The evolutionarily conserved peripheral benzodiazepine receptor (PBR), or 18-kDa translocator protein (TSPO), is thought to be essential for cholesterol transport and steroidogenesis, and thus life. TSPO has been proposed as a biomarker of neuroinflammation and a new drug target in neurological diseases ranging from Alzheimer’s disease to anxiety. Here we show that global C57BL/6-Tspotm1GuWu(GuwiyangWurra)-knockout mice are viable with normal growth, lifespan, cholesterol transport, blood pregnenolone concentration, protoporphyrin IX metabolism, fertility and behaviour. However, while the activation of microglia after neuronal injury appears to be unimpaired, microglia from GuwiyangWurraTSPO knockouts produce significantly less ATP, suggesting reduced metabolic activity. Using the isoquinoline PK11195, the ligand originally used for the pharmacological and structural characterization of the PBR/TSPO, and the imidazopyridines CLINDE and PBR111, we demonstrate the utility of GuwiyangWurraTSPO knockouts to provide robust data on drug specificity and selectivity, both in vitro and in vivo, as well as the mechanism of action of putative TSPO-targeting drugs.

The 18 kDa translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, is expressed in the injured brain. It has become known as an imaging marker of "neuroinflammation" indicating active disease, and is best interpreted as a nondiagnostic biomarker and disease staging tool that refers to histopathology rather than disease etiology. The therapeutic potential of TSPO as a drug target is mostly based on the understanding that it is an outer mitochondrial membrane protein required for the translocation of cholesterol, which thus regulates the rate of steroid synthesis. This pivotal role together with the evolutionary conservation of TSPO has underpinned the belief that any loss or mutation of TSPO should be associated with significant physiological deficits or be outright incompatible with life. However, against prediction, full Tspo knockout mice are viable and across their lifespan do not show the phenotype expected if cholesterol transport and steroid synthesis were significantly impaired. Thus, the "translocation" function of TSPO remains to be better substantiated. Here, we discuss the literature before and after the introduction of the new nomenclature for TSPO and review some of the newer findings. In light of the controversy surrounding the function of TSPO, we emphasize the continued importance of identifying compounds with confirmed selectivity and suggest that TSPO expression is analyzed within specific disease contexts rather than merely equated with the reified concept of "neuroinflammation."

Optimization of [(18)F]radiolabeling conditions and subsequent stability analysis in mobile phase, PBS buffer, and rat serum of 12 aryl sulfonyl chloride precursors with various substituents (electron-withdrawing groups, electron-donating groups, increased steric bulk, heterocyclic) were performed using an Advion NanoTek Microfluidic Synthesis System. A comparison of radiochemical yields and reaction times for a microfluidics device versus a conventional reaction vessel is reported. [(18)F]Radiolabeling of sulfonyl chlorides in the presence of competing nucleophiles, H-bond donors, and water was also assessed and demonstrated the versatility and potential utility of [(18)F]sulfonyl fluorides as synthons for indirect radiolabeling.

BackgroundRecently, inflammatory cascades have been suggested as a target for epilepsy therapy. Positron emission tomography (PET) imaging offers the unique possibility to evaluate brain inflammation longitudinally in a non-invasive translational manner. This study investigated brain inflammation during early epileptogenesis in the post-kainic acid-induced status epilepticus (KASE) model with post-mortem histology and in vivo with [18F]-PBR111 PET.MethodsStatus epilepticus (SE) was induced (N = 13) by low-dose injections of KA, while controls (N = 9) received saline. Translocator protein (TSPO) expression and microglia activation were assessed with [125I]-CLINDE autoradiography and OX-42 immunohistochemistry, respectively, 7 days post-SE. In a subgroup of rats, [18F]-PBR111 PET imaging with metabolite-corrected input function was performed before post-mortem evaluation. [18F]-PBR111 volume of distribution (Vt) in volume of interests (VOIs) was quantified by means of kinetic modelling and a VOI/metabolite-corrected plasma activity ratio.ResultsAnimals with substantial SE showed huge overexpression of TSPO in vitro in relevant brain regions such as the hippocampus and amygdala (P < 0.001), while animals with mild symptoms displayed a smaller increase in TSPO in amygdala only (P < 0.001). TSPO expression was associated with OX-42 signal but without obvious cell loss. Similar in vivo [18F]-PBR111 increases in Vt and the simplified ratio were found in key regions such as the hippocampus (P < 0.05) and amygdala (P < 0.01).ConclusionBoth post-mortem and in vivo methods substantiate that the brain regions important in seizure generation display significant brain inflammation during epileptogenesis in the KASE model. This work enables future longitudinal investigation of the role of brain inflammation during epileptogenesis and evaluation of anti-inflammatory treatments.

