Causal role of apoptosis-inducing factor for neuronal cell death following traumatic brain injury.

Traumatic brain injury (TBI) consists of two phases: an immediate phase in which damage is caused as a direct result of the mechanical impact; and a late phase of altered biochemical events that results in delayed tissue damage and is therefore amenable to therapeutic treatment. Because the molecular mechanisms of delayed post-traumatic neuronal cell death are still poorly understood, we investigated whether apoptosis-inducing factor (AIF), a pro-apoptotic mitochondrial molecule and the key factor in the caspase-independent, cell death signaling pathway, plays a causal role in neuronal death following TBI. Using an in vitro model of neuronal stretch injury, we demonstrated that AIF translocated from mitochondria to the nucleus of neurons displaying axonal disruption, chromatin condensation, and nuclear pyknosis in a caspase-independent manner, whereas astrocytes remained unaffected. Similar findings were observed following experimental TBI in mice, where AIF translocation to the nucleus coincided with delayed neuronal cell death in both cortical and hippocampal neurons. Down-regulation of AIF in vitro by siRNA significantly reduced stretch-induced neuronal cell death by 67%, a finding corroborated in vivo using AIF-deficient harlequin mutant mice, where secondary contusion expansion was significantly reduced by 44%. Hence, our current findings demonstrate that caspase-independent, AIF-mediated signaling pathways significantly contribute to post-traumatic neuronal cell death and may therefore represent novel therapeutic targets for the treatment of TBI.

Bone marrow stromal cells mediate protection through stimulation of PI3-K/Akt and MAPK signaling in neurons.

Application of adult bone marrow stromal cells (BMSC) improves functional outcome in animal models of cerebral ischemia, traumatic brain injury, and spinal cord injury. Accumulating evidence suggests that such functional recovery after BMSC treatment is mediated by enhanced trophic support of the injured neurons and improved neuronal plasticity rather than tissue replacement by bone marrow-derived stem cells. Therefore, the aim of the present study was to explore the potential of non-hematopoietic BMSC to stimulate signaling pathways in neurons that mediate trophic effects and neuroprotection. In primary embryonic rat neurons, BMSC conditioned medium (CM) attenuated staurosporine (STS) or amyloid-beta peptide-induced apoptosis in a concentration-dependent manner. The neuroprotective effect of CM required several hours of pretreatment and was abolished by heating over 90 degrees C. Immunoblot analyses revealed that CM enhanced Erk1/2 and Akt phosphorylation in neurons, and the specific MEK1 inhibitor PD98059 or the phosphoinositide-3 kinase (PI3-K) inhibitor Ly294002 abolished the neuroprotective effect of CM. Further, double-conditioned medium (DCM) obtained from BMSC previously stimulated by medium from STS-challenged neurons showed a more potent anti-apoptotic effect compared to the single-conditioned medium. Overall, these findings demonstrate that BMSC trigger endogenous survival signaling pathways in neurons that mediate protection against apoptotic insults. Moreover, the interaction between stressed neurons and BMSC further amplifies the observed neuroprotective effect.

Molecular insights into mechanisms of the cell death program: role in the progression of neurodegenerative disorders.

