Klas Blomgren

Regulation of autophagy by cytoplasmic p53

Multiple cellular stressors, including activation of the tumour suppressor p53, can stimulate autophagy. Here we show that deletion, depletion or inhibition of p53 can induce autophagy in human, mouse and nematode cells subjected to knockout, knockdown or pharmacological inhibition of p53. Enhanced autophagy improved the survival of p53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels. Inhibition of p53 led to autophagy in enucleated cells, and cytoplasmic, not nuclear, p53 was able to repress the enhanced autophagy of p53−/− cells. Many different inducers of autophagy (for example, starvation, rapamycin and toxins affecting the endoplasmic reticulum) stimulated proteasome-mediated degradation of p53 through a pathway relying on the E3 ubiquitin ligase HDM2. Inhibition of p53 degradation prevented the activation of autophagy in several cell lines, in response to several distinct stimuli. These results provide evidence of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53.

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

Brain development in rodents and humans: Identifying benchmarks of maturation and vulnerability to injury across species.

Hypoxic-ischemic and traumatic brain injuries are leading causes of long-term mortality and disability in infants and children. Although several preclinical models using rodents of different ages have been developed, species differences in the timing of key brain maturation events can render comparisons of vulnerability and regenerative capacities difficult to interpret. Traditional models of developmental brain injury have utilized rodents at postnatal day 7-10 as being roughly equivalent to a term human infant, based historically on the measurement of post-mortem brain weights during the 1970s. Here we will examine fundamental brain development processes that occur in both rodents and humans, to delineate a comparable time course of postnatal brain development across species. We consider the timing of neurogenesis, synaptogenesis, gliogenesis, oligodendrocyte maturation and age-dependent behaviors that coincide with developmentally regulated molecular and biochemical changes. In general, while the time scale is considerably different, the sequence of key events in brain maturation is largely consistent between humans and rodents. Further, there are distinct parallels in regional vulnerability as well as functional consequences in response to brain injuries. With a focus on developmental hypoxic-ischemic encephalopathy and traumatic brain injury, this review offers guidelines for researchers when considering the most appropriate rodent age for the developmental stage or process of interest to approximate human brain development.

Bacterial endotoxin sensitizes the immature brain to hypoxic–ischaemic injury

Epidemiological studies show a markedly increased risk of cerebral palsy following the combined exposure of infection and birth asphyxia. However, the underlying mechanisms of this increased vulnerability remain unclear. We have examined the effects of a low dose of bacterial endotoxin on hypoxic–ischaemic injury in the immature brain of rats. Bacterial endotoxin (lipopolysaccharide 0.3 mg/kg) was administered to 7‐day‐old rats 4 h prior to unilateral hypoxia–ischaemia and the neurological outcome was determined 3 days later. Rectal temperature and cerebral blood flow was measured during the study and the expression of CD14 and toll‐like receptor‐4 mRNA in the brain was examined. We found that a low dose of endotoxin dramatically sensitizes the immature brain to injury and induces cerebral infarction in response to short periods of hypoxia–ischaemia that by themselves caused no or little injury. This effect could not be explained by a reduction in cerebral blood flow or hyperthermia. In association with the sensitization of injury we found an altered expression of CD14 mRNA and toll‐like receptor‐4 mRNA in the brain. These results suggest that the innate immune system may be involved in the vulnerability of the immature brain following the combination of infection and hypoxia–ischaemia.

The influence of age on apoptotic and other mechanisms of cell death after cerebral hypoxia–ischemia

Unilateral hypoxia–ischemia (HI) was induced in C57/BL6 male mice on postnatal day (P) 5, 9, 21 and 60, corresponding developmentally to premature, term, juvenile and adult human brains, respectively. HI duration was adjusted to obtain a similar extent of brain injury at all ages. Apoptotic mechanisms (nuclear translocation of apoptosis-inducing factor, cytochrome c release and caspase-3 activation) were several-fold more pronounced in immature than in juvenile and adult brains. Necrosis-related calpain activation was similar at all ages. The CA1 subfield shifted from apoptosis-related neuronal death at P5 and P9 to necrosis-related calpain activation at P21 and P60. Oxidative stress (nitrotyrosine formation) was also similar at all ages. Autophagy, as judged by the autophagosome-related marker LC-3 II, was more pronounced in adult brains. To our knowledge, this is the first report demonstrating developmental regulation of AIF-mediated cell death as well as involvement of autophagy in a model of brain injury.

Involvement of caspase-3 in cell death after hypoxia-ischemia declines during brain maturation.

The involvement of caspase-3 in cell death after hypoxia–ischemia (HI) was studied during brain maturation. Unilateral HI was produced in rats at postnatal day 7 (P7), 15 (P15), 26 (P26), and 60 (P60) by a combination of left carotid artery ligation and systemic hypoxia (8% O2). Activation of caspase-3 and cell death was examined in situ by high-resolution confocal microscopy with anti-active caspase-3 antibody and propidium iodide and by biochemical analysis. The active caspase-3 positive neurons were composed of more than 90% HI damaged striatal and neocortical neurons in P7 pups, but that number was reduced to approximately 65% in striatum and 34% in the neocortex of P15 pups, and approximately 26% in striatum and 2% in neocortex of P26 rats. In P60 rats, less than 4% of the damaged neurons in striatum and less than 1% in neocortex were positive for active caspase-3. Western blot analysis demonstrated that the level of inactive caspase-3 in normal forebrain tissue gradually declined from a high level in young pups to very low levels in adult rats. Concomitantly, HI-induced active caspase-3 was reduced from a relatively high level in P7, to moderate levels in P15 and P26, to a barely detectable level in P60 rats. The authors conclude that the involvement of caspase-3 in the pathogenesis of cell death after HI declines during neuronal maturation. The authors hypothesize that caspase-3 may play a major role in cell death in immature neurons but a minor role in cell death in mature neurons after brain injury.

