Concentration, distribution, and influence of aging on the 18 kDa translocator protein in human brain: Implications for brain imaging studies

Abstract

Positron emission tomography (PET) imaging of the translocator protein (TSPO) is widely used as a biomarker of microglial activation. However, TSPO protein concentration in human brain has not been optimally quantified nor has its regional distribution been compared to TSPO binding. We determined TSPO protein concentration, change with age, and regional distribution by quantitative immunoblotting in autopsied human brain. Brain TSPO protein concentration (>0.1 ng/µg protein) was higher than those reported by in vitro binding assays by at least 2 to 70 fold. TSPO protein distributed widely in both gray and white matter regions, with distribution in major gray matter areas ranked generally similar to that of PET binding in second-generation radiotracer studies. TSPO protein concentration in frontal cortex was high at birth, declined precipitously during the first three months, and increased modestly during adulthood/senescence (10%/decade; vs. 30% for comparison astrocytic marker GFAP). As expected, TSPO protein levels were significantly increased (+114%) in degenerating putamen in multiple system atrophy, providing further circumstantial support for TSPO as a gliosis marker. Overall, findings show some similarities between TSPO protein and PET binding characteristics in the human brain but also suggest that part of the TSPO protein pool might be less available for radioligand binding.

Keywords: Translocator protein TSPO; aging; multiple system atrophy; positron emission tomography; postmortem human brain.

Figure 1.

Quantification of TSPO protein in autopsied human brain. (a) Immunoblots of TSPO in the pooled tissue standards, in the commercial N-His-tagged recombinant human TSPO-3462H and in human adrenal samples with monoclonal (EPR5384, 1:100K and MA5-24844, 1:30K dilution) and polyclonal (PA5-75544, 1:10K) antibodies. (b) Standard curves for the recombinant TSPO. Note more abundant TSPO in adrenal than in brain for both EPR5384 and MA5-24844, polymer protein bands of the recombinant TSPO, high molecular weight non-specific reactions for PA5-75544, and detection of a TSPO fragment but not 18 kDa TSPO by PA5-75544 in human adrenal samples.

Figure 2.

Representative immunoblots of the regional distribution of TSPO and GFAP in autopsied human brain. A23: cingulate gyrus posterior; A24: cingulate gyrus anterior; A25: paraolfactory/subgenual gyrus; CCc: corpus callosum caudal; CCr: corpus callosum rostral; cereb: cerebellar cortex; CN: caudate; CNA: hippocampal Ammon’s horn; CSTH: subthalamic nucleus; GD: dentate gyrus; GH: hippocampal gyrus; GPe: globus pallidus external; GPi: globus pallidus internal; GUNC: gyrus of uncus; hypothal: hypothalamus; ICr: internal capsule rostral; MDTH: mediodorsal thalamus; NAM: amygdala; NAV: anterior ventral nucleus of thalamus; N. basalis: nucleus basalis; NL: nucleus lateralis of thalamus; NLV: lateral ventral nucleus of thalamus; NPM: medial pulvinar of thalamus; PUT: putamen; RN: red nucleus; SBI: substantia innominata; SNpc: substantia nigra pars compacta.

Figure 3.

Correlations (Pearson) between regional protein levels of TSPO determined in this study vs. outcome measures of binding reported in the literature (p < 0.05 for solid lines and p > 0.05 for dashed lines). To assess the correlation between TSPO protein vs. TSPO binding amongst different brain areas, the following regions were employed, with subregional averages when necessary: thalamus, midbrain, globus pallidus, hippocampus, amygdala, cerebral cortical areas (cingulate, insula, frontal, temporal, parietal and occipital cortices), cerebellar cortex, and striatum (caudate and putamen). (a) TSPO protein versus [³H]PK11195 binding density determined by autoradiography in autopsied human brain reported by Doble et al. and [¹¹C](R)-PK11195 binding potential acquired by positron emission tomography (PET) reported by Cagnin et al. Note the lack of correlation between the regional distribution of TSPO protein in our autopsied human brain study vs. the regional distribution of binding of the first generation TSPO tracer, PK11195. (b, c) TSPO protein regional distribution in autopsied human brain versus PET total distribution volume (V_T) of five second generation TSPO radiotracers that are influenced by the single nucleotide genetic polymorphism (rs6971, HAB-high affinity binder and MAB-mixed affinity binder), including [F]-FEPPA reported by Atwells et al., [¹¹C]PBR28 reported by Rizzo et al., [¹⁸F]DPA-714 reported by Lavisse et al., [¹⁸F]PBR-111 reported by De Picker et al., and [¹⁸F]GE-180 reported by Fan et al. Note that in contrast to the lack of correlation with the first generation ligand, there was positive (r = 0.72–0.79, p < 0.05) or a trend for a positive correlation (r = 0.45–0.55, p = 0.10–0.16) between the regional distribution of TSPO protein in autopsied brain vs. those of binding of the second generation tracers in PET imaging studies, with the exception of [¹¹C]PBR28 in MAB (r = −0.09, p = 0.82).

Figure 4.

Aging and neonatal developmental changes of levels of TSPO and GFAP in autopsied human brain. The inset shows enlarged portion below 1 year of age for TSPO. The linear correlations are for adults (18–99 years). The dashed line in the GFAP graph shows the linear correlation in the full age range (21 hours to 99 years of age) excluding five outliers (solid circles). Note high levels of TSPO during first three months of birth, its exponential drop thereafter, and slow increase during adult aging and senescence; in contrast, GFAP levels were low at birth, other than in the five outliers, and increased steadily throughout life.

Figure 5.

Increased levels of TSPO protein in putamen of patients with multiple system atrophy (MSA) as compared to control subjects. ***p < 0.001 (two-tailed t-test). Also shown are representative immunoblots of TSPO and an astroglial marker vimentin probed at the same time. Note greater increases in levels of vimentin as compared to that of TSPO in MSA (M1 and M2) versus controls (C1 and C2).