SLAMF1-derived peptide exhibits cardio protection after permanent left anterior descending artery ligation in mice.

Acute myocardial infarction (MI) results in tissue damage to affected areas of the myocardium. The initial inflammatory response is the most damaging for residual cardiac function, while at later stages inflammation is a prerequisite for proper healing and scar formation. Balancing the extent and duration of inflammation during various stages after MI is thus pivotal for preserving cardiac function. Recently, a signaling lymphocytic activation molecule 1 (SLAMF1)-derived peptide (P7) was shown to reduce the secretion of inflammatory cytokines and protected against acute lipopolysaccharide-induced death in mice. In the present study, we experimentally induced MI by permanent ligation of the left anterior descending artery (LAD) in mice and explored the beneficial effect of immediately administering P7, with the aim of dampening the initial inflammatory phase without compromising the healing and remodeling phase. Blood samples taken 9 h post-LAD surgery and P7 administration dampened the secretion of inflammatory cytokines, but this dampening effect of P7 was diminished after 3 days. Echocardiography revealed less deterioration of cardiac contraction in mice receiving P7. In line with this, less myocardial damage was observed histologically in P7-treated mice. In conclusion, the administration of a SLAMF1-derived peptide (P7) immediately after induction of MI reduces the initial myocardial inflammation, reduces infarct expansion, and leads to less deterioration of cardiac contraction.

PINK1 Content in Mitochondria is Regulated by ER-Associated Degradation.

Maintaining a pool of functional mitochondria requires degradation of damaged ones within the cell. PINK1 is critical in this quality-control process: loss of mitochondrial membrane potential causes PINK1 to accumulate on the mitochondrial surface, triggering mitophagy. However, little is known about how PINK1 is regulated. Recently, we showed that PINK1 content is kept low in healthy mitochondria by continuous ubiquitination and proteasomal degradation of its mature form via a mechanism inconsistent with the proposed N-end rule process. Using both human female and monkey cell lines, we now demonstrate that once generated within the mitochondria, 52 kDa PINK1 adopts a mitochondrial topology most consistent with it being at the mitochondrial-endoplasmic reticulum (ER) interface. From this particular submitochondrial location, PINK1 interacts with components of the ER-associated degradation pathway, such as the E3 ligases gp78 and HRD1, which cooperate to catalyze PINK1 ubiquitination. The valosin-containing protein and its cofactor, UFD1, then target ubiquitinated PINK1 for proteasomal degradation. Our data show that PINK1 in healthy mitochondria is negatively regulated via an interplay between mitochondria and ER, and shed light on how this mitochondrial protein gains access to the proteasome.SIGNIFICANCE STATEMENT Regulation of mitochondrial content of PINK1, a contributor to mitophagy, is an important area of research. Recently, we found that PINK1 content is kept low in healthy mitochondria by continuous ubiquitination and proteasomal degradation. We now extend and refine this novel finding by showing that PINK1 localizes at the mitochondrial-endoplasmic reticulum (ER) interface, from where it interacts with the ER-associated degradation machinery, which catalyzes its ubiquitination and transfer to the proteasome. Thus, these data show that PINK1 in healthy mitochondria is negatively regulated via a mitochondria and ER interplay, and how this mitochondrial protein gains access to the proteasome.

Lactate receptor sites link neurotransmission, neurovascular coupling, and brain energy metabolism.

The G-protein-coupled lactate receptor, GPR81 (HCA1), is known to promote lipid storage in adipocytes by downregulating cAMP levels. Here, we show that GPR81 is also present in the mammalian brain, including regions of the cerebral neocortex and hippocampus, where it can be activated by physiological concentrations of lactate and by the specific GPR81 agonist 3,5-dihydroxybenzoate to reduce cAMP. Cerebral GPR81 is concentrated on the synaptic membranes of excitatory synapses, with a postsynaptic predominance. GPR81 is also enriched at the blood-brain-barrier: the GPR81 densities at endothelial cell membranes are about twice the GPR81 density at membranes of perivascular astrocytic processes, but about one-seventh of that on synaptic membranes. There is only a slight signal in perisynaptic processes of astrocytes. In synaptic spines, as well as in adipocytes, GPR81 immunoreactivity is located on subplasmalemmal vesicular organelles, suggesting trafficking of the protein to and from the plasma membrane. The results indicate roles of lactate in brain signaling, including a neuronal glucose and glycogen saving response to the supply of lactate. We propose that lactate, through activation of GPR81 receptors, can act as a volume transmitter that links neuronal activity, cerebral energy metabolism and energy substrate availability.

