Characterization of the 18 kDa translocator protein (TSPO) expression in post-mortem normal and Alzheimer's disease brains.
ABSTRACT: The 18 kDa translocator protein (TSPO) is a widely used target for microglial PET imaging radioligands, but its expression in post-mortem normal and diseased human brain is not well described. We aimed at characterizing the TSPO expression in human control (CTRL) and Alzheimer's disease (AD) brains. Specifically, we sought to: (1) define the cell type(s) expressing TSPO; (2) compare tspo mRNA and TSPO levels between AD and CTRL brains; (3) correlate TSPO levels with quantitative neuropathological measures of reactive glia and AD neuropathological changes; and (4) investigate the effects of the TSPO rs6971 SNP on tspo mRNA and TSPO levels, glial responses and AD neuropathological changes. We performed quantitative immunohistochemistry and Western blot in post-mortem brain samples from CTRL and AD subjects, as well as analysis of publicly available mouse and human brain RNA-Seq datasets. We found that: (1) TSPO is expressed not just in microglia, but also in astrocytes, endothelial cells and vascular smooth muscle cells; (2) there is substantial overlap of tspo mRNA and TSPO levels between AD and CTRL subjects and in TSPO levels between temporal neocortex and white matter in both groups; (3) TSPO cortical burden does not correlate with the burden of activated microglia or reactive astrocytes, A? plaques or neurofibrillary tangles, or the cortical thickness; (4) the TSPO rs6971 SNP does not significantly impact tspo mRNA or TSPO levels, the magnitude of glial responses, the cortical thickness, or the burden of AD neuropathological changes. These results could inform ongoing efforts toward the development of reactive glia-specific PET radioligands.
Project description:[(18)F]-FEPPA binds to the 18-kDa translocator protein (TSPO) and is used in positron emission tomography (PET) to detect microglial activation. However, quantitative interpretations of the PET signal with new generation TSPO PET radioligands are confounded by large interindividual variability in binding affinity. This presents as a trimodal distribution, reflecting high-affinity binders (HABs), low-affinity binder (LAB), and mixed-affinity binders (MABs). Here, we show that one polymorphism (rs6971) located in exon 4 of the TSPO gene, which results in a nonconservative amino-acid substitution from alanine to threonine (Ala147Thr) in the TSPO protein, predicts [(18)F]-FEPPA total distribution volume in human brains. In addition, [(18)F]-FEPPA exhibits clearly different features in the shape of the time activity curves between genetic groups. Testing for the rs6971 polymorphism may allow quantitative interpretation of TSPO PET studies with new generation of TSPO PET radioligands.
Project description:The imaging of translocator 18 kDa protein (TSPO) in living human brain with radioligands by positron emission tomography (PET) has become an important means for the study of neuroinflammatory conditions occurring in several neuropsychiatric disorders. The widely used prototypical PET radioligand [(11)C](R)-PK 11195 ([(11)C](R)-1; [N-methyl-(11)C](R)-N-sec-butyl-1-(2-chlorophenyl)-N-methylisoquinoline-3-carboxamide) gives a low PET signal and is difficult to quantify, whereas later generation radioligands have binding sensitivity to a human single nucleotide polymorphism (SNP) rs6971, which imposes limitations on their utility for comparative quantitative PET studies of normal and diseased subjects. Recently, azaisosteres of 1 have been developed with improved drug-like properties, including enhanced TSPO affinity accompanied by moderated lipophilicity. Here we selected three of these new ligands (7-9) for labeling with carbon-11 and for evaluation in monkey as candidate PET radioligands for imaging brain TSPO. Each radioligand was readily prepared by (11)C-methylation of an N-desmethyl precursor and was found to give a high proportion of TSPO-specific binding in monkey brain. One of these radioligands, [(11)C]7, the direct 4-azaisostere of 1, presents many radioligand properties that are superior to those reported for [(11)C]1, including higher affinity, lower lipophilicity, and stable quantifiable PET signal. Importantly, 7 was also found to show very low sensitivity to the human SNP rs6971 in vitro. Therefore, [(11)C]7 now warrants evaluation in human subjects with PET to assess its utility for imaging TSPO in human brain, irrespective of subject genotype.
