Mfn2 downregulation in excitotoxicity causes mitochondrial dysfunction and delayed neuronal death.

Mitochondrial fusion and fission is a dynamic process critical for the maintenance of mitochondrial function and cell viability. During excitotoxicity neuronal mitochondria are fragmented, but the mechanism underlying this process is poorly understood. Here, we show that Mfn2 is the only member of the mitochondrial fusion/fission machinery whose expression is reduced in in vitro and in vivo models of excitotoxicity. Whereas in cortical primary cultures, Drp1 recruitment to mitochondria plays a primordial role in mitochondrial fragmentation in an early phase that can be reversed once the insult has ceased, Mfn2 downregulation intervenes in a delayed mitochondrial fragmentation phase that progresses even when the insult has ceased. Downregulation of Mfn2 causes mitochondrial dysfunction, altered calcium homeostasis, and enhanced Bax translocation to mitochondria, resulting in delayed neuronal death. We found that transcription factor MEF2 regulates basal Mfn2 expression in neurons and that excitotoxicity-dependent degradation of MEF2 causes Mfn2 downregulation. Thus, Mfn2 reduction is a late event in excitotoxicity and its targeting may help to reduce excitotoxic damage and increase the currently short therapeutic window in stroke

Mfn2 downregulation in excitotoxicity causes mitochondrial dysfunction and delayed neuronal death

Mitochondrial fusion and fission is a dynamic process critical for the maintenance of mitochondrial function and cell viability. During excitotoxicity neuronal mitochondria are fragmented but the mechanism underlying this process is poorly understood. Here we show that Mfn2 is the only member of the mitochondrial fusion/fission machinery whose expression is reduced in in vitro and in vivo models of excitotoxicity. Whereas in cortical primary cultures Drp1 recruitment to mitochondria plays a primordial role in mitochondrial fragmentation in an early phase that can be reversed once the insult has ceased, Mfn2 downregulation intervenes in a delayed mitochondrial fragmentation phase that progresses even when the insult has ceased. Downregulation of Mfn2 causes mitochondrial dysfunction, altered calcium homeostasis and enhanced Bax translocation to mitochondria, resulting in delayed neuronal death. We found that transcription factor MEF2 regulates basal Mfn2 expression in neurons, and that excitotoxicity-dependent degradation of MEF2 causes Mfn2 downregulation. Thus, Mfn2 reduction is a late event in excitotoxicity and its targeting may help to reduce excitotoxic damage and increase the currently short therapeutic window in stroke

Lactoferrin during lactation protects the immature hypoxic-ischemic rat brain

Lactoferrin (Lf) is an iron-binding glycoprotein secreted in maternal milk presenting anti-inflammatory and antioxidant properties. It shows efficient absorption into the brain from nutritional source. Brain injury frequently resulting from cerebral hypoxia-ischemia (HI) has a high incidence in premature infants with ensuing neurodevelopmental disabilities. We investigated the neuroprotective effect of maternal nutritional supplementation with Lf during lactation in a rat model of preterm HI brain injury using magnetic resonance imaging (MRI), brain gene, and protein expression

Low glucose-induced LC3-II and p62 accumulation is principally due to a defect of autophagosome/lysosome fusion.

<p>661W cells were cultured as mentioned in material & methods, and then incubated at low (1 mM) or high (25 mM) glucose for 48 h in absence or in presence of fusion inhibitors (10 µg/ml Pepstatin/E64 and 75 µM Chloroquine) during the last 4 h. <b>A)</b> Representative western blot analysis of LC3-II protein expression and quantification. Results are expressed as mean ± SEM of 4 experiments, *p<0.03 (1 mM <i>vs.</i> 25 mM without inhibitors). <b>B)</b> Transfection of 661W cells with a lentivirus expressing the GFP-LC3 chimeric protein and incubation for 48 hrs at 1 mM (a-d) and 25 mM (e-h) glucose in absence (a and e) or in presence of 50 nM Bafilomycin (b and f), 10 µg/ml Pepstatine/E64 (c and g) and 75 µM Chloroquine (d and h) for the last 4 h. <b>C)</b> Transfection of 661W cells with a lentivirus vector expressing the mRFP-GFP-LC3 chimeric protein and incubation for 48 h at 1 mM (a–f) and 25 mM (g–l) glucose in absence (a–c and g–i) or in presence (d–f and j–l) of 75 µM chloroquine during the last 4 h.</p

3-MA chemical inhibition of autophagy increases low glucose-induced cell death and caspase 3 activity.

