Orlando: It’s Getting Hot around CDK5, Has the Field Noticed Yet?
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In a sparsely attended slide presentation yesterday at the Neuroscience meeting, Inez Vincent of the University of Washington, Seattle, presented a feat no one seems to have pulled off before: With an experimental small-molecule compound that inhibits the kinase cdk5, she almost completely reversed neurodegeneration in a mouse model that may be obscure, but whose neurofibrillary pathology is very similar to that seen in human AD.
Vincent studied a natural mouse model of Niemann-Pick Type C (NPC). This human disease was so named because of its lysosomal storage defect, which causes ballooning neurons filled with lipid, spheroid structures in axons, and neuronal loss. Trying to understand how this pathology comes about, Vincent studied phosphorylation of cytoskeletal proteins from human disease and the mouse model. She found that tau was hyperphosphorylated in both human and mouse, and correlated with increased cdk5 activity and conversion of p35 to p25. This started at one month of age in mice. P25 and cdk5 accumulated together with hyperphosphorylated cytoskeletal proteins in axon spheroids.
The kicker, however, lies in the treatment experiment. Vincent treated four- to six-week-old mice with the cdk5/p25 inhibitor roscovitine, originally developed by a French group. The mice received intraventricular infusion driven by an osmotic pump for two weeks. Not only did the treated mice lose less weight and improve their locomotor scores compared to controls, but their protein phosphorylation decreased by about 90 percent in immunoblot, ELISA, and immunohistochemistry assays. The axonal spheroids partly resolved and neuronal loss decreased markedly. The authors conclude that cdk5/p25 mediates Niemann-Pick C neuropathology, and that inhibitors of this enzyme may treat this rare disease. The obvious implication is whether this cdk5/p25 mechanism is at work in AD, which also features tau hyperphosphorylation and neuronal degeneration. This apparently has not been tested. The drug could be tried in tau transgenic models, as well as in combined tau-APP transgenic models, which feature a more severe, combined phenotype.
Proteins Behaving Badly: P25/CDK5 Consorts with Pathologic Substrates.
Today, J.C. Cruz in Li-Huei Tsai’s lab presented an extension of Tsai’s presentation on Sunday (see related news story), in which she had suggested that hyperphosphorylation of APP’s cytoplasmic domain by p25/cdk5 influences the sorting of APP away from α-cleavage in the cell membrane and instead toward BACE cleavage in endosomes (see related news story). To examine this question in vivo, Crux et al. generated inducible transgenic mice that express p25-GFP under the control of the CamKinase2 promoter, so that the transgene is expressed only in the forebrain. With a panel of different phospho-specific antibodies, Crux found that with the induced p25, cdk5 no longer phosphorylated its normal targets, for example, the postsynaptic protein PSD-95. Instead, cdk5 now hyperphosphorylated APP, tau, and neurofilament. The authors suggest that the two regulatory proteins-p35 and p25-direct cdk5 to different sets of targets, one normal, one pathological.
The idea connecting all these dots, speculates Tsai, would be that p25/cdk5 phosphorylates tau, leading to axonal transport blockages and later to tangle deposition, and also phosphorylates APP, leading to Aβ generation in endosomes. How is that evil p25 made? The enzyme calpain, which has been implicated in neuronal death, cleaves p35 to generate p25 (see related news story). So what activates calpain? This is where the argument becomes circular, as Aβ 42 has been shown to activate. However, other toxic conditions do, as well, for example, oxidative stress and increased intracellular calcium. This ion has long been a suspect in AD, and recent studies indicate that presenilin-1 mutations, besides revving up Aβ generation, also disrupt the restorative flows of calcium between the inside and the outside of the cell in such a way that intracellular calcium rises to dangerous levels. Perhaps one way in which PS1 mutations might induce tau pathology is via this connection?
Does this simplified speculation hold water in humans? The p25-transgenic mice do exhibit neurodegeneration, and yet, does the overexpression of p25 make this an artificial, irrelevant finding? Tsai says that a mouse overexpressing an endogenous mouse protein may not be more artificial than a mouse overexpressing a human protein. Her original finding that p25 protein levels were elevated in the brains of AD patients (see news) did not address whether that varied between regions. Today, B.A. Samuels, and others in Tsai’s group report a follow-up study on another series of human autopsy samples from multiple brain regions. Working with Yong Shen of the Sun Health Research Institute in Sun City, Arizona, Samuels found that frontal cortex shows the strongest overproduction of p25 relative to p35.-Gabrielle Strobel.