Project description:To date, ¹¹C-(R)-PK11195 has been the most widely used TSPO PET imaging probe, although it suffers from high non-specific binding and low signal to noise. A significant number of 2nd generation TSPO radioligands have been developed with higher affinity and/or lower non-specific binding, however there is substantial inter-subject variation in their affinity for the TSPO. TSPO from human tissue samples binds 2nd generation TSPO radioligands with either high affinity (high affinity binders, HABs), or low affinity (LABs) or expresses both HAB and LAB binding sites (mixed affinity binders, MABs). The expression of these different TSPO binding sites in human is encoded by the rs6971 polymorphism in the TSPO gene. Here, we use a predictive biomathematical model to estimate the in vivo performances of three of these 2nd generation radioligands (¹?F-PBR111, ¹¹C-PBR28, ¹¹C-DPA713) and ¹¹C-(R)-PK11195 in humans. The biomathematical model only relies on in silico, in vitro and genetic data (polymorphism frequencies in different ethnic groups) to predict the radioactivity time course in vivo. In particular, we provide estimates of the performances of these ligands in within-subject (e.g. longitudinal studies) and between-subject (e.g. disease characterisation) PET studies, with and without knowledge of the TSPO binding class. This enables an assessment of the different radioligands prior to radiolabelling or acquisition of any in vivo data. The within-subject performance was characterised in terms of the reproducibility of the in vivo binding potential (%COV[BP(ND)]) for each separate TSPO binding class in normal and diseased states (50% to 400% increase in TSPO density), whilst the between-subject performance was characterised in terms of the number of subjects required to distinguish between different populations. The results indicated that the within-subject variability for ¹?F-PBR111, ¹¹C-PBR28 and ¹¹C-DPA713 (0.9% to 2.2%) was significantly lower than ¹¹C-(R)-PK11195 (16% to 36%) for HABs and MABs in both normal and diseased states. For between-subject studies, sample sizes required to detect 50% differences in TSPO density with the 2nd generation tracers are approximately half that required with ¹¹C-(R)-PK11195 when binding class information is known a priori. As binding class can be identified using a simple genetic test or from peripheral blood assays, the combination of binding class information with 2nd generation TSPO imaging data should provide superior tools to investigate inflammatory processes in humans in vivo.
Project description:Second-generation radioligands for translocator protein (TSPO), an inflammation marker, are confounded by the codominant rs6971 polymorphism that affects binding affinity. The resulting three groups are homozygous for high-affinity state (HH), homozygous for low-affinity state (LL), or heterozygous (HL). We tested if in vitro binding to leukocytes distinguished TSPO genotypes and if genotype could affect clinical studies using the TSPO radioligand [(11)C]PBR28. In vitro binding to leukocytes and [(11)C]PBR28 brain imaging were performed in 27 human subjects with known TSPO genotype. Specific [(3)H]PBR28 binding was measured in prefrontal cortex of 45 schizophrenia patients and 47 controls. Leukocyte binding to PBR28 predicted genotype in all subjects. Brain uptake was ?40% higher in HH than HL subjects. Specific [(3)H]PBR28 binding in LL controls was negligible, while HH controls had ?80% higher binding than HL controls. After excluding LL subjects, specific binding was 16% greater in schizophrenia patients than controls. This difference was insignificant by itself (P=0.085), but was significant after correcting for TSPO genotype (P=0.011). Our results show that TSPO genotype influences PBR28 binding in vitro and in vivo. Correcting for this genotype increased statistical power in our postmortem study and is recommended for in vivo positron emission tomography studies.
