Inhibition of N-Methyl-D-aspartate-induced Retinal Neuronal Death by Polyarginine Peptides Is Linked to the Attenuation of Stress-induced Hyperpolarization of the Inner Mitochondrial Membrane Potential

It is widely accepted that overactivation of NMDA receptors, resulting in calcium overload and consequent mitochondrial dysfunction in retinal ganglion neurons, plays a significant role in promoting neurodegenerative disorders such as glaucoma. Calcium has been shown to initiate a transient hyperpolarization of the mitochondrial membrane potential triggering a burst of reactive oxygen species leading to apoptosis. Strategies that enhance cell survival signaling pathways aimed at preventing this adverse hyperpolarization of the mitochondrial membrane potential may provide a novel therapeutic intervention in retinal disease. In the retina, brain-derived neurotrophic factor has been shown to be neuroprotective, and our group previously reported a PSD-95/PDZ-binding cyclic peptide (CN2097) that augments brain-derived neurotrophic factor-induced pro-survival signaling. Here, we examined the neuroprotective properties of CN2097 using an established retinal in vivo NMDA toxicity model. CN2097 completely attenuated NMDA-induced caspase 3-dependent and -independent cell death and PARP-1 activation pathways, blocked necrosis, and fully prevented the loss of long term ganglion cell viability. Although neuroprotection was partially dependent upon CN2097 binding to the PDZ domain of PSD-95, our results show that the polyarginine-rich transport moiety C-R(7), linked to the PDZ-PSD-95-binding cyclic peptide, was sufficient to mediate short and long term protection via a mitochondrial targeting mechanism. C-R(7) localized to mitochondria and was found to reduce mitochondrial respiration, mitochondrial membrane hyperpolarization, and the generation of reactive oxygen species, promoting survival of retinal neurons

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: Implications for AMP Kinase

Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc iso- lated from kidneys is phosphorylated on Thr28, leading to a par- tial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing supe- rior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type un- phosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (����m), and ROS levels are reduced compared with wild type. As we show by high resolu- tion crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a cen- tral position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kid- ney, is the most likely candidate to phosphorylate Thr28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissue- specific manner and leads to regulation of electron transport chain flux via “controlled respiration,” preventing ����m hyperpolarization, a known cause of ROS and trigger of apoptosis

Cox4i2, Ifit2, and Prdm11 Mutant Mice:Effective Selection of Genes Predisposing to an Altered Airway Inflammatory Response from a Large Compendium of Mutant Mouse Lines

We established a selection strategy to identify new models for an altered airway inflammatory response from a large compendium of mutant mouse lines that were systemically phenotyped in the German Mouse Clinic (GMC). As selection criteria we included published gene functional data, as well as immunological and transcriptome data from GMC phenotyping screens under standard conditions. Applying these criteria we identified a few from several hundred mutant mouse lines and further characterized the Cox4i2tm1Hutt, Ifit2tm1.1Ebsb, and Prdm11tm1.1ahl lines following ovalbumin (OVA) sensitization and repeated OVA airway challenge. Challenged Prdm11tm1.1ahl mice exhibited changes in B cell counts, CD4+ T cell counts, and in the number of neutrophils in bronchoalveolar lavages, whereas challenged Ifit2tm1.1Ebsb mice displayed alterations in plasma IgE, IgG1, IgG3, and IgM levels compared to the challenged wild type littermates. In contrast, challenged Cox4i2tm1Hutt mutant mice did not show alterations in the humoral or cellular immune response compared to challenged wild type mice. Transcriptome analyses from lungs of the challenged mutant mouse lines showed extensive changes in gene expression in Prdm11tm1.1ahl mice. Functional annotations of regulated genes of all three mutant mouse lines were primarily related to inflammation and airway smooth muscle (ASM) remodeling. We were thus able to define an effective selection strategy to identify new candidate genes for the predisposition to an altered airway inflammatory response under OVA challenge conditions. Similar selection strategies may be used for the analysis of additional genotype-envirotype interactions for other diseases

In vivo induction of cytochrome <i>c</i> Tyr97 phosphorylation in rat brain by insulin treatment.

<p>(A) Cyt<i>c</i> was isolated from rat brain after global brain ischemia (lane 1), from untreated control rats (lane 2), from sham-operated animals after insulin treatment (lane 3), and from rat brain after ischemia with insulin treatment (lane 4). Top, Western blot with an anti-phosphotyrosine antibody (4G10) reveals no detectable tyrosine phosphorylation of brain Cyt<i>c</i> under control conditions and after global brain ischemia (lanes 2 and 1, respectively), whereas tyrosine phosphorylation of Cyt<i>c</i> is strongly induced after insulin treatment (lane 3), but slightly reduced by ischemic stress (lane 4). Bottom, Coomassie gel shows equal loading (1 µg per lane) and purity of the isolated Cyt<i>c</i> species. (B) Nano-LC/ESI-MS/MS analysis of rat brain Cyt<i>c</i> after insulin treatment (corresponding to lane 3 in Fig. 5A) unambiguously identifies Tyr97 phosphorylation by fragment ions y3, y4, y5, and y6. The sequence of the peptide was definitively assigned by b3, b4, y2, y3, y4, y5, and y6. (C) Nano-LC/ESI-MS/MS analysis of rat brain Cyt<i>c</i> after global brain ischemia and insulin treatment (corresponding to lane 4 in Fig. 5A) unambiguously identifies Tyr97 phosphorylation by fragment ions y3, y4, y6, and y7. The sequence of the peptide was definitively assigned by b3, b4, y3, y4, y6, and y7.</p

Cytochrome <i>c</i> release is inhibited by insulin administration.

<p>(A) Sham operated control animals (Sham, n = 3) show faint detectable amounts of Cyt<i>c</i> in the cytosolic fraction of hippocampal CA1 neurons. After 8 min of ischemia followed by 24 h of reperfusion (R24, n = 5) cytosolic Cyt<i>c</i> increases ∼4 fold (p < 0.05) and there is a trend toward mitochondrial Cyt<i>c</i> decrease (p = 0.08), both indicative of mitochondrial Cyt<i>c</i> release. Animals exposed to 8 min of ischemia followed by 24 h of reperfusion with a single bolus of IV insulin at the onset of reperfusion (T24, n = 5) demonstrate a reduction of cytosolic Cyt<i>c</i> (p < 0.05) and increased mitochondrial Cyt<i>c</i> compared to the R24 controls. (B and C) Densitometric analyses of Western blot data (Mean +/−SEM, *p < 0.05).</p

Insulin prevents neuronal cell death in the CA1 hippocampus following brain ischemia.

<p>Cresyl violet stained sections (top row) show CA1 hippocampus densely populated with pyramidal neurons in sham-operated controls (Sham), and triple-label immunofluorescence (bottom row) shows these cells to be NeuN-positive (red). After 8 min of global brain ischemia followed by 14 days of reperfusion (I/R, n = 5) there is a 90% loss of CA1 neurons and an increase in Iba-1-positive microglia and GFAP-positive astrocytes (green and magenta, respectively). Animals exposed to 8 min of ischemia followed by 14 days of reperfusion with a single bolus of insulin given at the onset of reperfusion (I/R + Ins, n = 4) demonstrate a 49% increase in NeuN-positive neurons (p < 0.05) while Iba-1 and GFAP-positive cells remain unchanged.</p