Since several different pathways are involved in cerebral ischemia/reperfusion injury, combination therapy rather than monotherapy may be required for efficient neuroprotection. In this study, we examined the protective effects of an apoptosis inhibitor Gly 14-humanin (HNG) and a necroptosis inhibitor necrostatin-1 (Nec-1) on hypoxia/ischemia/reperfusion injury. Cultured mouse primary cortical neurons were incubated with Nec-1, HNG or both in a hypoxia chamber for 60 min. Cell viability was determined by MTS assay at 24 h after oxygen-glucose deprivation (OGD) treatment. Mice underwent middle cerebral artery occlusion for 75 min followed by 24 h reperfusion. Mice were administered HNG and/or Nec-1 (i.c.v.) at 4 h after reperfusion. Neurological deficits were evaluated and the cerebral infarct volume was determined by TTC staining. Nec-1 or HNG alone had protective effects on OGD-induced cell death. Combined treatment with Nec-1 and HNG resulted in more neuroprotection than Nec-1 or HNG alone. Treatment with HNG or Nec-1 reduced cerebral infarct volume from 59.3 ± 2.6% to 47.0 ± 2.3% and 47.1 ± 1.5%, respectively. Combined treatment with HNG and Nec-1 improved neurological scores and decreased infarct volume to 38.6 ± 1.5%. In summary, we demonstrated that the combination treatment of HNG and Nec-1 conferred synergistic neuroprotection on hypoxia/ischemia/reperfusion injury in vitro and in vivo. These findings provide a novel therapeutic strategy for the treatment of stroke by combining anti-apoptosis and anti-necroptosis therapy.

Humanin (HN) is an anti-apoptotic peptide that suppresses neuronal cell death induced by Alzheimer's disease, prion protein fragments, and serum deprivation. Recently, we demonstrated that Gly14-HN (HNG), a variant of HN in which the 14th amino acid serine is replaced with glycine, can decrease apoptotic neuronal death and reduce infarct volume in a focal cerebral ischemia/reperfusion mouse model. In this study, we postulate that the mechanism of HNG's neuroprotective effect is mediated by the PI3K/Akt pathway. Oxygen-glucose deprivation (OGD) was performed in cultured mouse primary cortical neurons for 60 min. The effect of HNG and PI3K/Akt inhibitors on OGD-induced cell death was examined at 24 h after reperfusion. HNG increased cell viability after OGD in primary cortical neurons, whereas the PI3K/Akt inhibitors wortmannin and Akti-1/2 attenuated the protective effect of HNG. HNG rapidly increased Akt phosphorylation, an effect that was inhibited by wortmannin and Akti-1/2. Mouse brains were injected intraventricularly with HNG before being subjected to middle cerebral artery occlusion (MCAO). HNG treatment significantly elevated p-Akt levels after cerebral I/R injury and decreased infarct volume. The protective effect of HNG on infarct size was attenuated by wortmannin and Akti-1/2. Taken as a whole, these results suggest that PI3K/Akt activation mediates HNG's protective effect against hypoxia/ischemia reperfusion injury.

BACKGROUND AND PURPOSE - Humanin (HN) is a 24-amino acid peptide best known for its ability to protect neurons from damage caused by Alzheimer disease-related proteins. This study examines the neuroprotective effects of HNG (a potent form of HN) on focal cerebral ischemia/reperfusion injury in mice. METHODS - Mice underwent middle cerebral artery occlusion for 75 minutes followed by 24-hour reperfusion. Mice were pretreated with 0.1 μg HNG (intracerebroventricularly) 30 minutes before ischemia; posttreated at 0, 2, 4, and 6 hours after ischemia; or pretreated with 1 μg HNG (intraperitoneally) 1 hour before ischemia. Neurological deficits and cerebral infarct volume were evaluated. Neuronal apoptosis and activated poly(ADP-ribose) polymerase expression were measured by TUNEL and Western blot analysis, respectively. Activated ERKs were examined by Western blot analysis. RESULTS - Pretreatment with 0.1 μg HNG (intracerebroventricularly) 30 minutes before ischemia reduced cerebral infarct volume from 56.2±3.0% to 26.1±1.4% (P<0.01). HNG posttreatment after 4 hours of reperfusion reduced cerebral infarct volume to 45.6±2.6% (P<0.05). Pretreatment with 1 μg HNG (intraperitoneally) 1 hour before ischemia or posttreatment after 2 hours of reperfusion reduced cerebral infarct volume significantly. HNG also significantly improved neurological function and inhibited both neuronal apoptosis as well as poly(ADP-ribose) polymerase activation. A significant decrease of phospho-ERK was observed in mice treated with HNG, whereas phospho-JNK and phospho-p38 levels were not altered. CONCLUSIONS - Our results demonstrate that HNG protects against cerebral ischemia/reperfusion injury in mice. HNG offers neuroprotection in vivo at least in part by inhibiting ERK activation. These findings suggest a potential therapeutic role for HNG in the treatment of stroke.

