Monocytes/macrophages (MMs), mononuclear phagocytes, have been implicated in stroke-induced inflammation and injury. However, the presence of pro-inflammatory Ly-6Chigh and antiinflammatory Ly-6Clow monocyte subsets raises uncertainty regarding their role in stroke pathologic assessment. With recent identification of the spleen as an immediate reservoir of MMs, this current study addresses whether the spleen-derived MMs are required for stroke pathologic assessment. We observed that the spleen was contracted in poststroke animals and the contraction was accompanied by decreased number of Ly-6Chigh and Ly-6Clow subsets in the spleen. The deployment of these subsets from the spleen temporally coincided with respective increases in the ischemic brain. Compared to mice with the spleen, mice receiving a splenectomy just before the stroke displayed less accumulation of Ly-6Chigh and Ly-6Clow MMs in the brain. Despite the reduced accumulation of both subsets, infarct size and swelling were not reduced in the asplenic mice. The dissociative findings of infarct size and extent of MM infiltration in the postischemic brain indicate minimal involvement of spleen-derived total MMs in acute infarct development. Selective Ly-6Chigh or Ly-6Clow MM targeting is suggested to address the contribution of the individual subset to acute stroke pathologic assessment.

Infiltrating monocyte-derived macrophages (M-M

Brain injury from stroke is typically considered an event exclusive to the CNS, but injury progression and repair processes are profoundly influenced by peripheral immunity. Stroke stimulates an acute inflammatory response that results in a massive infiltration of peripheral immune cells into the ischemic area. While these cells contribute to the development of brain injury, their recruitment has been considered as a key step for tissue repair. The paradoxical role of inflammatory monocytes in stroke raises the possibility that the manipulation of peripheral immune cells before infiltration into the brain could influence stroke outcome. One such manipulation is remote ischemic limb conditioning (RLC), which triggers an endogenous tolerance mechanism. We observed that mice subjected to poststroke RLC shifted circulating monocytes to a CCR2 ϩ proinflammatory monocyte subset and had reduced acute brain injury, swelling, and improved motor/gait function in chronic stroke. The RLC benefits were observed regardless of injury severity, with a greater shift to a CCR2 ϩ subset in severe stroke. Adoptive transfer of CCR2-deficient monocytes abolished RLC-mediated protection. The study demonstrates the importance of RLC-induced shift of monocytes to a CCR2 ϩ proinflammatory subset in attenuating acute injury and promoting functional recovery in chronic stroke. The defined immune-mediated mechanism underlying RLC benefits allows for an evidence-based framework for the development of immune-based therapeutic strategies for stroke patients.

Running title: CD36 inhibition improves metabolic dysfunction in obese miceKey words: CD36, diabetes, insulin resistance, obese, salvianolic acid B AbstractObesity-induced metabolic dysfunctions increase risk of vascular diseases including type II diabetes and stroke. While managing obesity is an interest to address the worldwide health problem, how genetic variability affects human obesity development and specific targets for obesity-related metabolic disease have not been thoroughly studied. A single nucleotide polymorphism (SNP) in the brain derived neurotropic factor (BDNF) gene that results in the substitution of a valine to a methionine at codon 66 (val66met) occurs with a high frequency in humans. This study addressed the effect of genetic variability in developing obesity and the efficacy of the inhibition of CD36, a multifunctional receptor implicated in obesity and insulin resistance, in wild-type mice and mice with the bdnf val66met variant. CD36 inhibition by salvionolic acid B (SAB) in diet-induced obese wild-type mice reduced visceral fat accumulation and improved insulin resistance. The benefit of SAB was aborted in CD36 knockout mice, showing the specificity of SAB. In addition, mice with the val66met variant in both alleles (BDNF M/M ) fed high-fat diet exhibited extreme obesity with increased CD36 gene and protein levels in macrophages. Chronic SAB treatment in BDNF M/M mice significantly decreased visceral fat accumulation and improved insulin resistance. Notably, the effect of SAB was greater in the extreme obese BDNF M/M mice compared to the wild-type mice. The study demonstrated a link between bdnf val66met and elevated CD36 expression, and suggested that CD36 inhibition may be a novel strategy to improve metabolic dysfunctions and to normalize risk factors for vascular diseases in the obese population.

