beta amyloid precursor protein (beta APP). Amino acids centered around the alpha-secretase cleavage site within the A beta sequence were replaced with residues comprising an epitope for which high-affinity monoclonal antibodies are commercially available. The resulting mutant beta APP cDNAs were expressed in human embryonic kidney cells (HEK 293). Cleavage of labeled beta APP by beta- and gamma-secretase(s) results in the release of an epitope-tagged A beta peptide, whereas cleavage by alpha-secretase results in destruction of the epitope. Highly sensitive and specific immunoassays were developed to study processing of this labeled beta APP via the amyloidogenic pathway. Secretion of epitope-tagged A beta was prevented by MDL 28170, a previously described gamma-secretase inhibitor. Confocal microscopic studies revealed that processing and cellular trafficking of epitope-tagged beta APP was not different from wild-type beta APP. These results suggest that positive-negative epitope-tagged beta APP is normally processed within the cell and may be used to identify secretase inhibitors as therapeutics for Alzheimer's disease.

Topics: Alzheimer Disease; Amino Acid Sequence; Amino Acid Substitution; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Antibodies, Monoclonal; Aspartic Acid Endopeptidases; Blotting, Western; Cell Line; Culture Media, Conditioned; Dipeptides; Endopeptidases; Enzyme-Linked Immunosorbent Assay; Epitopes; Humans; Immunohistochemistry; Isoenzymes; Protease Inhibitors; Protein Processing, Post-Translational; Recombinant Fusion Proteins; Sensitivity and Specificity; Transfection

Cerebral deposition of beta-amyloid protein is a pathological feature central to Alzheimer's disease. Production of beta-amyloid by proteolytic processing of the beta-amyloid precursor protein (beta APP) is a critical initial step in beta-amyloidogenesis. We use an inhibitor of beta APP processing to block beta-amyloid peptide formation. Application of the inhibitor to cultured cells results in an accumulation of proteolytic intermediates of beta APP, enabling a precursor-product relationship between beta APP carboxy-terminal fragments and beta-amyloid peptides to be demonstrated directly. In the presence of inhibitor, these amyloidogenic carboxy-terminal fragments can be degraded to nonamyloidogenic products. The catabolism of beta APP carboxy-terminal intermediates and the formation of beta-amyloid peptides are likely to involve an early endosomal compartment as the subcellular site of processing.

Topics: Alzheimer Disease; Amino Acid Sequence; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Brain; CHO Cells; Cricetinae; Dipeptides; Humans; Kinetics; Molecular Sequence Data; Peptide Fragments; Protease Inhibitors; Protein Processing, Post-Translational; Recombinant Proteins; Transfection