Michael K. Ahlijanian

Cdk5 Is a Key Factor in Tau Aggregation and Tangle Formation In Vivo

Tau aggregation is a common feature of neurodegenerative diseases such as Alzheimers disease, and hyperphosphorylation of tau has been implicated as a fundamental pathogenic mechanism in this process. To examine the impact of cdk5 in tau aggregation and tangle formation, we crossed transgenic mice overexpressing the cdk5 activator p25, with transgenic mice overexpressing mutant (P301L) human tau. Tau was hyperphosphorylated at several sites in the double transgenics, and there was a highly significant accumulation of aggregated tau in brainstem and cortex. This was accompanied by increased numbers of silver-stained neurofibrillary tangles (NFTs). Insoluble tau was also associated with active GSK. Thus, cdk5 can initiate a major impact on tau pathology progression that probably involves several kinases. Kinase inhibitors may thus be beneficial therapeutically.

APP processing is regulated by cytoplasmic phosphorylation.

Amyloid-β peptide (Aβ) aggregate in senile plaque is a key characteristic of Alzheimers disease (AD). Here, we show that phosphorylation of amyloid precursor protein (APP) on threonine 668 (P-APP) may play a role in APP metabolism. In AD brains, P-APP accumulates in large vesicular structures in afflicted hippocampal pyramidal neurons that costain with antibodies against endosome markers and the β-secretase, BACE1. Western blot analysis reveals increased levels of T668-phosphorylated APP COOH-terminal fragments in hippocampal lysates from many AD but not control subjects. Importantly, P-APP cofractionates with endosome markers and BACE1 in an iodixanol gradient and displays extensive colocalization with BACE1 in rat primary cortical neurons. Furthermore, APP COOH-terminal fragments generated by BACE1 are preferentially phosphorylated on T668 verses those produced by α-secretase. The production of Aβ is significantly reduced when phosphorylation of T668 is either abolished by mutation or inhibited by T668 kinase inhibitors. Together, these results suggest that T668 phosphorylation may facilitate the BACE1 cleavage of APP to increase Aβ generation.

Role of cdk5 in the Pathogenesis of Alzheimer’s Disease

Alzheimer’s disease (AD) is characterized by two pathological hallmarks, namely, senile plaques and neurofibrillary tangles (NFTs). The former are mainly composed of amyloid-β peptides (Aβ) while the latter consists mainly of filaments of hyperphosphorylated tau. Cyclin-dependent kinase 5 (cdk5) has been implicated not only in the tangle pathology, but recent data also implicate cdk5 in the generation of Aβ peptides. Since both Aβ peptides and NFTs are believed to play a role in neurodegeneration in AD, this proline-directed serine/threonine protein kinase is likely to contribute to the pathogenesis of AD. In vitro and in vivo animal data demonstrate the ability of cdk5 to induce phosphorylation and aggregation of tau, and NFT deposition and neurodegeneration. Findings from AD brain samples also show an elevated cdk5 activity and conditions that support the activation of cdk5. Evidence for the role of cdk5 in regulating Aβ production is just emerging. The mechanisms for this potentially damaging activity of cdk5 are largely unknown although amyloid precursor protein and presenilin-1 are both cdk5 substrates.

Partial Rescue of the p35−/− Brain Phenotype by Low Expression of a Neuronal-Specific Enolase p25 Transgene

Cyclin-dependent kinase 5 (Cdk5) is activated on binding of activator proteins p35 and p39. A N-terminally truncated p35, termed p25, is generated through cleavage by the Ca2+-dependent protease calpain after induction of ischemia in rat brain. p25 has been shown to accumulate in brains of patients with Alzheimers disease and may contribute to A-β peptide-mediated toxicity. Studies from transfected neurons as well as p35 and p25 transgenic mice have indicated that Cdk5, when activated by p25, gains some toxic function compared with p35/Cdk5. It remains unclear, however, whether p25/Cdk5 signaling additionally channels into pathways usually used by p35/Cdk5 and whether p25 is associated with a loss of p35 function. To clarify these issues, we have generated p25-transgenic mice in a p35-null background. We find that low levels of p25 during development induce a partial rescue of the p35−/− phenotype in several brain regions analyzed, including a rescue of cell positioning of a subset of neurons in the neocortex. In accordance with the partial rescue of brain anatomy, phosphorylation of the Cdk5 substrate mouse disabled 1 is partially restored during development. Besides this, p25/Cdk5 fails to phosphorylate other substrates that are normally phosphorylated by p35/Cdk5. Our results show that p25 can substitute for p35/Cdk5 under certain circumstances during development. In addition, they suggest that p25 may have lost some functions of p35.

Potent and cellularly active 4-aminoimidazole inhibitors of cyclin-dependent kinase 5/p25 for the treatment of Alzheimer's disease.

Utilizing structure-based drug design, a 4-aminoimidazole heterocyclic core was synthesized as a replacement for a 2-aminothiazole due to potential metabolically mediated toxicity. The synthetic route utilized allowed for ready synthesis of 1-substituted-4-aminoimidazoles. SAR exploration resulted in the identification of a novel cis-substituted cyclobutyl group that gave improved enzyme and cellular potency against cdk5/p25 with up to 30-fold selectivity over cdk2/cyclin E.

Pharmacokinetic and pharmacodynamic analysis of the gamma-secretase modulator (GSM) EVP-0015962

Background: CAD106 is an active immunotherapy developed for Alzheimer’s disease. It comprises amino acids 1-6 of Ab coupled to a virus-like particle derived from E. coli bacteriophage Qb. In APP overexpressing mice, administration of CAD106 induced Ab antibodies as well as a reduction of amyloid accumulation in the brain. Infusion of antibodies or active immunization against Ab have been shown to increase the level of total Ab in plasma indicating target engagement by the antibodies. In the present study we have investigated the active Ab immunotherapy CAD106 for its effect on plasma Ab. Methods: Cynomolgus monkeys aged 3 to 6 years were administered 600 mg CAD106 per subcutaneous or intramuscular injection at days 1, 14, 28 and every 4 weeks thereafter for a total of 24 weeks. Each dose contained either Alum or MF58 as adjuvant. Controls received adjuvant only. Total plasma Ab was determined at baseline and 12 to 14 days after the injections at week 8 and 24 using two different ELISAs and Western blotting. To minimize an interference of the CAD106-induced antibodies samples were SDSdenatured prior to quantification with an ELISA using a N-terminal capturing antibody. Alternatively, an assay combining C-terminal capture and mid-region detection antibodies was used. In addition, results were verified by direct SDS-PAGE separation of plasma followed by Western blotting. Results: Following active immunotherapy with CAD106, total plasma Ab levels increased compared to baseline and further between the intermediate and final time points of the study. Individual animals showed increases of up to about 50-fold over baseline. Median group increases reached up to 25-fold. Very similar results were obtained with the different assays. Overall the Ab levels in plasma correlated well with the Ab antibody titers. Conclusions: The increase in plasma Ab indicates an interaction of CAD106 induced antibodies with Ab in vivo. Its extent is in line with observations made with Nterminal monoclonal antibodies.