Genetic variants of Leucine-Rich Repeat Kinase 2 (LRRK2) are associated with a significantly enhanced risk for Parkinson disease, the second most common human neurodegenerative disorder. Despite major efforts, our understanding of LRRK2 biological function and regulation remains rudimentary. In the present study we analyze LRRK2 mRNA and protein expression in sub-populations of human peripheral blood mononuclear cells (PBMCs). LRRK2 mRNA and protein was found in circulating CD19+ B cells and in CD14+ monocytes, whereas CD4+ and CD8+ T cells were devoid of LRRK2 mRNA. Within CD14+ cells the CD14+CD16+ sub-population of monocytes exhibited high levels of LRRK2 protein, in contrast to CD14+CD16- cells. However both populations expressed LRRK2 mRNA. As CD14+CD16+ cells represent a more mature subset of monocytes, we monitored LRRK2 expression after in vitro treatment with various stress factors known to induce monocyte activation. We found that IFN-γ in particular robustly increased LRRK2 mRNA and protein levels in monocytes concomitant with a shift of CD14+CD16− cells towards CD14+CD16+cells. Interestingly, the recently described LRRK2 inhibitor IN-1 attenuated this shift towards CD14+CD16+ after IFN-γ stimulation. Based on these findings we speculate that LRRK2 might have a role in monocyte maturation. Our results provide further evidence for the emerging role of LRRK2 in immune cells and regulation at the transcriptional and translational level. Our data might also reflect an involvement of peripheral and brain immune cells in the disease course of PD, in line with increasing awareness of the role of the immune system in PD.

It is well established that lactate can be used as an energy substrate by the brain by conversion to pyruvate and a subsequent oxidation in the mitochondria. Knowing the need for readily metabolizable substrates directly after ischemia and the protective effect of lactate after excitotoxicity, the aim of this study was to investigate whether lactate administration directly after ischemia could be neuroprotective. In vitro, the addition of 4 mmol/L L-lactate to the medium of rat organotypic hippocampal slices, directly after oxygen and glucose deprivation (OGD), protected against neuronal death, whereas a higher dose of 20 mmol/L was toxic. In vivo, after middle cerebral artery occlusion in the mouse, an intracerebroventricular injection of 2 microL of 100 mmol/L L-lactate, immediately after reperfusion, led to a significant decrease in lesion size, which was more pronounced in the striatum, and an improvement in neurologic outcome. A later injection 1 h after reperfusion did not reduce lesion size, but significantly improved neurologic outcome, which is an important point in the context of a potential clinical application. Therefore, a moderate increase in lactate after ischemia may be a therapeutic tool.

Medium-chain triglycerides have been used as part of a ketogenic diet effective in reducing epileptic episodes. The health benefits of the derived medium-chain fatty acids (MCFAs) are thought to result from the stimulation of liver ketogenesis providing fuel for the brain. We tested whether MCFAs have direct effects on energy metabolism in induced pluripotent stem cell-derived human astrocytes and neurons. Using single-cell imaging, we observed an acute pronounced reduction of the mitochondrial electrical potential and a concomitant drop of the NAD(P)H signal in astrocytes, but not in neurons. Despite the observed effects on mitochondrial function, MCFAs did not lower intracellular ATP levels or activate the energy sensor AMP-activated protein kinase. ATP concentrations in astrocytes were unaltered, even when blocking the respiratory chain, suggesting compensation through accelerated glycolysis. The MCFA decanoic acid (300 μM) promoted glycolysis and augmented lactate formation by 49.6%. The shorter fatty acid octanoic acid (300 μM) did not affect glycolysis but increased the rates of astrocyte ketogenesis 2.17-fold compared with that of control cells. MCFAs may have brain health benefits through the modulation of astrocyte metabolism leading to activation of shuttle systems that provide fuel to neighboring neurons in the form of lactate and ketone bodies.-Thevenet, J., De Marchi, U., Santo Domingo, J., Christinat, N., Bultot, L., Lefebvre, G., Sakamoto, K., Descombes, P., Masoodi, M., Wiederkehr, A. Medium-chain fatty acids inhibit mitochondrial metabolism in astrocytes promoting astrocyte-neuron lactate and ketone body shuttle systems.