Alterations of monocarboxylate transporter densities during hypoxia in brain and breast tumour cells.

BACKGROUND: Tumour cells are characterized by aerobic glycolysis, which provides biomass for tumour proliferation and leads to extracellular acidification through efflux of lactate via monocarboxylate transporters (MCTs). Deficient and spasm-prone tumour vasculature causes variable hypoxia, which favours tumour cell survival and metastases. Brain metastases frequently occur in patients with advanced breast cancer.Effective treatment strategies are therefore needed against brain metastasis from breast carcinoma. MATERIAL AND METHODS: In order to identify differences in the capacity for lactate exchange, human T-47D breast cancer cells and human glioblastoma T98G cells were grown under 4 % or 20 % oxygen conditions and examined for MCT1, MCT2 and MCT4 expression on plasma membranes by quantitative post embedding immunogold electron microscopy. Whereas previous studies on MCT expression in tumours have recorded mRNA and protein levels in cell extracts, we examined concentrations of the proteins in the microvillous plasma membrane protrusions specialized for transmembrane transport. RESULTS: In normoxia, both tumour cell types highly expressed the low affinity transporter MCT4, which is thought to mainly mediate monocarboxylate efflux, while for high affinity transport the breast tumour cells preferentially expressed MCT1 and the brain tumour cells resembled brain neurons in expressing MCT2, rather than MCT1. The expressions of MCT1 and MCT4 were upregulated in hypoxic conditions in both breast and brain tumour cells. The expression of MCT2 also increased in hypoxic breast cancer cells, but decreased in hypoxic brain tumour cells. Quantitative immunoblots showed similar hypoxia induced changes in the protein levels. CONCLUSION: The differential expression and regulation of MCTs in the surface membranes of hypoxic and normoxic tumour cells of different types provide a foundation for innovation in tumour therapy through the selective targeting of MCTs. Selective inhibition of various MCTs could be an efficient way to quench an important energy source in both original breast tumour and metastatic cancer tissue in the brain.

Modeling the impact of mitochondrial DNA damage in forebrain neurons and beyond.

We have generated an inducible transgenic mouse model, which expresses a mutated version of UNG1 (mutUNG1) that removes thymine, in addition to uracil from mitochondrial DNA. The abasic-sites (AP-sites) generated by removal of thymine or uracil are a threat to genomic integrity, and are particularly harmful in mitochondria due to inhibition of mitochondrial DNA polymerase. MutUNG1, accompanied by a luciferase reporter-gene, is controlled by the Tet-on system. Transgene expression is spatially regulated by the forebrain specific CaMKIIα-promoter, and temporally by the addition of doxycycline. Mice harboring this transgene develop compromised mitochondrial dynamics, neurodegeneration and impaired behavior.

Repair deficient mice reveal mABH2 as the primary oxidative demethylase for repairing 1meA and 3meC lesions in DNA.

Two human homologs of the Escherichia coli AlkB protein, denoted hABH2 and hABH3, were recently shown to directly reverse 1-methyladenine (1meA) and 3-methylcytosine (3meC) damages in DNA. We demonstrate that mice lacking functional mABH2 or mABH3 genes, or both, are viable and without overt phenotypes. Neither were histopathological changes observed in the gene-targeted mice. However, in the absence of any exogenous exposure to methylating agents, mice lacking mABH2, but not mABH3 defective mice, accumulate significant levels of 1meA in the genome, suggesting the presence of a biologically relevant endogenous source of methylating agent. Furthermore, embryonal fibroblasts from mABH2-deficient mice are unable to remove methyl methane sulfate (MMS)-induced 1meA from genomic DNA and display increased cytotoxicity after MMS exposure. In agreement with these results, we found that in vitro repair of 1meA and 3meC in double-stranded DNA by nuclear extracts depended primarily, if not solely, on mABH2. Our data suggest that mABH2 and mABH3 have different roles in the defense against alkylating agents.