<p>661W cells were cultured as mentioned in material & methods, and then incubated at low (1 mM) or high (25 mM) glucose for different periods of time (8 or 48 h). <b>A)</b> TUNEL assay was performed in absence (a, d) or in presence (b, c and e, f) of 600 µM 3-MA, at 1 mM (a, b, c) or 25 mM (d, e, f) glucose concentrations, for different periods of time as indicated to the left. White arrows show TUNEL positive cells. Quantification of TUNEL positive cells was performed in three different experiments, *p<0.05; #p<0.0001 and **p<0.002 <b>B)</b> Measures of Caspase 3 activity, results are expressed as mean ± SEM of 3 experiments (n = 13), *p<0.04 and #p<0.0002, and immunostaining of cleaved Caspase 3 in 661W cells incubated at 1 mM (a and b) or 25 mM (c and d) glucose concentrations in absence (a and c) or presence of 600 µM 3-MA (b and d).</p

Low glucose induces 661W cell death via the BCL2/BAX pathway.

<p>661W cells were cultured as mentioned in material & methods and then incubated at 1 mM glucose for different time periods (2, 4, 8, 24 and 48 h) or for 48 h at high (25 mM) glucose concentrations. <b>A)</b> Graphic representation of <i>Bcl2</i>, <i>Bcl-XL</i> and <i>Bax</i> mRNA expression normalized by housekeeping genes. Results are expressed as mean ± SEM of 2 experiments performed in triplicate. <b>B)</b> Western blot of BCL2, BCL-XL and BAX proteins after exposure to 1 mM or 25 mM glucose concentration during 24 or 48 h. Protein amounts were normalized with TUBULIN and compared to 25 mM condition set to 100%. Results are expressed as mean ± SEM of 3 experiments, *p<0.040. <b>C)</b> Immunoprecipitation analysis of active BAX from cells exposed to 1 mM or 25 mM glucose during 48 h. Left panel showed the expression of total BAX in whole cell lysates, while right panel showed the expression of immunoprecipitated active BAX (black arrow). Immunofluorescence of active BAX (green) is observed in cells incubated at low glucose during 48 h but not at high glucose conditions.</p

Low glucose induces autophagic flux through AMPK/RAPTOR/mTOR pathway.

<p>661W cells were cultured as mentioned in material & methods, and then incubated at low (1 mM) or high (25 mM) glucose for 24 or 48 h. <b>A)</b> Western blot showed the phosphorylation and the expression of mTOR and is representative of three different experiments. <b>B)</b> Western blot showed the phosphorylation and the expression of AMPK. Quantifications of pAMPK/AMPK ratio are expressed as mean ± SEM of 3 experiments (n = 8), * p<0.035. <b>C)</b> Western blot showed the phosphorylation and the expression of RAPTOR. Oligomycin (Olig) was used at for positive control induction of pRAPTOR. Quantifications of pRAPTOR/TUBULIN ratio are expressed as mean ± SEM of 3 experiments (n = 8), # p<0.019.</p

Specific ATG5 or ATG7 inhibition induces cell death and caspase 3 activity.

<p>661W cells were cultured as mentioned in material & methods. <b>A)</b> Each clonal cell colony was cultured at 1 mM (g, h, i) or 25 mM (j, k, l) glucose for 48 h prior cell death analysis by TUNEL assay. White arrows show TUNEL positive dying cells and quantification is representative of three distinct experiments, *p<0.005; and #p<0.0001. <b>B)</b> Representative western blot and quantification showing cleaved Caspase 3 expression in ATG5 and ATG7 down-regulated clonal cell cultured at 1 mM and 25 mM glucose concentrations. Western blot is representative of three distinct experiments and quantification expressed as 100% of control (25 mM), **p<0.005 and #p<0.0001.</p