Involvement of apoptosis‐inducing factor in neuronal death after hypoxia‐ischemia in the neonatal rat brain

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% O2) 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 AIF‐positive 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.

Chemokine and Inflammatory Cell Response to Hypoxia-Ischemia in Immature Rats

Hypoxia-ischemia induces an inflammatory response in the immature central nervous system that may be important for development of brain injury. Recent data implicate that chemoattractant cytokines, chemokines, are involved in the recruitment of immune cells. The aim was to study α- and β-chemokines in relation to the temporal activation of inflammatory cells after hypoxia-ischemia in immature rats. Hypoxia-ischemia was induced in 7-day-old rats (left carotid artery occlusion + 7.7% oxygen). The pups were decapitated at different times after the insult. Immunohistochemistry was used for evaluation of the inflammatory cell response and RT-PCR to analyze the cytokine mRNA and chemokine mRNA expression. A distinct interleukin-1β and tumor necrosis factor-α cytokine expression was found 0-24 h after hypoxia-ischemia that was accompanied by induction of α-chemokines (growth related gene and macrophage inflammatory protein-2). In the next phase, the β2-integrin expression was increased (12 h and onward) and neutrophils transiently invaded the vessels and tissue in the infarct region. The mRNA induction for the β-chemokines macrophage inflammatory protein-1α, macrophage inflammatory protein-1β, and RANTES preceded the expression of markers for lymphocytes [cluster of differentiation (CD)4, CD8], microglia/macrophages (MHC I), and natural killer cells in the infarct area. The activation of microglia/macrophages, CD4 lymphocytes, and astroglia persisted up to at least 42 d of postnatal age implicating a chronic component of immunoinflammatory activation. The expression of mRNA for α- and β-chemokines preceded the appearance of immune cells suggesting that these molecules may have a role in the inflammatory response to insults in the immature central nervous system.

Apoptosis-Inducing Factor Triggered by Poly(ADP-Ribose) Polymerase and Bid Mediates Neuronal Cell Death after Oxygen-Glucose Deprivation and Focal Cerebral Ischemia

Delayed neuronal cell death occurring hours after reperfusion is a hallmark of ischemic stroke and a primary target for neuroprotective strategies. In the present study, we investigated whether apoptosis-inducing factor (AIF), a caspase-independent proapoptotic protein, is responsible for neuronal cell death after glutamate toxicity and oxygen-glucose deprivation (OGD) in vitro and after experimental stroke in vivo. AIF translocated to the nucleus in which it colocalized with DNA fragmentation and nuclear apoptotic morphology after exposure to glutamate or OGD in cultured neurons or after transient middle cerebral artery occlusion (MCAo) in mice. Small inhibitory RNA-mediated downregulation of AIF reduced glutamate- and OGD-induced neuronal apoptosis by 37 and 60%, respectively (p < 0.01). Moreover, Harlequin mutant mice, which express AIF at low levels (∼20% of wild-type mice), displayed smaller infarct volumes (-43%; p < 0.03) and showed dramatically reduced cell death in the ischemic penumbra after 45 min of MCAo compared with wild-type littermates. Inhibition of poly(ADP-ribose) polymerase and Bid reduced nuclear AIF translocation. These results provide the first evidence for a causal role of AIF in ischemic neuronal cell death. Therefore, caspase-independent cell death signaling may provide a promising novel target for therapeutic interventions in cerebrovascular diseases.

Erythropoietin Improved Neurologic Outcomes in Newborns With Hypoxic-Ischemic Encephalopathy

OBJECTIVE: The purpose of this study was to evaluate the efficacy and safety of erythropoietin in neonatal hypoxic-ischemic encephalopathy (HIE), by using a randomized, prospective study design. METHODS: A total of 167 term infants with moderate/severe HIE were assigned randomly to receive either erythropoietin (N = 83) or conventional treatment (N = 84). Recombinant human erythropoietin, at either 300 U/kg (N = 52) or 500 U/kg (N = 31), was administered every other day for 2 weeks, starting <48 hours after birth. The primary outcome was death or disability. Neurodevelopmental outcomes were assessed at 18 months of age. RESULTS: Complete outcome data were available for 153 infants. Nine patients dropped out during treatment, and 5 patients were lost to follow-up monitoring. Death or moderate/severe disability occurred for 35 (43.8%) of 80 infants in the control group and 18 (24.6%) of 73 infants in the erythropoietin group (P = .017) at 18 months. The primary outcomes were not different between the 2 erythropoietin doses. Subgroup analyses indicated that erythropoietin improved long-term outcomes only for infants with moderate HIE (P = .001) and not those with severe HIE (P = .227). No negative hematopoietic side effects were observed. CONCLUSION: Repeated, low-dose, recombinant human erythropoietin treatment reduced the risk of disability for infants with moderate HIE, without apparent side effects.