[11C]PBR28 PET imaging is sensitive to neuroinflammation in the aged rat

Neuroinflammation in the aging rat brain was investigated using [¹¹C]PBR28 microPET (positron emission tomography) imaging. Normal rats were studied alongside LRRK2 p.G2019S transgenic rats; this mutation increases the risk of Parkinson''s disease in humans. Seventy [¹¹C]PBR28 PET scans were acquired. Arterial blood sampling enabled tracer kinetic modeling and estimation of V_T. In vitro autoradiography was also performed. PBR28 uptake increased with age, without differences between nontransgenic and transgenic rats. In 12 months of aging (4 to 16 months), standard uptake value (SUV) increased by 56% from 0.44 to 0.69 g/mL, whereas V_T increased by 91% from 30 to 57 mL/cm³. Standard uptake value and V_T were strongly correlated (r=0.52, 95% confidence interval (CI)=0.31 to 0.69, n=37). The plasma free fraction, f_p, was 0.21±0.03 (mean±standard deviation, n=53). In vitro binding increased by 19% in 16 months of aging (4 to 20 months). The SUV was less variable across rats than V_T; coefficients of variation were 13% (n=27) and 29% (n=12). The intraclass correlation coefficient for SUV was 0.53, but was effectively zero for V_T. These data show that [¹¹C]PBR28 brain uptake increases with age, implying increased microglial activation in the aged brain.     

Immunopurification of human plasma kininogens

Human plasma kininogens were purified by immunoadsorption on Sepharose columns using two different approaches, either removing protein impurities with the respective immunospecific polymers or applying an anti-kininogen-specific immunoadsorbent column. An anti-kininogen serum developed and investigated in this laboratory in earlier studies was used. This antiserum recognizes the native conformational determinants in the kininogen heavy chain, the common denominator in plasma kininogens, and reacts with three heterogeneous molecular forms of high mol. wt kininogen (mol. wts 103,000, 92,000 and 90,000) as well as with low mol. wt kininogen. Heterogeneity of kininogens was shown by SDS gel electrophoresis and immunoelectrophoresis. With the antibody-specific polymers the yield was 80-100% compared to 75% or lower when several consecutive immunoadsorption steps were applied to remove impurities. Both methods serve the purpose of preparing immunologically pure kininogens suitable for immunization.     

Systemic administration of alcohol to adult rats inhibits leydig cell activity: Time course of effect and role of nitric oxide

BACKGROUND: Alcohol has been shown to interfere with testosterone (T) release from Leydig cells. However, the mechanisms responsible for this phenomenon, which may include decreased activity of the luteinizing hormone-releasing hormone (LHRH)-LH axis, as well as a direct influence of the drug on the testes, are not fully understood. In this work, we investigated the influence of alcohol, administered intragastrically (i.g.) or delivered via vapors, on Leydig cell activity and T release. Leydig cell function was studied by measuring changes in the levels of the steroidogenic proteins steroidogenic acute regulatory (StAR), the peripheral-type benzodiazepine receptor (PBR), and the cytochrome P450 side-chain cleavage enzyme (P450scc). Testosterone release was studied under basal conditions or in response to human chorionic gonadotropin (hCG). Finally, to identify potential factors mediating the influence of alcohol, we measured the testicular variant of the neuronal nitric oxide (NO) synthase (NOS), TnNOS, in semipurified Leydig cells. METHODS: Adult male Sprague-Dawley rats were either injected with alcohol i.g. once or exposed to alcohol vapors (4 h/d) for 1 or 5 days. Controls received the vehicle (i.g. model) or were kept in boxes through which no vapors were circulated. Following these treatments, one series of experiments was devoted to investigate Leydig cell responsiveness by measuring plasma T levels before or after the intravenous injection of hCG (1 U/kg). In another series of experiments, we used semipurified Leydig cell preparations to measure StAR, PBR, P450scc, and TnNOS by Western blot analysis. RESULTS: In the i.g. model, the T response to hCG was blunted for 12 hours following alcohol injection, but showed a rebound at 48 hours. Levels of StAR protein and of PBR, but not of P450scc, were significantly decreased within 10 minutes of drug administration. While StAR then remained depressed for 24 hours, PBR values were variable over this time course. By 48 hours, StAR, PBR, and P450scc levels had increased above control values. Both StAR and PBR levels showed correlations with plasma T levels. In the alcohol vapor models, both regimens of the drug also significantly depressed StAR and PBR protein concentrations, blunted the T response to hCG, and did not alter P450scc. Finally, we observed that alcohol delivered i.g. or via vapors up-regulated TnNOS levels in Leydig cells, but that blockade of NO formation failed to restore a normal T response to hCG. CONCLUSIONS: Collectively, these results suggest that (a) the ability of Leydig cells to release T does not show a simple correlation with changes in StAR, PBR, and P450scc levels; (b) the time course of the alcohol-induced changes were protein-specific; and (c) despite the ability of alcohol to stimulate TnNOS expression, NO does not appear to mediate the inhibitory influence of this drug on testicular steroidogenesis in the models that we studied.     