Oligodendrocytes are the predominant cell type in white matter and are highly vulnerable to ischemic injury. The role of oligodendrocyte dysfunction in ischemic brain injury is unknown. In this study, we used a 24-amino acid peptide S14G-Humanin (HNG) to examine oligodendrogenesis and neurological functional recovery in a hypoxic/ischemic (H/I) neonatal model. Intraperitoneal HNG pre-treatment decreased infarct volume following H/I injury. Delayed HNG treatment 24 h after H/I injury did not reduce infarct volume but did decrease neurological deficits and brain atrophy. Delayed HNG treatment did not attenuate axonal demyelination at 48 h after H/I injury. However, at 14 d after H/I injury, delayed HNG treatment increased axonal remyelination, the thickness of corpus callosum at the midline, the number of Olig2+/BrdU+ cells, and levels of brain-derived neurotrophic factor (BDNF). Our results suggest that targeting oligodendrogenesis via delayed HNG treatment may represent a promising approach for the treatment of stroke.

Stroke is the third leading cause of death and disability in the United States. At present, intravenous administration of tissue plasminogen activator (t-PA) is the only thrombolytic therapy approved by the FDA for the treatment of acute ischemic stroke. There are no other effective treatments available so far. The discovery of new drugs and new treatments for stroke to reduce mortality and disability is an urgent medical research priority. In this study, the protective effects and mechanisms of two novel agents Gly14 humanin (HNG) and necrostatin-1 (Nec-1) were examined. HNG, a highly potent neuropeptide against amyloid toxicity, exhibited anti-apoptotic properties on cerebral ischemia injury. HNG reduced infarct volume after ischemia/reperfusion injury with pre-treatment or post-treatment (i.c.v. and i.p.) in a middle cerebral artery occlusion model in mice and decreased neurological deficits induced by ischemia. The protection of HNG was mediated by inhibiting ERK activation and activating PI3K/Akt pathway. Inhibition of the PI3K/Akt pathway blocked the protective effects of HNG. Nec-1 is a specific inhibitor of necroptosis, a newly identified cell death, and was reported to reduce infarct volume even when it was administered at 6 h post-ischemia in a mouse stroke model. Interestingly, this small molecule protected against glutamate-induced oxidative toxicity in a hippocampal HT-22 cell line. It inhibited the translocation of apoptosis-inducing factor from the mitochondria to the nucleus, increased the cellular glutathione level, and decreased free radical formation after glutamate treatment. More importantly, Nec-1 inhibited BNIP3-mediated caspase-independent cell death. Cerebral ischemia/reperfusion injury involves the activation of different pathways that lead to neuronal cell death. Given this multifactorial pathnogenesis, it is possible that a cocktail of neuroprotective agents would be superior to monotherapy. In this study, a cocktail of HNG and Nec-1 was examined in vitro and in vivo. HNG and Nec-1 exerted synergistic neuroprotection on oxygen-glucose deprivation-induced cell death and cerebral ischemia/reperfusion injury. This study provided a new therapeutic strategy for the treatment of stroke by the combination of anti-apoptosis and anti-necroptosis therapy.

