In the September 21 online Nature Neuroscience, YouMing Lu and colleagues at the University of Calgary, Canada, report that the serine-threonine kinase Cdk5, which is known to phosphorylate the neurofibrillary tangle protein tau, is essential for ischemia-provoked death of hippocampal CA1 neurons. Lu was prompted to investigate the role of the cyclin-dependent kinase because of a previous report that p25, a truncated, persistent form of the Cdk5 activator p35, is found in rat forebrain following cerebral ischemia (see ARF related news story). Significantly, p25 has also been shown to accumulate in the brains of Alzheimer's patients, leading to tau phosphorylation(see ARF related news story).

To test the relationships among p25, Cdk5, and cell death, first author Jian Wang and colleagues quantified the amounts of the two proteins in CA1, CA3, and dentate gyrus extracts taken from either control mice or those that had suffered from transient forebrain ischemia. Only the CA1 neurons from ischemic animals showed accumulation of p25. In addition, levels of activated Cdk5 increased in these neurons fourfold as compared to samples from sham-treated animals. Next, to probe the role played by Ckd5 in neuronal death, the authors infected the animals with a virus carrying a dominant-negative form of the kinase. When these animals were subjected to ischemia, the CA1 neurons were protected as judged by Fluoro-Jade and NeuN staining, which reveal degenerating and surviving neurons, respectively. Without the dominant-negative protein, the numbers of dying neurons skyrocketed from about 250 per square millimeter to almost 2,500.

How might Cdk5 contribute to the death of neurons? It is known that in vitro, the kinase can phosphorylate the NR2A subunit of the N-methyl-D-aspartate (NMDA) receptor. To test if this is also true in vivo, the authors raised an antibody that recognizes specifically Cdk5 phosphorylated NR2A (on serine 1232). This antibody allowed them to detect a ~3.5-fold increase in ser1232 phosphorylated receptor in CA1 neurons following ischemia.

The data indicate that ischemia sets off a chain of events that includes the accumulation of p25, activation of Cdk5, and phosphorylation of the NMDA channel, which in turn leads to enhanced channel activity, a known contributor to neuronal death.

The events following stroke show striking parallels with those that occur in AD, where p25 causes phosphorylation of tau by Cdk5. Pressing questions now include: Why does p25 accumulate, and what explains the exquisite neuronal specificity of these cascades?—Tom Fagan

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  1. In this study, YM Lu and colleagues have unambiguously demonstrated the significance of S1232 phosphorylation of the NMDA receptor subunit NR2A by the p25/Cdk5 kinase in ischemic-induced CA1 neuronal death. They elegantly show that inhibition of p25/Cdk5 by expression of dominant-negative mutants of Cdk5 protects CA1 neurons from ischemic injury. They also highlight the role of S1232 phosphorylation in this process with experimental data showing that expression of the mutant NR2A harboring alanine1232 fully protects neurons from ischemic injury. It should be noted that this study as well as a previous report from H Pant's laboratory (Li et al., 2001) suggest that S1232 phosphorylation upregulates NMDA receptor channel activity. Interestingly, p25 accumulation and Cdk5 activation following ischemia seem to occur upstream of the modification in NMDA receptor function; this is because MK-801, a specific noncompetitive antagonist of NMDA receptors, prevents neuronal death but does not affect p25 accumulation and Cdk5 activation. As such, what is the mechanism accountable for Ca++ entry that leads to calpain activation and p35 to p25 conversion? Lu and colleagues suggest that AMPA receptor channels are likely to be involved in the ischemic response, as systemic administration of the AMPA receptor antagonist NBQX prior to insult inhibits p25 accumulation and Cdk5 activation, and this ultimately exerts a protective effect on CA1 neuronal death. Taken together, this study offers a signaling cascade underlying ischemic brain injury that goes like this:

    Ischemic injuries --> AMPA receptors activation --> Ca++ entry --> Calpain activation --> p25 accumulation --> Aberrant Cdk5 activation --> Elevated NR2A S1232 phosphorylation --> Sustained increase in NMDA receptor activity --> Neuronal death

    Recently, a p25 transgenic mouse model was reported that expresses very low levels of p25 (about 30 percent of endogenous p35) in the hippocampus (Angelo et al., 2003). Interestingly, these animals exhibited improved reversal learning and altered fear conditioning. Perhaps this phenotype reflects a slightly increased S1232 phosphorylation of NR2A and thus a sustained increase in NMDAR activity in the hippocampus of these transgenic mice.