Synaptic degeneration and death of neurons in limbic and cortical brain regions are the fundamental processes responsible for the manifestation of cognitive dysfunction and behavioural abnormalities in Alzheimer's disease (AD). Despite the various genetic and environmental factors, and the aging process itself that may lead to the manifestation of AD, multiple evidence from studies in experimental models and in AD brain tissue demonstrate that the underlying neurodegeneration is associated with morphological and biochemical features of apoptosis. At the cellular level, neuronal apoptosis in AD may be initiated by oxidative stress and related DNA damage, disruption of cellular calcium homeostasis, or endoplasmic reticulum (ER) stress. The molecular mechanisms of the biochemical cascades of apoptosis are beginning to be understood and involve upstream effectors such as Par-4, p53, and pro-apoptotic Bcl-2 family members, which mediate mitochondrial dysfunction and subsequent release of pro-apoptotic proteins, such as cytochrome c or apoptosis inducing factor (AIF), and subsequent caspase-dependent and -independent pathways which finally result in degradation of proteins and nuclear DNA. The regulation of apoptotic cascades is complex and involves transcriptional control as well as posttranscriptional protein modifications, such as protease-mediated cleavage, ubiquitination or poly(ADP-ribosylation). More recently, the regulation of protein phosphorylation by kinases and phosphatases is emerging as a prerequisite mechanism in the control of the apoptotic cell death program. A better understanding of the molecular underpinnings of neuronal apoptosis will lead to novel preventive and therapeutic approaches to the neurodegenerative processes in Alzheimer's disease and other neurological disorders where programmed cell death is prominent.

Structure-activity relationships by interligand NOE-based design and synthesis of antiapoptotic compounds targeting Bid.

Bcl-2 family proteins play a crucial role in tissue homeostasis and apoptosis (programmed cell death). Bid is a proapoptotic member of the Bcl-2 family, promoting cell death when activated by caspase-8. Following an NMR-based approach (structure-activity relationships by interligand NOE) we were able to identify two chemical fragments that bind on the surface of Bid. Covalent linkage of the two fragments led to high-affinity bidentate derivatives. In vitro and in-cell assays demonstrate that the compounds prevent tBid translocation to the mitochondrial membrane and the subsequent release of proapoptotic stimuli and inhibit neuronal apoptosis in the low micromolar range. Therefore, by using a rational chemical-biology approach, we derived antiapoptotic compounds that may have a therapeutic potential for disorders associated with Bid activation, e.g., neurodegenerative diseases, cerebral ischemia, or brain trauma.

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 (approximately 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.

Nitric oxide donors induce neurotrophin-like survival signaling and protect neurons against apoptosis.

Our previous results showed that inhibition of protein tyrosine phosphatases (PTP) by orthovanadate is an appropriate strategy to mimic nerve growth factor (NGF) effects in neurons, including enhanced phosphorylation of TrkA, stimulation of downstream survival signaling pathways, and protection against apoptotic stress. In this study, we wanted to trigger such NGF-like survival signaling in primary hippocampal neurons with the more specific PTP inhibitors ethyl-3,4-dephostatin (DPN), 4-O-methyl-ethyl-3,4-dephostatin (Me-DPN), and methoxime-3,4-dephostatin. It was striking that only the nitric oxide (NO)-releasing dephostatin analogs DPN and Me-DPN, but not the nitrosamine-free methoxime derivative (which did not release NO), enhanced TrkA phosphorylation and protected the neurons against staurosporine (STS)-induced apoptosis. The established NO donor S-nitroso-N-acetylpenicillamine (SNAP) also enhanced TrkA phosphorylation and prevented apoptosis similarly to DPN and Me-DPN. Analysis of the major signaling pathways downstream of TrkA revealed that both SNAP and DPN enhanced phosphorylation of Akt and the mitogen-activated kinases (MAPK) Erk1/2. Blocking of these signaling pathways by the PI3-K inhibitor wortmannin or the MAPK kinase inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene] equally abolished the neuroprotective effect of the NO donors. It was striking that inhibition of the soluble guanylyl cyclase (sGC) by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or protein kinase G (PKG) inhibition by (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo-[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester (KT5823) also blocked the neuroprotective effect of the NO donors, and ODQ clearly attenuated SNAP-induced phosphorylation of TrkA, Akt, and MAPK. In conclusion, NO release by the dephostatin derivatives and subsequent stimulation of sGC and PKG is essential for their neuroprotective effects. In primary neurons, such NO-activated survival signaling involves NGF-like effects, including enhanced phosphorylation of TrkA and activation of PI3-K/Akt and MAPK pathways.