Background Microglia-specific genetic variants are enriched in several neurodegenerative diseases, including Alzheimer’s disease (AD), implicating a central role for alterations of the innate immune system in the disease etiology. A rare coding variant in the PLCG2 gene (rs72824905, p.P522R) expressed in myeloid lineage cells was recently identified and shown to reduce the risk for AD. Methods To assess the role of the protective variant in the context of immune cell functions, we generated a Plcγ2-P522R knock-in (KI) mouse model using CRISPR/Cas9 gene editing. Results Functional analyses of macrophages derived from homozygous KI mice and wild type (WT) littermates revealed that the P522R variant potentiates the primary function of Plcγ2 as a Pip2-metabolizing enzyme. This was associated with improved survival and increased acute inflammatory response of the KI macrophages. Enhanced phagocytosis was observed in mouse BV2 microglia-like cells overexpressing human PLCγ2-P522R, but not in PLCγ2-WT expressing cells. Immunohistochemical analyses did not reveal changes in the number or morphology of microglia in the cortex of Plcγ2-P522R KI mice. However, the brain mRNA signature together with microglia-related PET imaging suggested enhanced microglial functions in Plcγ2-P522R KI mice. Conclusion The AD-associated protective Plcγ2-P522R variant promotes protective functions associated with TREM2 signaling. Our findings provide further support for the idea that pharmacological modulation of microglia via TREM2-PLCγ2 pathway-dependent stimulation may be a novel therapeutic option for the treatment of AD.

Three benzimidazole derivatives (13–15) have been synthetized as potential positron emission tomography (PET) imaging ligands for mGluR2 in the brain. Of these compounds, 13 exhibits potent binding affinity (IC50 = 7.6 ± 0.9 nM), positive allosteric modulator (PAM) activity (EC50 = 51.2 nM), and excellent selectivity against other mGluR subtypes (>100-fold). [11C]13 was synthesized via O-[11C]­methylation of its phenol precursor 25 with [11C]­methyl iodide. The achieved radiochemical yield was 20 ± 2% (n = 10, decay-corrected) based on [11C]­CO2 with a radiochemical purity of >98% and molar activity of 98 ± 30 GBq/μmol EOS. Ex vivo biodistribution studies revealed reversible accumulation of [11C]13 and hepatobiliary and urinary excretions. PET imaging studies in rats demonstrated that [11C]13 accumulated in the mGluR2-rich brain regions. Pre-administration of mGluR2-selective PAM, 17 reduced the brain uptake of [11C]13, indicating a selective binding. Therefore, [11C]13 is a potential PET imaging ligand for mGluR2 in different central nervous system-related conditions.

Luciferase reporter assays are commonly used in high-throughput screening methods. Here, we report new firefly luciferase (FLuc) inhibitors based on 5-benzyl-3-phenyl-4,5-dihydroisoxazoles and 5-benzyl-3-phenyl-1,4,2-dioxazoles, which showed up as "false positives" in a luciferase reporter gene-based assay for nuclear receptor antagonists. The inhibition was shown to be noncompetitive for both natural enzyme substrates (d-luciferin and ATP) and selective to FLuc and proven to arise from a direct interaction between the enzyme and the inhibitor. Of the 63 evaluated compounds, 28 showed significantly better inhibition potency than the well-known inhibitor resveratrol (IC(50) = 59 nM), with five compounds having distinctly subnanomolar IC(50) values. The most efficient compounds inhibited the luminescence at concentrations lower than (1)/(100) in comparison to resveratrol (lowest IC(50) = 0.26 nM) and can thus be considered to belong to the most potent FLuc inhibitors reported thus far. Overall, the novel inhibitors form a unique molecular library for structure-activity relationship (SAR) analyses.

There has been a growing interest toward mitochondrial fatty acid synthesis (mtFAS) since the recent discovery of a neurodegenerative human disorder termed MEPAN (mitochondrial enoyl reductase protein associated neurodegeneration), which is caused by mutations in the mitochondrial enoyl-CoA/ACP (acyl carrier protein) reductase (MECR) carrying out the last step of mtFAS. We show here that MECR protein is highly expressed in mouse Purkinje cells (PCs). To elucidate mtFAS function in neural tissue, here, we generated a mouse line with a PC-specific knock-out (KO) of Mecr, leading to inactivation of mtFAS confined to this cell type. Both sexes were studied. The mitochondria in KO PCs displayed abnormal morphology, loss of protein lipoylation, and reduced respiratory chain enzymatic activities by the time these mice were 6 months of age, followed by nearly complete loss of PCs by 9 months of age. These animals exhibited balancing difficulties ϳ7 months of age and ataxic symptoms were evident from 8 -9 months of age on. Our data show that impairment of mtFAS results in functional and ultrastructural changes in mitochondria followed by death of PCs, mimicking aspects of the clinical phenotype. This KO mouse represents a new model for impaired mitochondrial lipid metabolism and cerebellar ataxia with a distinct and well trackable cellular phenotype. This mouse model will allow the future investigation of the feasibility of metabolite supplementation approaches toward the prevention of neurodegeneration due to dysfunctional mtFAS.We have recently reported a novel neurodegenerative disorder in humans termed MEPAN (mitochondrial enoyl reductase protein associated neurodegeneration) (Heimer et al., 2016). The cause of neuron degeneration in MEPAN patients is the dysfunction of the highly conserved mitochondrial fatty acid synthesis (mtFAS) pathway due to mutations in MECR, encoding mitochondrial 2-enoyl-CoA/ACP reductase. The report presented here describes the analysis of the first mouse model suffering from mtFASdefect-induced neurodegenerative changes due to specific disruption of the Mecr gene in Purkinje cells. Our work sheds a light on the mechanisms of neurodegeneration caused by mtFAS deficiency and provides a test bed for future treatment approaches.

We have synthesized and characterized [ 18 F]-N-(4-chloro-3-((fluoromethyl-d 2)thio)phenyl)picolinamide ([ 18 F]15) as a potential ligand for the PET imaging of mGluR4 in the brain. Radioligand [ 18 F]15 displays CNS drug-like properties, including mGluR4 affinity, potent mGluR4 PAM activity and selectivity against other mGluRs, as well as sufficient metabolic stability. Radiosynthesis was carried out in two steps. The radiochemical yield of [ 18 F]15 was 11.6 ± 2.9% (n = 7, decay corrected) with a purity of 99% and a molar activity of 84.1 ± 11.8 GBq/ μmol. Ex vivo biodistribution studies showed reversible binding of [ 18 F]15 in all investigated tissues including the brain, liver, heart, lungs, and kidneys. PET imaging studies in male Sprague-Dawley rats showed that [ 18 F]15 accumulates in the brain regions known to express mGluR4. Pretreatment with the unlabeled mGluR4 PAM compounds 13 (methylthio analog) and 15 showed significant dose-dependent blocking effects. These results suggest that [ 18 F]15 is a promising radioligand for PET imaging mGluR4 in the brain.