Apoptosis-inducing factor (AIF) triggers apoptosis in a caspase-independent manner. Here we report for the first time involvement of AIF in neuronal death induced by cerebral ischemia. Unilateral cerebral hypoxia-ischemia (HI) was induced in 7-day-old rats by ligation of the left carotid artery and hypoxia (7.7% O 2 ) for 55 min. AIF release from mitochondria and AIF translocation to nuclei was detected immediately after HI, and only in damaged areas, as judged by the concurrent loss of MAP-2. AIF release was detected earlier than that of cytochrome c. Cells with AIF-positive nuclei displayed nuclear condensation and signs of DNA damage. The number of AIFpositive nuclei showed a positive correlation with the infarct volume 72 h post-HI, and this was not changed by treating the animals with boc-Asp-fmk (BAF), a multicaspase inhibitor. BAF treatment reduced the activity of caspase-3, -2 and -9 (78, 73 and 33%, respectively), and prevented caspase-dependent fodrin cleavage in vivo, but did not affect AIF release from mitochondria or the frequency of positive nuclear AIF or DNA damage 72 h post-HI, indicating that these processes occurred in a caspase-independent fashion. In summary, AIF-mediated cell death may be an important mechanism of HI-induced neuronal loss in the immature brain.

Long recognized as an important regulatory mechanism in biosignal processes, modulation of the phosphorylation state of proteins has emerged as the most important mechanism for understanding signal transduction. In contrast to the multitude of protein kinases and the clear signal transduction pathways, relatively few protein phosphatases are known and their regulation is unclear. Among them, calcineurin, a calcium/calmodulin-dependent phosphatase (PP2B), is the best enzyme to unveil the phosphatase function, because it was shown to be the direct target for immunosuppressants CsA and FK506, which are powerful tools for understanding this function in diseases as well as in several tissues and organs. Although calcineurin has been found in the highest concentrations in brain, it has also been detected in many other mammalian tissues. Well characterized in T cell activation by analysing the transcription factor NFAT, the function of calcineurin, however, was less well understood in other tissues and organs. Since the mid-1990s, several novel functions of this phosphatase have been reported, revealing that it plays important roles as a multifunctional regulator under the direct regulation of calcium signaling.

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In a model of cerebral hypoxia-ischemia in the immature rat, widespread brain injury is produced in the ipsilateral hemisphere, whereas the contralateral hemisphere is left undamaged. Previously, we found that calpains were equally translocated to cellular membranes (a prerequisite for protease activation) in the ipsilateral and contralateral hemispheres. However, activation, as judged by degradation of fodrin, occurred only in the ipsilateral hemisphere. In this study we demonstrate that calpastatin, the specific, endogenous inhibitor protein to calpain, is up-regulated in response to hypoxia and may be responsible for the halted calpain activation in the contralateral hemisphere. Concomitantly, extensive degradation of calpastatin occurred in the ipsilateral hemisphere, as demonstrated by the appearance of a membrane-bound 50-kDa calpastatin breakdown product. The calpastatin breakdown product accumulated in the synaptosomal fraction, displaying a peak 24 h post-insult, but was not detectable in the cytosolic fraction. The degradation of calpastatin was blocked by administration of CX295, a calpain inhibitor, indicating that calpastatin acts as a suicide substrate to calpain during hypoxia-ischemia. In summary, calpastatin was up-regulated in areas that remain undamaged and degraded in areas where excessive activation of calpains and infarction occurs.Hypoxic-ischemic brain damage is an important contributor to long term neurological sequelae in term and pre-term infants (1-3). The intracellular calcium concentration increases during anoxia/ischemia (4) followed by a secondary phase of cellular calcium overload simultaneous with or slightly preceding development of hypoxic-ischemic neuronal damage (5, 6). Activation of calcium-dependent enzymes is considered to be an early feature in this process (7). Calpains (EC 3.4.22.17) are calciumactivated, nonlysosomal, neutral cysteine proteases proposed to participate in many important intracellular processes, such as turnover of cytoskeletal proteins and regulation of kinase activity and transcription factors (8, 9). The activity of calpains is strictly regulated by calcium concentrations and interaction with calpastatin (the endogenous inhibitor protein), membrane phospholipids, and a multitude of other factors. The ubiquitous distribution of calpains and the complex regulation of their activity indicate that these proteases play important roles under both physiological and pathological conditions. Calpain activation, as judged by the appearance of specific fodrin (10) (also called brain spectrin (11)) breakdown products (FBDP) 1 has previously been demonstrated in adult (12-16) and neonatal (17, 18) models of ischemia and hypoxia. Activation of calpains and selective degradation of preferred substrates precede neuronal degeneration, indicating that these proteases are activated during hypoxia-ischemia (HI) and in the early phase of reperfusion after HI, preceding neuronal death (12, 13, 16 -19). In vivo administration of inhibitors of calpain activity has been sho...

No abstract

Neural transplantation is an experimental treatment for Parkinson's disease. Widespread clinical application of the grafting technique is hampered by a relatively poor survival (around 10%) of implanted embryonic dopamine neurones. Earlier animal studies have indicated that a large proportion of the grafted cells die during graft tissue preparation and within the first few days after intracerebral implantation. The present study was designed to reveal the prevalence of cell death in rat intrastriatal grafts at 90 min, 1, 3, 6 and 42 days after implantation. We examined apoptotic cell death using semithin and paraffin sections stained with methylene blue and an antibody against activated caspase 3, respectively. We identified abundant apoptotic cell death up to 3 days after transplantation. In addition, we studied calpain activation using an antibody specific for calpain-cleaved fodrin. We report a peak in calpain activity 90 min after grafting. Surprisingly, we did not observe any significant difference in the number of dopaminergic neurones over time. The present results imply that grafted cells may be victims of either an early necrotic or a later apoptotic cell death and that there is substantial cell death as early as 90 min after implantation.

The aim was to study the effects of an NMDA receptor antagonist on caspase-3 activation and DNA fragmentation after hypoxia-ischemia (HI) in 7-day-old rats. Animals were treated with vehicle or MK-801 (0.5 mg/kg) directly after HI and sacrificed 8, 24 or 72h later. MK-801 reduced injury (by 53%), cells positive for active caspase-3 (by 39%) and DNA fragmentation (by 79%) in the cerebral cortex. Furthermore, MK-801 significantly decreased caspase-3 activity, and Western blots revealed a tendency towards decreased proteolytic cleavage of the caspase-3 proform. The data imply that NMDA receptors are involved in the activation of apoptotic processes in the immature brain after HI.

No abstract