Mitochondrial dynamics, elimination and biogenesis during post-ischemic recovery in ischemia-resistant and ischemia-vulnerable gerbil hippocampal regions.

Transient ischemic attacks (TIA) result from a temporary blockage in blood circulation in the brain. As TIAs cause disabilities and often precede full-scale strokes, the effects of TIA are investigated to develop neuroprotective therapies. We analyzed changes in mitochondrial network dynamics, mitophagy and biogenesis in sections of gerbil hippocampus characterized by a different neuronal survival rate after 5-minute ischemia-reperfusion (I/R) insult. Our research revealed a significantly greater mtDNA/nDNA ratio in CA2-3, DG hippocampal regions (5.8 ± 1.4 vs 3.6 ± 0.8 in CA1) that corresponded to a neuronal resistance to I/R. During reperfusion, an increase of pro-fission (phospho-Ser616-Drp1/Drp1) and pro-fusion proteins (1.6 ± 0.5 and 1.4 ± 0.3 for Mfn2 and Opa1, respectively) was observed in CA2-3, DG. Selective autophagy markers, PINK1 and SQSTM1/p62, were elevated 24-96 h after I/R and accompanied by significant elevation of transcription factors proteins PGC-1α and Nrf1 (1.2 ± 0.4, 1.78 ± 0.6, respectively) and increased respiratory chain proteins (e.g., 1.5 ± 0.3 for complex IV at I/R 96 h). Contrastingly, decreased enzymatic activity of citrate synthase, reduced Hsp60 protein level and electron transport chain subunits (0.88 ± 0.03, 0.74 ± 0.1 and 0.71 ± 0.1 for complex IV at I/R 96 h, respectively) were observed in I/R-vulnerable CA1. The phospho-Ser616-Drp1/Drp1 was increased while Mfn2 and total Opa1 reduced to 0.88 ± 0.1 and 0.77 ± 0.17, respectively. General autophagy, measured as LC3-II/I ratio, was activated 3 h after reperfusion reaching 2.37 ± 0.9 of control. This study demonstrated that enhanced mitochondrial fusion, followed by late and selective mitophagy and mitochondrial biogenesis might together contribute to reduced susceptibility to TIA.

Mitofusin 2 Integrates Mitochondrial Network Remodelling, Mitophagy and Renewal of Respiratory Chain Proteins in Neurons after Oxygen and Glucose Deprivation.

In attempts to develop effective therapeutic strategies to limit post-ischemic injury, mitochondria emerge as a key element determining neuronal fate. Mitochondrial damage can be alleviated by various mechanisms including mitochondrial network remodelling, mitochondrial elimination and mitochondrial protein biogenesis. However, the mechanisms regulating relationships between these phenomena are poorly understood. We hypothesized that mitofusin 2 (Mfn2), a mitochondrial GTPase involved in mitochondrial fusion, mitochondria trafficking and mitochondria and endoplasmic reticulum (ER) tethering, may act as one of linking and regulatory factors in neurons following ischemic insult. To verify this assumption, we performed temporal oxygen and glucose deprivation (OGD/R) on rat cortical primary culture to determine whether Mfn2 protein reduction affected the onset of mitophagy, subsequent mitochondrial biogenesis and thus neuronal survival. We found that Mfn2 knockdown increased neuronal susceptibility to OGD/R, prevented mitochondrial network remodelling and resulted in prolonged mitophagosomes formation in response to the insult. Next, Mfn2 knockdown was observed to be accompanied by reduced Parkin protein levels and increased Parkin accumulation on mitochondria. As for wild-type neurons, OGD/R insult was followed by an elevated mtDNA content and an increase in respiratory chain proteins. Neither of these phenomena were observed for Mfn2 knockdown neurons. Collectively, our findings showed that Mfn2 in neurons affected their response to mild and transient OGD stress, balancing the extent of defective mitochondria elimination and positively influencing mitochondrial respiratory protein levels. Our study suggests that Mfn2 is one of essential elements for neuronal response to ischemic insult, necessary for neuronal survival.

ECTV Abolishes the Ability of GM-BM Cells to Stimulate Allogeneic CD4 T Cells in a Mouse Strain-Independent Manner.

Ectromelia virus (ECTV) is the etiological agent of mousepox, an acute and systemic disease with high mortality rates in susceptible strains of mice. Resistance and susceptibility to mousepox are triggered by the dichotomous T-helper (Th) immune response generated in infected animals, with strong protective Th1 or nonprotective Th2 profile, respectively. Th1/Th2 balance is influenced by dendritic cells (DCs), which were shown to differ in their ability to polarize naïve CD4+ T cells in different mouse strains. Therefore, we have studied the inner-strain differences in the ability of conventional DCs (cDCs), generated from resistant (C57BL/6) and susceptible (BALB/c) mice, to stimulate proliferation and activation of Th cells upon ECTV infection. We found that ECTV infection of GM-CSF-derived bone marrow (GM-BM) cells, composed of cDCs and macrophages, affected initiation of allogeneic CD4+ T cells proliferation in a mouse strain-independent manner. Moreover, infected GM-BM cells from both mouse strains failed to induce and even inhibited the production of Th1 (IFN-γ and IL-2), Th2 (IL-4 and IL-10) and Th17 (IL-17A) cytokines by allogeneic CD4+ T cells. These results indicate that in in vitro conditions ECTV compromises the ability of cDCs to initiate/polarize adaptive antiviral immune response independently of the host strain resistance/susceptibility to lethal infection.