Cyclin-dependent kinases (CDKs) are commonly known to regulate cell proliferation. However, previous reports suggest that in cultured postmitotic neurons, activation of CDKs is a signal for death rather than cell division. We determined whether CDK activation occurs in mature adult neurons during focal stroke in vivo and whether this signal was required for neuronal death after reperfusion injury. Cdk4͞cyclin D1 levels and phosphorylation of its substrate retinoblastoma protein (pRb) increase after stroke. Deregulated levels of E2F1, a transcription factor regulated by pRb, are also observed. Administration of a CDK inhibitor blocks pRb phosphorylation and the increase in E2F1 levels and dramatically reduces neuronal death by 80%. These results indicate that CDKs are an important therapeutic target for the treatment of reperfusion injury after ischemia.T he mechanism by which stroke-induced neuronal death occurs is complex and is likely dependent upon the severity and duration of ischemic insult and an elaborate interplay between ischemic death initiators such as excitotoxicity, oxidative stress, DNA damage, and inflammatory responses (1-3). Neurons that survive the acute ischemic injury undergo a delayed cell death that exhibits some characteristics of apoptosis (1-3). This delayed cell death is dependent upon selected death-signaling elements such as caspases, poly(ADP-ribose) polymerase, and p53 (4). The identification of signaling molecules that control delayed neuronal death has led to the hope that some of these death-signaling elements may serve as useful therapeutic targets for the reduction of neuropathology and behavioral deficits associated with stroke injury. The mechanism by which stroke-evoked delayed death occurs, however, is not fully understood.The cell cycle is a highly coordinated process regulated by the appropriate and timely activation of cyclin-dependent kinases (CDKs) (5). Regulation of CDKs is complex and includes binding to their obligate cyclin partner, activating and inhibitory phosphorylation events, and endogenous inhibitors of CDK activity. Distinct CDKs regulate progressive phases of the cell cycle. Generally, it is thought that the Cdk4͞6͞cyclin D1 complex regulates the G 0 to G 1 , Cdk2͞cyclin E and Cdk3 control G 1 to S, and Cdk2͞cyclin A and Cdk1͞cyclin B control G 2 and M progressions. Although the downstream targets of CDKs are not fully characterized, one important substrate is the tumor suppresser retinoblastoma protein (pRb), which is phosphorylated by activated Cdk4͞6͞cyclin D complex (6). Once hyperphosphorylated, pRb is released from the transcription factor complex E2F͞DP, which then activates genes required for S phase transition (7-9).Paradoxically, increasing evidence suggests that CDKs may have functions beyond that of cell cycle regulation. Numerous reports indicate the requirement of CDK signals for death of cultured postmitotic neurons exposed to select death insults. For example, inappropriate cyclin B and cyclin D1 transcripts have been observed in neuronal P...

The neuronal apoptosis inhibitory protein (NAIP) was identified as a candidate gene for the inherited neurodegenerative disorder spinal muscular atrophy. NAIP is the founding member of a human protein family that is characterized by highly conserved N-terminal motifs called baculovirus inhibitor of apoptosis repeats (BIR). Five members of the human family of inhibitor of apoptosis proteins including NAIP have been shown to be antiapoptotic in various systems. To date, a mechanism for the antiapoptotic effect of NAIP has not been elucidated. To investigate NAIP function, we found cytoprotection of NAIP-expressing primary cortical neurons treated to undergo caspase-3-dependent apoptosis. The additional treatment of these neurons with the pancaspase inhibitor boc-aspartyl(OMe)-fluoromethylketone did not result in increased survival. Similar cytoprotective effects were obtained using HeLa cells transiently transfected with a NAIP N-terminal construct and treated to undergo a caspase-3-dependent cell death. To examine whether NAIP inhibits caspases directly, recombinant N-terminal NAIP protein containing BIR domains was overexpressed, purified, and tested for caspase inhibition potential. Our results demonstrate that inhibition of caspases is selective and restricted to the effector group of caspases, with K i values as low as ϳ14 nM for caspase-3 and ϳ45 nM for caspase-7. Additional investigations with NAIP fragments containing either one or two NAIP BIRs revealed that the second BIR and to a lesser extent the third BIR alone are sufficient to mediate full caspase inhibition.

Despite a wide range of clinical tools, the etiology of mental retardation and multiple congenital malformations remains unknown for many patients. Array-based comparative genomic hybridization (aCGH) has proven to be a valuable tool in these cases, as its pangenomic coverage allows the identification of chromosomal aberrations that are undetectable by other genetic methods targeting specific genomic regions. Therefore, aCGH is increasingly used in clinical genetics, both in the postnatal and the prenatal settings. While the diagnostic yield in the postnatal population has been established at 10-12%, studies investigating fetuses have reported variable results. We used whole-genome aCGH to investigate fetuses presenting at least one major malformation detected on ultrasound, but for whom standard genetic analyses (including karyotype) failed to provide a diagnosis. We identified a clinically significant chromosomal aberration in 8.2% of tested fetuses (4/49), and a result of unclear clinical significance in 12.2% of tested fetuses (6/49). Our results document the value of whole-genome aCGH as a prenatal diagnostic tool and highlight the interpretation difficulties associated with copy number variations of unclear significance.