31 March 2005. Exactly what makes p25/Cdk5 so deadly to neurons? A good place to start tracing the path of this rogue kinase would be retinoblastoma (Rb), a tumor suppressor protein positioned strategically at the crossroads between cell proliferation and death. This is the upshot of a poster presented at the 7th International AD/PD Conference earlier this month in Sorrento, Italy. Luc Buée and colleagues at INSERM in Lille, France, with collaborators at Aventis Pharma, reported that inducing p25 in neurons prompted an inactivating phosphorylation of the Rb protein, which then allowed the transcription of genes pushing otherwise differentiated cells to start dividing.
During the past decade, researchers in a growing number of labs, most prominently Inez Vincent’s, have implicated an aberrant reactivation of the cell cycle in the pathogenesis of Alzheimer disease, claiming that this process contributes early on to the neuronal loss seen in AD (see, for example, ARF related news story and ARF news story). One of the many questions surrounding this hypothesis is what happens upstream, i.e., what forces quiescent neurons to reenter the cell cycle?
Independently, other laboratories including Li-Huei Tsai’s have accused a pathogenic cleavage product of the physiological Cdk5 regulator p35 of playing a role in AD, ALS, and stroke. These investigators believe that noxious stimuli, ranging from Aβ and elevated calcium to H2O2 or glutamate, can activate the protease calpain, which then produces p25. Whereas scientists consider p35/Cdk5 a tightly controlled, physiological, membrane-tethered kinase, they view p25/Cdk5 as a hyperactive maniac engaged in a phosphorylating spree throughout the cytoplasm and the nucleus (see ARF related news story and ARF news story).
It’s established that p25/Cdk5 can trigger neurodegeneration, but how it does so is less clear. Scientists have focused on two possible explanations: phosphorylation and dysregulation of the microbutule binding protein tau, or something untoward happening with the cell cycle. Intrigued by a Live Discussion on the Alzheimer Research Forum, Buée, himself long a student of tau, decided to pursue the question of the cell cycle.
His group first established a stable neuronal cell line that expresses an inducible p25/Cdk5 activity (Hamdane et al., 2003). In Sorrento, he presented evidence that, as early as six hours after p25/Cdk5 induction, this kinase phosphorylates Rb. P-Rb then activates the transcription factor E2F and its downstream genes, some of which are cell cycle genes documented to be aberrantly active in AD. The kinases that normally phosphorylate Rb were not active in this system. Roscovitine, an inhibitor of Cdk5 but not most other cyclin-dependent kinases, abolished the p25/Cdk5-induced change in Rb. Finally, p25/Cdk5 isolated from the cells directly phosphorylated Rb in vitro, the researchers report.
The work suggests that Rb phosphorylation is an early event in p25/Cdk5-induced neurodegeneration, Buée said (see Hamdane et al., 2005). More broadly, this would imply that Cdk5 deregulation sets off an inappropriate phosphorylation of several different substrates, including Rb, placing Cdk5 upstream of cell cycle changes in the pathogenesis of AD.
The French scientists performed their latest work in differentiated neuroblastoma cells. Usually, inducing p25 in such cells causes apoptosis. To circumvent this problem, first author Malika Hamdane simultaneously overexpressed tau, deploying it as a phosphorylation reservoir to absorb some of the rampant p25 activity. This allowed the cells to survive long enough for Hamdane to detect the Rb phosphorylation and subsequent activation of cell cycle genes.
Buée readily acknowledges that pathway hypotheses generated from experiments with cultured cell lines and overexpressed genes all share the caveat that the cells used may well activate different signal transduction pathways than do aging human neurons. Therefore, Buée is planning to check if his lab’s finding holds up in vivo, for example, in p25-inducible mouse strains.—Gabrielle Strobel.