Background and Purpose-In 2 models of severe ischemic injury, we have evaluated the neuroprotective action of D-JNKI1, a cell-penetrating and protease-resistant peptide selectively inhibiting the c-Jun-N-terminal kinase (JNK). Methods-Hippocampal slices from newborn rats were subjected to oxygen (5%) and glucose (1 mmol/L) deprivation for 30 minutes. Cell death was evaluated with propidium iodide, and the evoked potential responses were recorded in the CA1 region after stimulation in CA3. Male ICR-CD1 mice were subjected to permanent endoluminal "suture" middle cerebral artery occlusion (MCAo). The lesion size was determined after 24 hours by triphenyl-tetrazolium chloride staining, and neurological scores and rotarod treadmill performance were used to evaluate the neurological outcome. Results-In vitro, D-JNKI administration 6 hours after oxygen glucose deprivation reduced cell death at 24 hours from 21%Ϯ8% (nϭ10) to 5%Ϯ3% (nϭ7, PϽ0.01). This protective effect was still seen at 48 hours, paralleled by an improved amplitude of the evoked potential response. In vivo in the mouse, D-JNKI1 administration 3 hours after ischemia significantly reduced the infarct volume from 162Ϯ27 mm 3 (nϭ14) to 85Ϯ27 mm 3 (nϭ9, PϽ0.001). The functional outcome was also improved. Conclusions-JNK inhibition prevents cell death induced by oxygen and glucose deprivation in hippocampal slice cultures in vitro and by permanent suture MCAo in vivo. D-JNKI1 is a powerful neuroprotectant in models of both mild and severe cerebral ischemia, with an extended therapeutic window. Further investigations are needed to identify the relevant JNK target(s) mediating ischemic neuronal death.

Background: Nutrients stimulate calcium dependent activation of energy metabolism, in pancreatic beta cells.Results: Glucose-induced ATP synthase-dependent respiration is strictly calcium-dependent, with little or no effect of calcium on the NAD(P)H response.Conclusion: Calcium coordinates oxidative metabolism and respiration in pancreatic beta cells.Significance: Calcium has novel mitochondrial targets downstream of mitochondrial dehydrogenases.

Mitochondrial acyl-coenzyme A species are emerging as important sources of protein modification and damage. Succinyl-CoA ligase (SCL) deficiency causes a mitochondrial encephalomyopathy of unknown pathomechanism. Here, we show that succinyl-CoA accumulates in cells derived from patients with recessive mutations in the tricarboxylic acid cycle (TCA) gene succinyl-CoA ligase subunit-β (SUCLA2), causing global protein hyper-succinylation. Using mass spectrometry, we quantify nearly 1,000 protein succinylation sites on 366 proteins from patient-derived fibroblasts and myotubes. Interestingly, hyper-succinylated proteins are distributed across cellular compartments, and many are known targets of the (NAD+)-dependent desuccinylase SIRT5. To test the contribution of hyper-succinylation to disease progression, we develop a zebrafish model of the SCL deficiency and find that SIRT5 gain-of-function reduces global protein succinylation and improves survival. Thus, increased succinyl-CoA levels contribute to the pathology of SCL deficiency through post-translational modifications.

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Thrombin is involved in mediating neuronal death in cerebral ischemia. We investigated its so far unknown mode of activation in ischemic neural tissue. We used an in vitro approach to distinguish the role of circulating coagulation factors from endogenous cerebral mechanisms. We modeled ischemic stroke by subjecting rat organotypic hippocampal slice cultures to 30‐min oxygen (5%) and glucose (1 mmol/L) deprivation (OGD). Perinuclear activated factor X (FXa) immunoreactivity was observed in CA1 neurons after OGD. Selective FXa inhibition by fondaparinux during and after OGD significantly reduced neuronal death in the CA1 after 48 h. Thrombin enzyme activity was increased in the medium 24 h after OGD and this increase was prevented by fondaparinux suggesting that FXa catalyzes the conversion of prothrombin to thrombin in neural tissue after ischemia in vitro. Treatment with SCH79797, a selective antagonist of the thrombin receptor protease‐activated receptor‐1 (PAR‐1), significantly decreased neuronal cell death indicating that thrombin signals ischemic damage via PAR‐1. The c‐Jun N‐terminal kinase (JNK) pathway plays an important role in excitotoxicity and cerebral ischemia and we observed activation of the JNK substrate, c‐Jun in our model. Both the FXa inhibitor, fondaparinux and the PAR‐1 antagonist SCH79797, decreased the level of phospho‐c‐Jun Ser73. These results indicate that FXa activates thrombin in cerebral ischemia, which leads via PAR‐1 to the activation of the JNK pathway resulting in neuronal death.