In light of repeated translational failures with preclinical neuroprotection-based strategies, this preclinical study reevaluates brain swelling as an important pathological event in diabetic stroke and investigates underlying mechanism of the comorbidity-enhanced brain edema formation. Type 2 (mild), type 1 (moderate), and mixed type 1/2 (severe) diabetic mice were subjected to transient focal ischemia. Infarct volume, brain swelling, and IgG extravasation were assessed at 3 days post-stroke. Expression of vascular endothelial growth factor (VEGF)-A, endothelial-specific molecule-1 (Esm1), and the VEGF receptor 2 (VEGFR2) was determined in the ischemic brain. Additionally, SU5416, a VEGFR2 inhibitor, was treated in the type 1/2 diabetic mice, and stroke outcomes were determined. All diabetic groups displayed bigger infarct volume and brain swelling compared to nondiabetic mice, and the increased swelling was disproportionately larger relative to infarct enlargement. Diabetic conditions significantly increased VEGF-A, Esm1, and VEGFR2 expressions in the ischemic brain compared to nondiabetic mice. Notably, in diabetic mice, VEGFR2 mRNA levels were positively correlated with brain swelling, but not with infarct volume. Treatment with SU5416 in diabetic mice significantly reduced brain swelling. The study shows that brain swelling is a predominant pathological event in diabetic stroke and that an underlying event for diabetes-enhanced brain swelling includes the activation of VEGF signaling. This study suggests consideration of stroke therapies aiming at primarily reducing brain swelling for subjects with diabetes.

Brain activities of the mitochondrial enzyme α-ketoglutarate dehydrogenase complex (KGDHC) are reduced in Alzheimer’s disease and other age-related neurodegenerative disorders. The goal of the present study was to test the consequences of mild impairment of KGDHC on the structure, protein signaling and dynamics (mitophagy, fusion, fission, biogenesis) of the mitochondria. Inhibition of KGDHC reduced its in situ activity by 23–53% in human neuroblastoma SH-SY5Y cells, but neither altered the mitochondrial membrane potential nor the ATP levels at any tested time-points. The attenuated KGDHC activity increased translocation of dynamin-related protein-1 (Drp1) and microtubule-associated protein 1A/1B-light chain 3 (LC3) from the cytosol to the mitochondria, and promoted mitochondrial cytochrome c release. Inhibition of KGDHC also increased the negative surface charges (anionic phospholipids as assessed by Annexin V binding) on the mitochondria. Morphological assessments of the mitochondria revealed increased fission and mitophagy. Taken together, our results suggest the existence of the regulation of the mitochondrial dynamism including fission and fusion by the mitochondrial KGDHC activity via the involvement of the cytosolic and mitochondrial protein signaling molecules. A better understanding of the link among mild impairment of metabolism, induction of mitophagy/autophagy and altered protein signaling will help to identify new mechanisms of neurodegeneration and reveal potential new therapeutic approaches.

No abstract

Discoba (Excavata) is an evolutionarily important group of eukaryotes that includes Jakobida, with the most bacterial-like mitochondrial genomes known, and Euglenozoa, many of which have extensively fragmented mitochondrial genomes. However, little is known about the mitochondrial genomes of Heterolobosea, the third main group of Discoba. Here, we studied two heteroloboseids—an undescribed amoeba “BB2” and Pharyngomonas kirbyi. Phylogenomic analysis revealed that they form a clade that is a sister group to all other Heterolobosea. We characterized the mitochondrial genomes of BB2 and P. kirbyi, which encoded 44 and 48 putative protein-coding genes respectively. Their gene contents were similar to that of Naegleria. In BB2, mitochondrially encoded RNAs were heavily edited, with ∼500 mononucleotide insertion events, mostly guanosines. These insertions always have the same identity as an adjacent nucleotide. Editing occurs in all ribosomal RNAs and protein-coding transcripts except one, and half of the transfer RNAs. Analysis of Illumina deep-sequencing data suggested that this RNA editing is very accurate and efficient, and most likely co-transcriptional. The dissimilarity of this editing process to other RNA editing phenomena in discobids, as well as its apparent absence in P. kirbyi, suggest that this remarkably extensive system of insertional editing evolved independently in the BB2 lineage, after its divergence from the P. kirbyi lineage.