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New York University School of Medicine/Nathan Kline Institute
The pathological cascade proposed in this news story—initiated by calpain activation and p35 cleavage to p25—is provocative, but should be viewed in the context of other calpain findings. The hypothesis that the inevitable outcome of p25-mediated phosphorylation of APP is increased Aβ generation in endosomes needs to be reconciled with the recent observations of Mathews at el.: that calpain inhibition, not activation, increases endosomal β-cleavage of APP and Aβ42 generation (Matthews et al., 2002). The phosphorylation of APP may well turn out to influence its trafficking and processing, but the calpain-p25 pathway is just one of several routes by which calpains might influence APP and cytoskeleton phosphorylation or processing and mediate cell death.
For example, data from our lab and others also implicate the calpain-PKC pathway in AD (Nixon 2000; Nixon and Mohan, 1999). Calpains are well-known to activate protein kinase C and, when persistently activated, they lower PKC levels. Lowered PKC activity, which is observed in the brains of Alzheimer’s patients, is associated with increased Aβ and reduced APPs production in neurons exposed to PKC inhibitors. That PKC also modulates endocytosis suggests a potential link to the endosomal abnormalities that develop very early in AD. Moreover, PKC activation promotes tau phosphorylation in part through mitogen-activated protein kinase (MAPK). Because both cdk 5 and jnk 3 phosphorylate T668 of APP (Iijima et al., 2000; Standen et al., 2001), it would not be surprising if APP is a potential target of other proline-directed kinases, including MAPK, under pathological conditions.
Calpain's known activity toward a broad array of substrates, including other kinases and phosphatases, may influence APP metabolism in still other ways. These pervasive effects explain why calpain activation can mediate neurodegeneration either directly (eg., Wallerian degeneration) or through several pathways leading to necrosis or apoptosis (Nixon 2000; Nixon and Mohan, 1999). In sum, the data emerging since we first showed a decade ago that brain calpains are activated in AD potentially link disparate aspects of Alzheimer’s pathobiology to this protease but do not support at present a mechanism of amyloidogenic APP processing based solely on calpain activation. These observations do not make the Tsai group’s findings any less intriguing, but they do illustrate that calpain's involvement in AD is multifaceted and unlikely to be explained by modulation through a single protein kinase pathway.
See also:
Nixon RA, Mohan PS. "Calpains in the pathogenesis of Alzheimer’s disease" In: Calpain: Pharmacology and Toxicology of Calcium-Dependent Protease. Wang K.K. ed. Taylor & Francis, Ann Arbor 1999, 267-291.
References:
Mathews PM, Jiang Y, Schmidt SD, Grbovic OM, Mercken M, Nixon RA. Calpain activity regulates the cell surface distribution of amyloid precursor protein. Inhibition of calpains enhances endosomal generation of beta-cleaved C-terminal APP fragments. J Biol Chem. 2002 Sep 27;277(39):36415-24. PubMed.
Nixon RA. A "protease activation cascade" in the pathogenesis of Alzheimer's disease. Ann N Y Acad Sci. 2000;924:117-31. PubMed.
Iijima K, Ando K, Takeda S, Satoh Y, Seki T, Itohara S, Greengard P, Kirino Y, Nairn AC, Suzuki T. Neuron-specific phosphorylation of Alzheimer's beta-amyloid precursor protein by cyclin-dependent kinase 5. J Neurochem. 2000 Sep;75(3):1085-91. PubMed.
Standen CL, Brownlees J, Grierson AJ, Kesavapany S, Lau KF, McLoughlin DM, Miller CC. Phosphorylation of thr(668) in the cytoplasmic domain of the Alzheimer's disease amyloid precursor protein by stress-activated protein kinase 1b (Jun N-terminal kinase-3). J Neurochem. 2001 Jan;76(1):316-20. PubMed.
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