phosphothreonine and Nerve-Degeneration

Cdk5 deregulation is highly neurotoxic in Alzheimer's disease (AD). We identified Mcl-1 as a direct Cdk5 substrate using an innovative chemical screen in mouse brain lysates. Our data demonstrate that Mcl-1 levels determine the threshold for cellular damage in response to neurotoxic insults. Mcl-1 is a disease-specific target of Cdk5, which associates with Cdk5 under basal conditions, but is not regulated by it. Neurotoxic insults hyperactivate Cdk5 causing Mcl-1 phosphorylation at T92. This phosphorylation event triggers Mcl-1 ubiquitylation, which directly correlates with mitochondrial dysfunction. Consequently, ectopic expression of phosphorylation-dead T92A-Mcl-1 fully prevents mitochondrial damage and subsequent cell death triggered by neurotoxic treatments in neuronal cells and primary cortical neurons. Notably, enhancing Mcl-1 levels offers comparable neuroprotection to that observed upon Cdk5 depletion, suggesting that Mcl-1 degradation by direct phosphorylation is a key mechanism by which Cdk5 promotes neurotoxicity in AD. The clinical significance of the Mcl-1-Cdk5 axis was investigated in human AD clinical specimens, revealing an inverse correlation between Mcl-1 levels and disease severity. These results emphasize the potential of Mcl-1 upregulation as an attractive therapeutic strategy for delaying or preventing neurodegeneration in AD.

Topics: Alzheimer Disease; Animals; Cell Nucleus; Cyclin-Dependent Kinase 5; Glutamic Acid; HEK293 Cells; Humans; Mice; Mitochondria; Models, Biological; Myeloid Cell Leukemia Sequence 1 Protein; Nerve Degeneration; Neurons; Neurotoxins; Phosphorylation; Phosphothreonine; Protein Transport; Proteolysis; Severity of Illness Index; Signal Transduction; Subcellular Fractions; Substrate Specificity; Ubiquitination

A number of recent findings have demonstrated re-expression of cell cycle-related proteins in vulnerable neurones in Alzheimer's disease. We hypothesize that this attempt by neurones to re-enter mitosis is a response to external growth stimuli that leads to an abortive re-entry into the cell cycle and, ultimately, neuronal degeneration. In this study, to further delineate the role of mitotic processes in the pathogenesis of Alzheimer's disease, we investigated p27, a cyclin-dependent kinase inhibitor that plays a negatively regulatory role in cell cycle progression that, once phosphorylated at Thr187, is degraded via an ubiquitin-proteasome pathway. Here we report that both p27 and phosphorylated p27 (Thr187) show increases in the cytoplasm of vulnerable neuronal populations in Alzheimer's disease vs. age-matched control subjects. Importantly, phosphorylated p27 (Thr187) shows considerable overlap with tau-positive neurofibrillary pathology, including neurofibrillary tangles, dystrophic neurites and neuropil threads. The findings presented here suggest that dysregulation of the cell cycle plays a crucial role in the pathogenesis of Alzheimer's disease that may provide a novel mechanistic basis for therapeutic intervention.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Cell Cycle; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p27; Cytoplasm; Female; Hippocampus; Humans; Male; Mitosis; Nerve Degeneration; Nerve Tissue Proteins; Neurites; Neurofibrillary Tangles; Neuropil; Phosphorylation; Phosphothreonine; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein Transport; Pyramidal Cells; tau Proteins; Temporal Lobe; Tumor Suppressor Proteins

Microtubule-associated protein tau contains a consensus motif for protein kinase B/Akt (Akt), which plays an essential role in anti-apoptotic signaling. The motif encompasses the AT100 double phospho-epitope (Thr212/Ser214), a specific marker for Alzheimer's disease (AD) and other neurodegenerations, raising the possibility that it could be generated by Akt. We studied Akt-dependent phosphorylation of tau protein in vitro. We found that Akt phosphorylated both Thr212 and Ser214 in the longest and shortest tau isoforms as determined using phospho site-specific antibodies against tau. Akt did not phosphorylate other tau epitopes, including Tau-1, AT8, AT180, 12E8 and PHF-1. The Akt-phosphorylated tau retained its initial electrophoretic mobility. Immunoprecipitation studies with phospho-specific Thr212 and Ser214 antibodies revealed that only one of the two sites is phosphorylated per single tau molecule, resulting in tau immunonegative for AT100. Mixed kinase studies showed that prior Ser214 phosphorylation by Akt blocked protein kinase A but not GSK3beta activity. On the other hand, GSK3beta selectively blocked Ser214 phosphorylation, which was prevented by lithium. The results suggest that Akt may be involved in AD-specific phosphorylation of tau at the AT100 epitope in conjunction with other kinases. Our data suggest that phosphorylation of tau by Akt may play specific role(s) in Akt-mediated anti-apoptotic signaling, particularly relevant to AD and other neurodegenerations.

Topics: Alzheimer Disease; Amino Acid Sequence; Biomarkers; Cloning, Molecular; Consensus Sequence; Escherichia coli; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Kinetics; Nerve Degeneration; Phosphorylation; Phosphoserine; Phosphothreonine; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Recombinant Proteins; Substrate Specificity; tau Proteins