The deletion of the gene for the regulatory subunit of protein kinase A (PKA) results in constitutively active PKA in the pkaR mutant. To investigate the role of PKA in the basic motile behavior and chemotaxis of Dictyostelium discoideum, pkaR mutant cells were subjected to computer-assisted two-and three-dimensional motion analysis. pkaR mutant cells crawled at only half the speed of wild-type cells in buffer, chemotaxed in spatial gradients of cyclic AMP (cAMP) but with reduced efficiency, were incapable of suppressing lateral pseudopods in the front of temporal waves of cAMP, a requirement for natural chemotaxis, did not exhibit the normal velocity surge in response to the front of a wave, and were incapable of chemotaxing toward an aggregation center in natural waves generated by wild-type cells that made up the majority of cells in mixed cultures. Many of the behavioral defects appeared to be the result of the constitutively ovoid shape of the pkaR mutant cells, which forced the dominant pseudopod off the substratum and to the top of the cell body. The behavioral abnormalities that pkaR mutant cells shared with regA mutant cells are discussed by considering the pathway ERK2 OԽ RegA OԽ [cAMP] 3 PKA, which emanates from the front of a wave. The results demonstrate that cells must suppress PKA activity in order to elongate along a substratum, suppress lateralpseudopod formation, and crawl and chemotax efficiently. The results also implicate PKA activation in dismantling cell polarity at the peak and in the back of a natural cAMP wave.As a population of Dictyostelium amoebae aggregate to a center, each amoeba must respond to the different phases of relayed, outwardly moving, nondissipating symmetric waves of the chemoattractant cyclic AMP (cAMP) (Fig. 1). In the front of each wave, cells experience a positive spatial gradient of cAMP (i.e., the concentration increases in the direction of the aggregation center) and an increasing temporal gradient of cAMP (i.e., the concentration increases with time). At the peak of each wave, cells experience a cAMP concentration that causes cellular depolarization, and in the back of each wave, cells experience a negative spatial gradient of cAMP (i.e., the concentration decreases in the direction of the aggregation center) and a decreasing temporal gradient of cAMP (i.e., the concentration decreases with time) (Fig. 1). Amoebae respond to each phase of a natural wave in a distinct and reproducible fashion, resulting in a sequence of behaviors that together represent the natural chemotactic response (29,32,37,40,41,47,48). In the absence of any external chemotactic signal, Dictyostelium amoebae translocate at near-maximum velocity and turn frequently (47,48). We propose that this basic motile behavior is modulated in the different phases of the natural wave, with the net result of directing cells into the aggregation center (29) (Fig. 1). During aggregation, cells are exposed to a series of waves with an average periodicity of 7 min (1). With each wave, translocation toward th...

SummaryShared, unique and redundant functions of three members of the class I myosins (MyoA, MyoB and MyoF) in motility and chemotaxis in Dictyostelium

The fluoroethoxy and fluoropropoxy substituted 2-(6-chloro-2-phenyl)imidazo[1,2- a]pyridin-3-yl)- N, N-diethylacetamides 8 (PBR102) and 12 (PBR111) and 2-phenyl-5,7-dimethylpyrazolo[1,5- a]pyrimidin-3-yl)- N, N-diethylacetamides 15 (PBR099) and 18 (PBR146) were synthesized and found to have high in vitro affinity and selectivity for the peripheral benzodiazepine receptors (PBRs) when compared with the central benzodiazepine receptors (CBRs). The corresponding radiolabeled compounds [ (18)F] 8 [ (18)F] 12, [ (18)F] 15, and [ (18)F] 18 were prepared from their p-toluenesulfonyl precursors in 50-85% radiochemical yield. In biodistribution studies in rats, the distribution of radioactivity of the [ (18)F]PBR compounds paralleled the known localization of PBRs. In the olfactory bulbs, where the uptake of radioactivity was higher than in the rest of the brain, PK11195 and Ro 5-4864 were able to significantly inhibit [ (18)F] 12, while little or no pharmacological action of these established PBR drugs were observed on the uptake of [ (18)F] 8, [ (18)F] 15, and [ (18)F] 18 compared to control animals. Hence, [ (18)F] 12 appeared to be the best candidate for evaluation as an imaging agent for PBR expression in neurodegenerative disorders.

Glial neuroinflammation is associated with the development and progression of multiple sclerosis. PET imaging offers a unique opportunity to evaluate neuroinflammatory processes longitudinally in a noninvasive and clinically translational manner. 18 F-PBR111 is a newly developed PET radiopharmaceutical with high affinity and selectivity for the translocator protein (TSPO), expressed on activated glia. This study aimed to investigate neuroinflammation at different phases of relapsing-remitting (RR) experimental autoimmune encephalomyelitis (EAE) in the brains of SJL/J mice by postmortem histologic analysis and in vivo by PET imaging with 18 F-PBR111. Methods: RR EAE was induced by immunization with PLP 139-151 peptide in complete Freund's adjuvant. Naive female SJL/J mice and mice immunized with saline-complete Freund's adjuvant were used as controls. The biodistribution of 18 F-PBR111 was measured in 13 areas of the central nervous system and compared with PET imaging results during different phases of RR EAE. The extents of TSPO expression and glial activation were assessed with immunohistochemistry, immunofluorescence, and a real-time polymerase chain reaction. Results: There was significant TSPO expression in all of the central nervous system areas studied at the peak of the first clinical episode and, importantly, at the preclinical stage. In contrast, only a few TSPO-positive cells were observed at the second episode. At the third episode, there was again an increase in TSPO expression. TSPO expression was associated with microglial cells or macrophages without obvious astrocyte labeling. The dynamics of 18 F-PBR111 uptake in the brain, as measured by in vivo PET imaging and biodistribution, followed the pattern of TSPO expression during RR EAE. Conclusion: PET imaging with the TSPO ligand 18 F-PBR111 clearly reflected the dynamics of microglial activation in the SJL/J mouse model of RR EAE. The results are the first to highlight the discrepancy between the clinical symptoms of EAE and TSPO expression in the brain, as measured by PET imaging at the peaks of various EAE episodes. The results suggest a significant role for PET imaging investigations of neuroinflammation in multiple sclerosis and allow for in vivo follow-up of antiinflammatory treatment strategies.