Les mitochondries, organelles d’origine bactérienne, sont trouvées dans les cellules de presque tous les organismes eucaryotes. Elles exercent des rôles centraux dans les fonctions cellulaires tels que la production d’énergie, la signalisation cellulaire et l’apoptose et ont aussi un impact sur le vieillissement ainsi que certains cancers et maladies neurodégénératives. Chez l’humain et les mammifères en général, le génome mitochondrial est une molécule d’ADN double brin circulaire composée de 37 gènes. Seulement 13 de ces gènes codent des protéines mitochondriales et les 24 autres produisent 22 ARNt (ARN de transfert) et 2 ARNr (ARN ribosomal) qui sont nécessaires à la traduction des 13 protéines mitochondriales. L’ADNmt (ADN mitochondrial) étant très compact, ceci suggère qu’il y a peu de possibilités pour des nouveautés évolutives. Cependant, de récentes recherches ont permis de révéler la présence de près d’une dizaine de petits ORF (cadres de lecture ouverts) fonctionnels à l’intérieur des gènes mitochondriaux 12S ARNr et 16S ARNr. Ceci remet en question la complexité du génome mitochondrial et montre que son potentiel codant a été sous-estimé. Une analyse approfondie du génome mitochondrial humain a révélé la présence de 227 séquences potentiellement traduites en protéines mitochondriales à travers l’ensemble du génome. Dans cette étude, nous avons sélectionné 9 de ces 227 séquences afin de déterminer si effectivement, elles produisent un peptide identifiable. Pour ce faire, des expériences d’immunobuvardage, d’immunofluorescence et d’immunoprécipitation ont été réalisées sur des cellules HeLa et des cellules HEK293T. Ces expériences ont permis d’identifier une protéine mitochondriale alternative nommée MTALTND4 dont la séquence codante est trouvée à l’intérieur du gène nd4, dans un cadre de lecture alternatif. MTALTND4 est traduite dans la mitochondrie et peut être exportée dans le cytoplasme ainsi qu’à l’extérieur de la cellule puisqu’elle a été retrouvée dans le plasma humain. Bien que la fonction de cette protéine n’ait pas encore été confirmée, des résultats préliminaires indiquent qu’elle a un impact sur la respiration cellulaire. MTALTND4 diminue la respiration mitochondriale et nos résultats suggèrent que son action serait induite par l’hypoxie. La découverte de ce nouveau gène mitochondrial humain confirme que le potentiel codant du génome mitochondrial est beaucoup plus vaste que ce que nous croyions. Il existe fort probablement encore plusieurs autres protéines mitochondriales dont les effets pourraient se révéler d’une grande importance. En effet, plusieurs des protéines dérivées du génome mitochondrial découvertes à ce jour ont des impacts majeurs au niveau du métabolisme et pourraient agir en tant que molécules thérapeutiques importantes. Nos résultats amènent à repenser l’évolution et les pressions de sélection exercées sur le génome mitochondrial et ouvrent la porte à de nombreuses recherches futures qui permettront de re-caractériser le génome mitochondrial et d’avoir une compréhension encore plus approfondie du rôle des mitochondries dans les fonctions cellulaires. / Mitochondria, organelles of bacterial origin, are found in almost every eukaryotic organism and play a central role in cellular functions such as energy production, cellular signaling and apoptosis and are also known to have an impact on aging, certain cancers and neurodegenerative diseases. In humans and mammals in general, the mitochondrial DNA is a small double-stranded circular molecule coding for only 37 genes. Only 13 of them code for mitochondrial proteins and the other 24 genes produce 22 tRNAs (transfer RNA) and 2 rRNAs (ribosomal RNA) necessary for the translation of the 13 protein coding genes. The extremely compact nature of mtDNA (mitochondrial DNA) suggests that there is little room for evolutionary novelties. However, recent research revealed the presence of about ten small functional ORFs inside the mitochondrial genes 12S rRNA and 16S rRNA. This calls into question the complexity of the mitochondrial genome and shows that its coding potential has been greatly underestimated. A thorough examination of the human mitochondrial genome revealed the presence of 227 sequences potentially translated into mitochondrial alternative proteins across the entire genome. In this study, we selected 9 of the 227 sequences to determine if they indeed produce identifiable peptides. This was done by immunoblotting, immunofluorescence and immunoprecipitation experiments on HeLa and HEK293T cells. These experiments allowed us to identify one alternative protein named MTALTND4 whose coding sequence is found inside the nd4 gene, in an alternative sequence. MTALTND4 is translated inside the mitochondria and can be exported in the cytoplasm as well as outside the cell since it has been found in human plasma. Although the function of this protein has not yet been confirmed preliminary results indicate its impact on cellular respiration. MTALTND4 decreases mitochondrial respiration and our results suggest that its action could be induced by hypoxia. The discovery of this new human mitochondrial gene confirms that the coding potential of the mitochondrial genome is much larger than we thought. There are most likely still many other mitochondrial proteins whose effects could prove to be of great importance. Indeed, several of the mitochondrial derived proteins discovered to date have major impacts on metabolism and could act as important therapeutic molecules. Our results lead to rethink the evolution and the selection pressures exerted on the mitochondrial genome and open the door to many future researches which will allow to re-characterize the mitochondrial genome and to have an even deeper understanding of the role of mitochondria in cellular functions.