Project description:For PET imaging of 18-kDa translocator protein (TSPO), a biomarker of neuroinflammation, most second-generation radioligands are sensitive to the single nucleotide polymorphism rs6971; however, this is probably not the case for the prototypical agent 11C-PK11195 (11C-labeled N-butan-2-yl-1-(2-chlorophenyl)-N-methylisoquinoline-3-carboxamide), which has a relatively lower signal-to-noise ratio. We recently found that 11C-ER176 (11C-(R)-N-sec-butyl-4-(2-chlorophenyl)-N-methylquinazoline-2-carboxamide), a new analog of 11C-(R)-PK11195, showed little sensitivity to rs6971 when tested in vitro and had high specific binding in monkey brain. This study sought, first, to determine whether the sensitivity of 11C-ER176 in humans is similar to the low sensitivity measured in vitro and, second, to measure the nondisplaceable binding potential (BPND, or the ratio of specific-to-nondisplaceable uptake) of 11C-ER176 in human brain.Nine healthy volunteers-3 high-affinity binders (HABs), 3 mixed-affinity binders (MABs), and 3 low-affinity binders (LABs)-were studied with whole-body 11C-ER176 PET imaging. SUVs from 60 to 120 min after injection derived from each organ were compared between genotypes. Eight separate healthy volunteers-3 HABs, 3 MABs, and 2 LABs-underwent brain PET imaging. The 3 HABs underwent a repeated brain scan after TSPO blockade with XBD173 (N-benzyl-N-ethyl-2-(7-methyl-8-oxo-2-phenylpurin-9-yl)acetamide) to determine nondisplaceable distribution volume (VND) via Lassen occupancy plotting and thereby estimate BPND in brain.Regional SUV averaged from 60 to 120 min after injection in brain and peripheral organs with high TSPO densities such as lung and spleen were greater in HABs than in LABs. On the basis of VND determined via the occupancy plot, the whole-brain BPND for LABs was estimated to be 1.4 ± 0.8, which was much lower than that for HABs (4.2 ± 1.3) but about the same as that for HABs with 11C-PBR28 ([methyl-11C]N-acetyl-N-(2-methoxybenzyl)-2-phenoxy-5-pyridinamine)) (?1.2).Obvious in vivo sensitivity to rs6971 was observed in 11C-ER176 that had not been expected from in vitro studies, suggesting that the future development of any improved radioligand for TSPO should consider the possibility that in vitro properties will not be reflected in vivo. We also found that 11C-ER176 has adequately high BPND for all rs6971 genotypes. Thus, the new radioligand would likely have greater sensitivity in detecting abnormalities in patients.
Project description:UNLABELLED:(11)C-PBR28 binds to the high-affinity state of the translocator protein 18 kDa (TSPO). A single-nucleotide polymorphism (rs6971) within the human TSPO gene determines the affinity state of the TSPO. The rs6971 genotype determines whether individuals express the high-, low-, or mixed-affinity phenotype of TSPO. The rs6971 genotype corresponds to in vivo (11)C-PBR28 binding, as measured quantitatively by total volume of distribution. However, it is not known whether standardized uptake value (SUV) can detect differences in brain (11)C-PBR28 uptake by TSPO genotype. METHODS:Thirty-two older adults (71.8 ± 7.94 y old) underwent (11)C-PBR28 PET scanning; rs6971 genotype was imputed after genomewide genotyping. SUV was extracted for several brain regions. The sample included 19 C/C carriers (high-affinity phenotype), 12 T/C carriers (mixed-affinity), and 1 T/T carrier (low-affinity) for rs6971. RESULTS:SUV was 30% lower in T/C subjects than in C/C subjects. CONCLUSION:The results indicate that brain (11)C-PBR28 SUV is sensitive to TSPO genotype.
Project description:Alzheimer's disease (AD) is the most common form of dementia. One of the neuropathological hallmarks of AD is the accumulation of amyloid-? plaques. Overexpression of human amyloid precursor protein in transgenic mice induces hippocampal and neocortical amyloid-? accumulation and plaque deposition that increases with age. The impact of these effects on neuronal population responses and network activity in sensory cortex is not well understood. We used Voltage Sensitive Dye Imaging, to investigate at high spatial and temporal resolution, the sensory evoked population responses in the barrel cortex of aged transgenic (Tg) mice and of age-matched non-transgenic littermate controls (Ctrl) mice. We found that a whisker deflection evoked abnormal sensory responses in the barrel cortex of Tg mice. The response amplitude and the spatial spread of the cortical responses were significantly larger in Tg than in Ctrl mice. At the network level, spontaneous activity was less synchronized over cortical space than in Ctrl mice, however synchronization during evoked responses induced by whisker deflection did not differ between the two groups. Thus, the presence of elevated A? and plaques may alter population responses and disrupts neural synchronization in large-scale networks, leading to abnormalities in sensory processing.
