Outer mitochondrial membrane localization of apoptosis-inducing factor: mechanistic implications for release

Poly(ADP-ribose) polymerase-1-dependent cell death (known as parthanatos) plays a pivotal role in many clinically important events including ischaemia/reperfusion injury and glutamate excitotoxicity. A recent study by us has shown that uncleaved AIF (apoptosis-inducing factor), but not calpain-hydrolysed truncated-AIF, was rapidly released from the mitochondria during parthanatos, implicating a second pool of AIF that might be present in brain mitochondria contributing to the rapid release. In the present study, a novel AIF pool is revealed in brain mitochondria by multiple biochemical analyses. Approx. 30% of AIF loosely associates with the outer mitochondrial membrane on the cytosolic side, in addition to its main localization in the mitochondrial intermembrane space attached to the inner membrane. Immunogold electron microscopic analysis of mouse brain further supports AIF association with the outer, as well as the inner, mitochondrial membrane in vivo. In line with these observations, approx. 20% of uncleaved AIF rapidly translocates to the nucleus and functionally causes neuronal death upon NMDA (N-methyl-d-aspartate) treatment. In the present study we show for the first time a second pool of AIF in brain mitochondria and demonstrate that this pool does not require cleavage and that it contributes to the rapid release of AIF. Moreover, these results suggest that this outer mitochondrial pool of AIF is sufficient to cause cell death during parthanatos. Interfering with the release of this outer mitochondrial pool of AIF during cell injury paradigms that use parthanatos hold particular promise for novel therapies to treat neurological disorders.     

Mechanisms underlying cell death in ischemia-like damage to the rat spinal cord in vitro

New spinal cord injury (SCI) cases are frequently due to non-traumatic causes, including vascular disorders. To develop mechanism-based neuroprotective strategies for acute SCI requires full understanding of the early pathophysiological changes to prevent disability and paralysis. The aim of our study was to identify the molecular and cellular mechanisms of cell death triggered by a pathological medium (PM) mimicking ischemia in the rat spinal cord in vitro. We previously showed that extracellular Mg²⁺ (1 mM) worsened PM-induced damage and inhibited locomotor function. The present study indicated that 1 h of PM+Mg²⁺ application induced delayed pyknosis chiefly in the spinal white matter via overactivation of poly (ADP-ribose) polymerase 1 (PARP1), suggesting cell death mediated by the process of parthanatos that was largely suppressed by pharmacological block of PARP-1. Gray matter damage was less intense and concentrated in dorsal horn neurons and motoneurons that became immunoreactive for the mitochondrial apoptosis-inducing factor (the intracellular effector of parthanatos) translocated into the nucleus to induce chromatin condensation and DNA fragmentation. Immunoreactivity to TRPM ion channels believed to be involved in ischemic brain damage was also investigated. TRPM2 channel expression was enhanced 24 h later in dorsal horn and motoneurons, whereas TRPM7 channel expression concomitantly decreased. Conversely, TRPM7 expression was found earlier (3 h) in white matter cells, whereas TRPM2 remained undetectable. Simulating acute ischemic-like damage in vitro in the presence of Mg²⁺ showed how, during the first 24 h, this divalent cation unveiled differential vulnerability of white matter cells and motoneurons, with distinct changes in their TRPM expression.     

PAAN/MIF nuclease inhibition prevents neurodegeneration in Parkinson’s disease

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Calpain activation is not required for AIF translocation in PARP-1-dependent cell death (parthanatos)

Apoptosis-inducing factor (AIF) is critical for poly(ADP-ribose) polymerase-1 (PARP-1)-dependent cell death (parthanatos). The molecular mechanism of mitochondrial AIF release to the nucleus remains obscure, although a possible role of calpain I has been suggested. Here we show that calpain is not required for mitochondrial AIF release in parthanatos. Although calpain I cleaved recombinant AIF in a cell-free system in intact cells under conditions where endogenous calpain was activated by either NMDA or N -methyl- N '-nitro- N -nitrosoguanidine (MNNG) administration, AIF was not cleaved, and it was released from mitochondria to the nucleus in its 62-kDa uncleaved form. Moreover, NMDA administration under conditions that failed to activate calpain still robustly induced AIF nuclear translocation. Inhibition of calpain with calpastatin or genetic knockout of the regulatory subunit of calpain failed to prevent NMDA- or MNNG-induced AIF nuclear translocation and subsequent cell death, respectively, which was markedly prevented by the PARP-1 inhibitor, 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-iso-quinolinone. Our study clearly shows that calpain activation is not required for AIF release during parthanatos, suggesting that other mechanisms rather than calpain are involved in mitochondrial AIF release in parthanatos.     

Reactive oxygen species‐induced parthanatos of immunocytes by human cytomegalovirus‐associated substance

Previous studies have examined various immune evasion strategies of human cytomegalovirus (HCMV) to gain understanding of its pathogenesis. Although the mechanism that underlies immunocyte destruction near HCMV‐infected lesions has yet to be established, it is here shown that substances produced by HCMV‐infected cells induce death in several types of immunocytes, but not in fibroblasts or astrocytomas. These substances contain HCMV proteins and were termed HCMV‐associated insoluble substance (HCMVAIS). The mechanism by which HCMVAIS induces cell death was characterized to improve understanding the death of immunocytes near HCMV‐infected lesions. HCMVAIS were found to trigger production of intracellular nicotinamide adenine dinucleotide phosphate oxidase‐derived reactive oxygen species (ROS), resulting in cell death, this effect being reversed following treatment with ROS inhibitors. Cell death was not induced in splenocytes from NOX‐2 knockout mice. It was hypothesized that DNA damage induced by oxidative stress initiates poly ADP‐ribose polymerase‐1 (PARP‐1)‐mediated cell death, or parthanatos. HCMVAIS‐induced cell death is accompanied by PARP‐1 activation in a caspase‐independent manner, nuclear translocation of apoptosis‐inducing factor (AIF), and DNA fragmentation, which are typical features of parthanatos. Treatment with an AIF inhibitor decreased the rate of HCMVAIS‐induced cell death, this being confirmed by hematoxylin and eosin staining; cell death in most HCMV‐positive foci in serial section samples of a large intestine with HCMV infection was TUNEL‐positive, cleaved caspase 3‐negative and CD45‐positive. Taken together, these data suggest that HCMV inhibits local immune responses via direct killing of immunocytes near HCMV‐infected cells through ROS‐induced parthanatos by HCMVAIS.     

一种新的细胞死亡形式——parthanatos研究进展

作为一种不同于凋亡的新型调控性细胞死亡形式,parthanatos与神经退行性疾病、中风、谷氨酸兴奋性中毒、活性氧(ROS)诱导的损伤和肿瘤等诸多疾病的发生发展密切相关.由于多聚ADP核糖聚合酶-1(ADP-ribose polymerase-1,PARP-1)的异常活化是诱发parthanatos发生的先决条件,所以...