Chez les cellules eucaryotes, la mitochondrie est une organelle impliquée dans plusieurs fonctions cellulaires (production d’énergie, apoptose, production de ROS, prolifération, signalisation cellulaire, vieillissement, immunité et plus encore) et possédant son propre génome, soit l’ADN mitochondrial (ADNmt). Chez l’humain, on croyait que l’ADNmt ne codait que pour 37 gènes impliqués dans la production d’énergie et la traduction mitochondriale. Cependant, le potentiel codant du génome mitochondrial aurait été sous-estimé. Il a récemment été démontré qu’à l’intérieur de ces principaux gènes connus pouvaient se cacher plusieurs petits gènes alternatifs. Ceux-ci se retrouvent au sein de régions non codantes ou possèdent des séquences d’initiation ou de terminaison de la traduction distinctes de celles du gène de référence dans lequel ils se retrouvent. Ils codent pour des micropeptides dérivés des mitochondries qui possèdent un large éventail de fonctions, s’ajoutant à la longue liste de fonctions dans lesquelles la mitochondrie est déjà impliquée. Parmi ces peptides, on retrouve l’Humanine, MOTS-c, SHLP1-6, Gau et SHMOOSE. Nous avons précédemment découvert un nouveau gène alternatif situé dans le gène nd4, nommé mtaltnd4. Dans cette étude, nous visions à clarifier les fonctions du peptide alternatif correspondant MTALTND4 en étudiant son patron d’expression dans les tissus humains, l’impact de plusieurs stress sur son expression, l’impact du peptide sur la transcription des gènes, et ses partenaires d’interaction. Nous avons découvert que MTALTND4 pourrait être une molécule de signalisation sécrétée par les cellules en réponse au stress et affectant la physiologie pour induire un état de dépression bioénergétique en réduisant les processus de production et de demande en ATP. Plusieurs autres indices révélés par nos expériences suggèrent que MTALTND4 pourrait être une protéine multifonctionnelle impliquée dans de nombreuses voies de régulation. / In eukaryotic cells, mitochondria are organelles involved in many cellular functions (energy production, apoptosis, ROS production, proliferation, cell signaling aging, immunity and more) and that possess their own genome, namely mitochondrial DNA (mtDNA). In humans, mtDNA was believed to encode only 37 genes involved in energy production and mitochondrial translation. However, the coding potential of the mitochondrial genome has been underestimated. It has recently been shown that within these main genes could hide several small alternative genes (i.e., genes withing non-coding regions or with translation initiation/termination sequences that are distinct from the reference gene sequences in which they are found). They code for mitochondrial-derived micropeptides (MDPs) that have a broad spectrum of functions, adding to the extensive list of functions in which mitochondria are already involved. These peptides include Humanine, MOTS-c, SHLP1-6, Gau and SHMOOSE. We have previously discovered a new alternative gene located in the nd4 gene, termed mtaltnd4. In this study, we aim to clarify the functions of the corresponding alternative peptide MTALTND4 by studying its expression pattern in human tissues, the impact of several stresses on its expression, the impact of the peptide on gene transcription, and its interaction partners. We have found that MTALTND4 could be a signaling molecule secreted by cells in response to stress and would affect physiology to induce a state of bioenergetic depression by reducing ATP-producing and ATP-demanding processes. Several other clues revealed by our experiments suggest that MTALTND4 could be a multifunctional protein involved in numerous regulatory pathways.