Neuronal death after perinatal cerebral hypoxia-ischemia: Focus on autophagy-mediated cell death.

Neonatal hypoxic-ischemic encephalopathy is a critical cerebral event occurring around birth with high mortality and neurological morbidity associated with long-term invalidating sequelae. In view of the great clinical importance of this condition and the lack of very efficacious neuroprotective strategies, it is urgent to better understand the different cell death mechanisms involved with the ultimate aim of developing new therapeutic approaches. The morphological features of three different cell death types can be observed in models of perinatal cerebral hypoxia-ischemia: necrotic, apoptotic and autophagic cell death. They may be combined in the same dying neuron. In the present review, we discuss the different cell death mechanisms involved in neonatal cerebral hypoxia-ischemia with a special focus on how autophagy may be involved in neuronal death, based: (1) on experimental models of perinatal hypoxia-ischemia and stroke, and (2) on the brains of human neonates who suffered from neonatal hypoxia-ischemia.

Nineteenth century research on cell death.

This paper reviews research on cell death in the 19th C. The first report of cell death was by Vogt in 1842, which was remarkably soon after the establishment of the cell theory by Schleiden and Schwann between 1838 and 1842. Initial studies on cell death, including that of Vogt, focused on its occurrence in metamorphosis (Vogt, 1842; Prévost and Lebert, 1844; Weismann, 1863-1866) or in blatant pathology (Virchow, 1858), but as histological techniques improved it was found to be involved in more subtle roles in numerous situations including endochondral ossification (Stieda, 1872), ovarian follicle atresia (Flemming, 1885), cell turnover (Nissen, 1886), the wholesale loss of a population of sensory neurons in fish (Beard, 1889), and the naturally occurring histogenetic death of myocytes (Felix, 1889) and neurons (Collin, 1906). The current categorization of cell death into about three main morphological types has 19th century roots in that apoptosis was well described by Flemming (1885), who called it chromatolysis, and various authors including Noetzel (1895) proposed a threefold classification. This article is part of a Special Issue entitled "Apoptosis: Four Decades Later".

JNK3 is abundant in insulin-secreting cells and protects against cytokine-induced apoptosis.

AIMS/HYPOTHESIS: In insulin-secreting cells, activation of the c-Jun NH(2)-terminal kinase (JNK) pathway triggers apoptosis. Whereas JNK1 and JNK2 are ubiquitously produced, JNK3 has been described exclusively in neurons. This report aims to characterise the expression and role in apoptosis of the three JNK isoforms in insulin-secreting cells exposed to cytokines. METHODS: Sections of human and mouse pancreases were used for immunohistochemistry studies with isoform-specific anti-JNK antibodies. Human, pig, mouse and rat pancreatic islets were isolated by enzymatic digestion and RNA or protein extracts were prepared. RNA and protein levels were determined by quantitative RT-PCR and western blotting respectively, using JNK-isoform-specific primers and isoform-specific antibodies; activities of the three JNK isoforms were determined by kinase assays following quantitative immunoprecipitation/depletion of JNK3. JNK silencing was performed with small interfering RNAs and apoptotic rates were determined in INS-1E cells by scoring cells displaying pycnotic nuclei. RESULTS: JNK3 and JNK2 mRNAs are the predominant isoforms expressed in human pancreatic islets. JNK3 is nuclear while JNK2 is also cytoplasmic. In INS-1E cells, JNK3 knockdown increases c-Jun levels and caspase-3 cleavage and sensitises cells to cytokine-induced apoptosis; in contrast, JNK1 or JNK2 knockdown is protective. CONCLUSIONS/INTERPRETATION: In insulin-secreting cells, JNK3 plays an active role in preserving pancreatic beta cell mass from cytokine attacks. The specific localisation of JNK3 in the nucleus, its recruitment by cytokines, and its effects on key transcription factors such as c-Jun, indicate that JNK3 is certainly an important player in the transcriptional control of genes expressed in insulin-secreting cells.