    Several questions remain to be answered. Is the NR2A subunit the only relevant target of p25/Cdk5 responsible for ischemia-induced neuronal death? Furthermore, how does phosphorylation of S1232 induce neuronal death? While it is clear that much effort is needed to elucidate these questions, this study significantly advances our current understanding of the mechanism underlying ischemic brain injury. In addition, it suggests that inhibition of S1232 phosphorylation may serve as a valid method to prevent neuronal death following ischemia.

    References:

    . Regulation of NMDA receptors by cyclin-dependent kinase-5. Proc Natl Acad Sci U S A. 2001 Oct 23;98(22):12742-7. PubMed.

    . Improved reversal learning and altered fear conditioning in transgenic mice with regionally restricted p25 expression. Eur J Neurosci. 2003 Jul;18(2):423-31. PubMed.

  2. Wang et al. suggest that ischemia-induced neuronal cell death may be mediated by activation of Cdk5. The authors propose that Cdk5 phosphorylates the NR2A subunit of the NMDA receptor complex and potentiates excitotoxicity mediated by this glutamate-gated ion channel. These data suggest that inhibition of Cdk5 may reduce ischemic injuries for patients.

    The present study has not only provided a novel mechanism for ischemia-induced neurodegeneration, but has also pointed to new directions for AD research. Cdk5 activity has been found to be elevated in AD brains (1,2). Mechanisms leading to Cdk5 activation are unclear. Despite inconsistent findings from different laboratories (3-6), an activator of Cdk5, p25, has been reported to be present at higher levels in AD compared to control brains (2,7). The protease responsible for p25 production, calpain, is activated in AD brains as well (6,8,9). Taking these data together, the present paper by Wang et al. would suggest that phosphorylation of the NR2A subunit on Ser1232 might be increased in AD brains. Therefore, examination of the phosphoepitope Ser1232 on the NR2A subunit should be highly valuable in further understanding mechanisms behind neurodegeneration in AD. However, protein phosphorylation in general in human brains could be problematic due to potential dephosphorylation during postmortem delay. In addition, since Wang et al. reported changes in NR2A phosphorylation only in the CA1 region but not CA3 and dentate gyrus, a detailed regional analysis of the brain may be warranted. Coincidentally, an NMDA receptor antagonist, memantine, has just been approved by the FDA advisory board for the treatment of moderate to severe forms of AD (see ARF related news story). Whether potential benefits from memantine are derived from its inhibition of Cdk5-enhanced NMDA receptor activity remains to be seen.

    References:

    . Elevated neuronal Cdc2-like kinase activity in the Alzheimer disease brain. Neurosci Res. 1999 May;34(1):21-9. PubMed.

    . Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration. Nature. 1999 Dec 9;402(6762):615-22. PubMed.

    . Brain levels of CDK5 activator p25 are not increased in Alzheimer's or other neurodegenerative diseases with neurofibrillary tangles. J Neurochem. 2003 Aug;86(3):572-81. PubMed.

    . Involvement of cyclin dependent kinase5 activator p25 on tau phosphorylation in mouse brain. Neurosci Lett. 2001 Jun 22;306(1-2):37-40. PubMed.

    . p25 protein in neurodegeneration. Nature. 2001 Jun 14;411(6839):763-4; discussion 764-5. PubMed.

    . Calpain-mediated degradation of p35 to p25 in postmortem human and rat brains. FEBS Lett. 2001 Jan 26;489(1):46-50. PubMed.

    . A survey of Cdk5 activator p35 and p25 levels in Alzheimer's disease brains. FEBS Lett. 2002 Jul 17;523(1-3):58-62. PubMed.

    . Widespread activation of calcium-activated neutral proteinase (calpain) in the brain in Alzheimer disease: a potential molecular basis for neuronal degeneration. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2628-32. PubMed.

    . Active site-directed antibodies identify calpain II as an early-appearing and pervasive component of neurofibrillary pathology in Alzheimer's disease. Brain Res. 1997 Jul 25;763(2):145-58. PubMed.

References

News Citations

  1. The Calpain Connection
  2. Enzyme Essential to Brain Development Found to Hyperphosphorylate Tau, Kill Neurons

Further Reading

Primary Papers

  1. . Cdk5 activation induces hippocampal CA1 cell death by directly phosphorylating NMDA receptors. Nat Neurosci. 2003 Oct;6(10):1039-47. PubMed.