The effect of midazolam on mouse Leydig cell steroidogenesis and apoptosis

The peripheral-type benzodiazepine receptor (PBR), a putative receptor in Leydig cells, modulates steroidogenesis. Since benzodiazepines are commonly used in regional anesthesia, their peripheral effects need to be defined. Therefore, this study set out to investigate in vitro effects of the benzodiazepine midazolam (MDZ) on Leydig cell steroidogenesis, and the possible underlying mechanisms. The effects of MDZ on steroidogenesis in primary mouse Leydig cells and MA-10 Leydig tumor cells were determined by radioimmunoassay. PBR, P450scc, 3β-HSD and StAR protein expression induced by MDZ was determined by Western blotting. Inhibitors of the signal transduction pathway and a MDZ antagonist were used to investigate the intracellular cascades activated by MDZ. In both cell types, MDZ-stimulated steroidogenesis in dose- and time-dependent manners, and induced the expression of PBR and StAR proteins, but had no effect on P450scc and 3β-HSD expressions. Moreover, H89 (PKA inhibitor) and GF109203X (PKC inhibitor) attenuated MDZ-stimulated steroid production. Interestingly, the MDZ antagonist (flumazenil) did not decrease MDZ-induced steroid production in both cell types. These results highly indicated that MDZ-induced steroidogenesis in mouse Leydig cells via PKA and PKC pathways, along with the expression of PBR and StAR proteins. In addition, MDZ at high dosages induced rounding-up, membrane blebbing, and then death in MA-10 cells. In conclusion, midazolam could induce Leydig tumor cell steroidogenesis, and high dose of midazolam could induce apoptosis in Leydig tumor cells.     

TSPO:外周苯二氮卓受体的新命名及其在中枢神经系统中的作用

早期外周苯二氮卓受体(PBR)是根据其在外周组织的广泛分布而命名的,用以区分中枢苯二氮卓受体(CBR)。但是近年来,随着研究的不断深入,人们发现PBR已不能反映该蛋白的分布、生理特性及其功能等,故2006年Papadopoulos等提出了18ku转位蛋白translocator protein(18ku),简称TSPO,将其作为PBR的新名称。该文将从TSPO的分子特性、定位分布、生理特性,特别是其在中枢神经系统疾病进程中所扮演的角色等方面对其研究进展做一综述。     

A pilot trial of RNS60 in amyotrophic lateral sclerosis

Introduction: RNS60 is a novel immune-modulatory agent that has shown neuroprotective effects in amytrophic lateral sclerosis (ALS) preclinical models. RNS60 is administered by weekly intravenous infusion and daily nebulization. The objective of this pilot open-label trial was to test the feasibility, safety, and tolerability of long-term RNS60 administration in ALS patients. Methods: The planned treatment duration was 23 weeks and the primary outcomes were safety and tolerability. Secondary outcomes included PBR28 positron emission tomography (PET) imaging and plasma biomarkers of inflammation. Results: Sixteen participants with ALS received RNS60 and 13 (81%) completed 23 weeks of RNS60 treatment. There were no serious adverse events and no participants withdrew from the trial due to drug-related adverse events. There were no significant changes in the biomarkers. Discussion: Long-term RNS60 administration was safe and well-tolerated. A large, multicenter, phase II trial of RNS60 is currently enrolling participants to test the effects of RNS60 on ALS biomarkers and disease progression. Muscle Nerve 59 :303–308, 2019     

Two binding sites for [3H]PBR28 in human brain: implications for TSPO PET imaging of neuroinflammation

[¹¹C]PBR28, a radioligand targeting the translocator protein (TSPO), does not produce a specific binding signal in approximately 14% of healthy volunteers. This phenomenon has not been reported for [¹¹C]PK11195, another TSPO radioligand. We measured the specific binding signals with [³H]PK11195 and [³H]PBR28 in brain tissue from 22 donors. Overall, 23% of the samples did not generate a visually detectable specific autoradiographic signal with [³H]PBR28, although all samples showed [³H]PK11195 binding. There was a marked reduction in the affinity of [³H]PBR28 for TSPO in samples with no visible [³H]PBR28 autoradiographic signal (K_i=188±15.6 nmol/L), relative to those showing normal signal (K_i=3.4±0.5 nmol/L, P<0.001). Of this latter group, [³H]PBR28 bound with a two-site fit in 40% of cases, with affinities (K_i) of 4.0±2.4 nmol/L (high-affinity site) and 313±77 nmol/L (low-affinity site). There was no difference in K_d or B_max for [³H]PK11195 in samples showing no [³H]PBR28 autoradiographic signal relative to those showing normal [³H]PBR28 autoradiographic signal. [³H]PK11195 bound with a single site for all samples. The existence of three different binding patterns with PBR28 (high-affinity binding (46%), low-affinity binding (23%), and two-site binding (31%)) suggests that a reduction in [¹¹C]PBR28 binding may not be interpreted simply as a reduction in TSPO density. The functional significance of differences in binding characteristics warrants further investigation.     

