The high affinity peripheral benzodiazepine receptor ligand DAA1106 binds to activated and infected brain macrophages in areas of synaptic degeneration: implications for PET imaging of neuroinflammation in lentiviral encephalitis

Abstract

HIV encephalitis (HIVE) is characterized by neurodegeneration mediated by toxins derived from infected and activated brain macrophages. Since the peripheral benzodiazepine receptor (PBR) is abundant on brain macrophages, we hypothesized that [(3)H]DAA1106, a new PBR ligand, can label infected and activated brain macrophages in HIVE. Using cell culture and postmortem brain tissues from HIVE and a macaque model of HIVE, we show that [(3)H]DAA1106 binds with high affinity to activated and infected macrophages in regions of synaptic damage. Further, binding affinity reflected by lower K(D) (dissociation constant) values and the B(max) (total number of binding sites) to K(D) ratios reflective of ligand-binding potential was significantly higher with [(3)H]DAA1106 compared to the extensively characterized PBR ligand [(3)H](R)-PK11195. These data suggest that DAA1106 binds with high affinity to activated and infected brain macrophages and possesses binding characteristics beneficial for in vivo use in the detection and clinical monitoring of HIVE using positron emission tomography.

Figure 1. [³H]DAA1106 binding is higher in HIVE compared to controls

Saturation filtration binding experiments were performed in basal ganglia tissues from HIVE (n=5), HIV non-encephalitis (HIV, n=6) and age-matched non-infected controls (n=3). A & B, Representative saturation binding curves (A) and schatchard plots (B, X-intercept represent B_max, and slope represents K_D) with [³H]DAA1106 from HIVE (black squares), HIV no-encephalitis (grey squares) and an age-matched control (clear squares). C, The B_max (fmols/mg) reflective of the total number of binding sites, with [³H]DAA1106 was significantly higher in HIVE (black bars) compared with HIV non-encephalitis (grey bars) and non-infected controls (clear bars), p=0.0003. D, The K_D (nM), reflective of the ligand binding affinity was not significantly different in all three conditions, p=0.0712. Data was analyzed using ANOVA.

Figure 2. [³H]DAA1106 binding is higher in SIVE compared to controls

A & B, [³H]DAA1106 autoradiographic binding assessed in the frontal cortex of macaques with SIVE corresponded to the distribution of microglial nodules (A) and was specific as it was displaced by 1 nM DAA1106 (B). C & D, B_max (fmols/mg) with [³H]DAA1106 was significantly higher in SIVE (n=3, black bars) compared with SIV non-encephalitis (n=4, grey bars) and non-infected controls (n=3, clear bars), p=0.0038 (C). The K_D (nM) was not significantly different in all three conditions, p=0.2492, (D). Data was analyzed using ANOVA.

Figure 3. Primary human microglia and macrophages, but not astrocytes show increased [³H]DAA1106 binding on activation

A, Primary human embryonic astrocytes activated with dB-cAMP show changes in morphology with the appearance of spindle shaped processes and increased GFAP staining compared to non-activated cultures (inset). B, Primary human macrophages activated with LPS show changes in morphology from a rounded shape (inset) to spindle shaped with increased CD68 staining in non-stimulated cultures. C, Primary human embryonic microglia were activated with LPS for 48 hrs show increased CD68 staining compared to non-activated cultures (inset). D, [³H]DAA1106 specific binding (fmols/mg mitochondrial protein) was significantly higher in mitochondrial preparations obtained from both macrophages (b) and microglia (c) activated with LPS (black bars) compared to unactivated controls (white bars) and astrocytes (a) with or without activation. Data was analyzed using ANOVA, n=3 in each group, ***p<0.0001.

Figure 4. [³H]DAA1106 binding corresponds to activated and infected macrophages in SIVE

A-C, Combined [³H]DAA1106 autoradiography (center panel, black grains) and immunostaining for astrocytes (A, GFAP, red), activated macrophages (B, CD68, green) and SIV infected cells (C, SIV gp110, green) was performed in cortical brain tissue obtained from macaques with SIVE. [³H]DAA1106 specific binding overlapped with CD68 labeled activated macrophages (C, merge) and SIV infected macrophages (D, merge), but not with GFAP immunostained astrocytes (A, Merge). Scale bar indicates 50 μm. D, Combined [³H]DAA1106 autoradiography (center panel, black grains) and immunostaining for HIV p24 (green) in HIVE basal ganglia showed [³H]DAA1106 specific binding overlapping with HIV infected macrophages (merge). Scale bar indicates 20 μm.

Figure 5. [³H]DAA1106 specific binding correlates with decreases in the presynaptic protein SYN and the postsynaptic protein MAP-2

A, The postsynaptic protein MAP-2 (Red) a marker for dendrites and neuronal cell bodies and presynaptic protein SYN (green) were lower in SIVE compared to SIV non-encephalitis (SIV). B-C, Quantification of these markers showed significant decreases in MAP-2 (B) and SYN (C) in SIVE (n=3, black bars) compared to SIV non-encephalitis (SIV, n=4, grey bars) and non-infected controls (Con, n=3, clear bars). Data was analyzed using student t test ANOVA, *p<0.05.

Figure 6. [³H]DAA1106 B_max/K_D ratios are significantly higher than that of [³H](R)-PK11195 in lentiviral encephalitis

A & B, The ratio of B_max/K_D (representing the binding potential) was significantly higher with either [³H]DAA1106 (black bars and line) or [³H](R)-PK11195 (clear bars and dotted line) in HIVE (n=5) compared to HIV non-encephalitis (HIV, n=6) and age-matched non-infected controls (Con, n=3) (A), and in SIVE (n=3) compared to SIV non-encephalitis (SIV, n=4) and non-infected controls (Con, n=3) (B). Data was analyzed using one-way ANOVA between the three conditions with either ligand. The B_max/ K_D ratio with [³H]DAA1106 was significantly higher compared to [³H](R)-PK11195 within each condition with the greatest obsereved differences in encephalitic tissues. Data was analyzed using student’s t test within the same condition, ***p<0.01**p<0.01, *p<0.05.