Project description:Core pathologies of Alzheimer's disease (AD) are aggregated amyloid-? peptides (A?) and tau, and the latter is also characteristic of diverse neurodegenerative tauopathies. These amyloid lesions provoke microglial activation, and recent neuroimaging technologies have enabled visualization of this response in living brains using radioligands for the peripheral benzodiazepine receptor also known as the 18 kDa translocator protein (TSPO). Here, we elucidated contributions of A? and tau deposits to in vivo TSPO signals in pursuit of mechanistic and diagnostic significance of TSPO imaging in AD and other tauopathies. A new antibody to human TSPO revealed induction of TSPO-positive microgliosis by tau fibrils in tauopathy brains. Emergence of TSPO signals before occurrence of brain atrophy and thioflavin-S-positive tau amyloidosis was also demonstrated in living mice transgenic for mutant tau by positron emission tomography (PET) with two classes of TSPO radioligands, [(11)C]AC-5216 and [(18)F]fluoroethoxy-DAA1106. Meanwhile, only modest TSPO elevation was observed in aged mice modeling A? plaque deposition, despite the notably enhanced in vivo binding of amyloid radiotracer, [(11)C]Pittsburgh Compound-B, to plaques. In these animals, [(11)C]AC-5216 yielded better TSPO contrasts than [(18)F]fluoroethoxy-DAA1106, supporting the possibility of capturing early neurotoxicity with high-performance TSPO probes. Furthermore, an additional line of mice modeling intraneuronal A? accumulation displayed elevated TSPO signals following noticeable neuronal loss, unlike TSPO upregulation heralding massive neuronal death in tauopathy model mice. Our data corroborate the utility of TSPO-PET imaging as a biomarker for tau-triggered toxicity, and as a complement to amyloid scans for diagnostic assessment of tauopathies with and without A? pathologies.
Project description:One of the cellular markers of neuroinflammation is increased microglia activation, characterized by overexpression of mitochondrial 18kDa Translocator Protein (TSPO). TSPO expression can be quantified in-vivo using the positron emission tomography (PET) radioligand [(18)F]-FEPPA. This study examined microglial activation as measured with [(18)F]-FEPPA PET across the adult lifespan in a group of healthy volunteers. We performed genotyping for the rs6971 TS.PO gene polymorphism to control for the known variability in binding affinity. Thirty-three healthy volunteers (age range: 19-82years; 22 high affinity binders (HAB), 11 mixed affinity binders (MAB)) underwent [(18)F]-FEPPA PET scans, acquired on the High Resolution Research Tomograph (HRRT) and analyzed using a 2-tissue compartment model. Regression analyses were performed to examine the effect of age adjusting for genetic status on [(18)F]-FEPPA total distribution volumes (VT) in the hippocampus, temporal, and prefrontal cortex. We found no significant effect of age on [(18)F]-FEPPA VT (F (1,30)=0.918; p=0.346), and a significant effect of genetic polymorphism (F (1,30)=8.767; p=0.006). This is the first in-vivo study to evaluate age-related changes in TSPO binding, using the new generation TSPO radioligands. Increased neuroinflammation, as measured with [(18)F]-FEPPA PET was not associated with normal aging, suggesting that healthy elderly individuals may serve as useful benchmark against patients with neurodegenerative disorders where neuroinflammation may be present.
Project description:The 18?kDa translocator protein (TSPO) is a ubiquitous conserved outer mitochondrial membrane protein implicated in numerous cell and tissue functions, including steroid hormone biosynthesis, respiration, cell proliferation, and apoptosis. TSPO binds with high affinity to cholesterol and numerous compounds, is expressed at high levels in steroid-synthesizing tissues, and mediates cholesterol import into mitochondria, which is the rate-limiting step in steroid formation. In humans, the rs6971 polymorphism on the TSPO gene leads to an amino acid substitution in the fifth transmembrane loop of the protein, which is where the cholesterol-binding domain of TSPO is located, and this polymorphism has been associated with anxiety-related disorders. However, recent knockout mouse models have provided inconsistent conclusions of whether TSPO is directly involved in steroid synthesis. In this report, we show that TSPO deletion mutations in rat and its corresponding rs6971 polymorphism in humans alter adrenocorticotropic hormone-induced plasma corticosteroid concentrations. Rat tissues examined show increased cholesteryl ester accumulation, and neurosteroid formation was undetectable in homozygous rats. These results also support a role for TSPO ligands in diseases with steroid-dependent stress and anxiety elements.