Development of new treatments for Alzheimer’s disease based on the modulation of translocator protein (TSPO)

The increase in life expectancy of the world population is associated with a higher prevalence of neurodegenerative diseases. Alzheimer’s Disease (AD) is the most common neurodegenerative disease, affecting currently 43 million people over the world. To date, most of the pharmacological interventions in AD are intended for the alleviation of some of its symptoms, and there are no effective treatments to inhibit the progression of the disease. Translocator protein (TSPO) is present in contact points between the outer and the inner mitochondrial membranes and is involved in the control of steroidogenesis, inflammation and apoptosis. In the last decade, studies have shown that TSPO ligands present neuroprotective effects in different experimental models of AD, both in vitro and in vivo. The aim of this review is to analyze the data provided by these studies and to discuss if TSPO could be a viable therapeutic target for the development of new treatments for AD.     

Efficient tritiation of the translocator protein (18 kDa) selective ligand DPA‐714

DPA‐714 (N,N‐diethyl‐2‐(2‐(4‐(2‐fluoroethoxy)phenyl)‐5,7‐dimethylpyrazolo[1,5‐a]pyrimidin‐3‐yl)acetamide) is a recently discovered fluorinated ligand of the translocator protein 18 kDa (TSPO). Labelled with the short‐lived positron emitter fluorine‐18, this structure is today the radioligand of reference for in vivo imaging of microglia activation and neuroinflammatory processes with positron emission tomography. In the present work, an isotopically tritium‐labelled version was developed ([³H]DPA‐714), in order to access high resolution in vitro and ex vivo microscopic autoradiography studies, repeated and long‐lasting receptor binding studies and in vivo pharmacokinetic determination at late time points. Briefly, DPA‐714 as reference, and its 3,5‐dibrominated derivative as precursor for labelling, were both prepared from DPA‐713 in nonoptimized 32% (two steps) and 10% (three steps) yields, respectively. Reductive debromination using deuterium gas and Pd/C as catalyst in methanol, performed at the micromolar scale, confirmed the regioselective introduction of two deuterium atoms at the meta positions of the phenyl ring. Tritiodebromination was analogously performed using no‐carrier tritium gas. HPLC purification provided >96% radiochemically pure [³H]DPA‐714 (7 GBq) with a 2.1 TBq/mmol specific radioactivity. Interestingly, additional hydrogen‐for‐tritium exchanges were also observed at the 5‐methyl and 7‐methyl positions of the pyrazolo[1,5‐a]pyrimidine, opening novel perspectives in the labelling of compounds featuring this heterocyclic core.     

Peripheral-type benzodiazepine receptor (PBR) and PBR drug ligands in fibroblast and fibrosarcoma cell proliferation: role of ERK, c-Jun and ligand-activated PBR-independent pathways

Peripheral-type benzodiazepine receptor (PBR) is a 18-kDa high-affinity drug and cholesterol binding protein, that has been implicated in several physiological processes, such as cholesterol transport and mitochondrial respiration. Specific PBR ligands regulate cell proliferation, although their action is controversial and probably cell-type specific. The aim of the present study was to examine the expression of PBR in cells of mesenchymal origin, i.e. human fibroblasts and fibrosarcoma cells, as well as its role in the regulation of their proliferation. Both mesenchymal cell types express high levels of PBR, localized exclusively in mitochondria. PBR-specific drug ligands, the isoquinoline carboxamide PK 11195 and the benzodiazepine Ro5-4864, at relative high concentrations (10(-4)M), exert a strong inhibitory effect on cell proliferation by arresting the cells at the G0/G1 phase of the cell cycle, while no apoptotic cell death was observed. In normal fibroblasts, this inhibition was correlated with a decrease in the activation of the cell cycle markers ERK and c-Jun. PBR knockdown by RNA inhibition did not affect the proliferation of either cell type and did not influence the inhibitory effect of PK 11195 and Ro5-4864 on cell growth. These data suggest that in fibroblasts and fibrosarcoma cells PBR drug ligands inhibit cell proliferation in a PBR-independent manner. These results are in contrast to data reported on cells of epithelial origin, suggesting that the origin of the cells is crucial in defining the role of PBR in their proliferation, and raise caution in the commonly made assumption that PBR mediates cell functions affected by PBR drug ligands.