Excitotoxicity-related endocytosis in cortical neurons.

Recent studies showed that endocytosis is enhanced in neurons exposed to an excitototoxic stimulus. We here confirm and analyze this new phenomenon using dissociated cortical neuronal cultures. NMDA-induced uptake (FITC-dextran or FITC or horseradish peroxidase) occurs in these cultures and is due to endocytosis, not to cell entry through damaged membranes; it requires an excitotoxic dose of NMDA and is dependent on extracellular calcium, but occurs early, while the neuron is still intact and viable. It involves two components, NMDA-induced and constitutive, with different characteristics. Neither component involves specific binding of the endocytosed molecules to a saturable receptor. Strikingly, molecules internalized by the NMDA-induced component are targeted to neuronal nuclei. This component, but not the constitutive one, is blocked by a c-Jun N-terminal protein kinase inhibitor. In conclusion, an excitotoxic dose of NMDA triggers c-Jun N-terminal protein kinase-dependent endocytosis in cortical neuronal cultures, providing an in vitro model of the excitotoxicity-induced endocytosis reported in intact tissues.

Role of the JNK pathway in NMDA-mediated excitotoxicity of cortical neurons.

Excitotoxic insults induce c-Jun N-terminal kinase (JNK) activation, which leads to neuronal death and contributes to many neurological conditions such as cerebral ischemia and neurodegenerative disorders. The action of JNK can be inhibited by the D-retro-inverso form of JNK inhibitor peptide (D-JNKI1), which totally prevents death induced by N-methyl-D-aspartate (NMDA) in vitro and strongly protects against different in vivo paradigms of excitotoxicity. To obtain optimal neuroprotection, it is imperative to elucidate the prosurvival action of D-JNKI1 and the death pathways that it inhibits. In cortical neuronal cultures, we first investigate the pathways by which NMDA induces JNK activation and show a rapid and selective phosphorylation of mitogen-activated protein kinase kinase 7 (MKK7), whereas the only other known JNK activator, mitogen-activated protein kinase kinase 4 (MKK4), was unaffected. We then analyze the action of D-JNKI1 on four JNK targets containing a JNK-binding domain: MAPK-activating death domain-containing protein/differentially expressed in normal and neoplastic cells (MADD/DENN), MKK7, MKK4 and JNK-interacting protein-1 (IB1/JIP-1).

Role of phosphoinositide 3-kinase in the autophagic death of serum-deprived PC12 cells.

The death of serum-deprived undifferentiated PC12 cells shows both autophagic and apoptotic features. Since it is still controversial whether the autophagy is instrumental in the cell death or a mere epiphenomenon, we tested the effects of inhibiting the autophagy by a variety of phosphoinositide 3-kinase inhibitors, and provided evidence that the autophagy, or a related trafficking event, is indeed instrumental in the cell death. Furthermore, by comparing the effects of PI3-K inhibition and caspase-inhibition on autophagic and apoptotic cellular events, we showed that in this case the autophagic and apoptotic mechanisms mediate cell death by parallel pathways and do not act in series.

Mathematical analysis of competition between sensory ganglion cells for neurotrophic factor in the skin.

A model is presented of competition between sensory axons for trophic molecules (e.g. a neurotrophin such as NGF), produced in a region of skin small enough to permit their free diffusion throughout it; e.g., a touch dome, or a vibrissal follicle hair sinus. The variables specified are the number of high affinity trophic factor receptors per axon terminal and the concentration of trophic factor in the extracellular space. Previous models of this class predicted the loss of all the axons innervating the region except the one requiring least trophic factor for its maintenance, even with high rates of trophic factor production. In the present model, we have imposed upper limits to axonal growth, thereby introducing new equilibria, and we show by a global analysis using LaSalle's theorem, and also by local analysis, that several axons can then coexist if the rate of production of trophic molecules is sufficiently high.