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. Author manuscript; available in PMC: 2009 Feb 1.
Published in final edited form as: Neurobiol Dis. 2007 Sep 7;29(2):232–241. doi: 10.1016/j.nbd.2007.08.016

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

Sriram Venneti 1, Guoji Wang 1, Clayton A Wiley 1
PMCID: PMC2258458  NIHMSID: NIHMS39293  PMID: 17920902

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 [3H]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 [3H]DAA1106 binds with high affinity to activated and infected macrophages in regions of synaptic damage. Further, binding affinity reflected by lower KD (dissociation constant) values and the Bmax (total number of binding sites) to KD ratios reflective of ligand-binding potential, were significantly higher with [3H]DAA1106 compared to the extensively characterized PBR ligand [3H](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.

Keywords: Peripheral benzodiazepine receptor, Macrophages, PET, DAA1106, HIV encephalitis, PK11195, SIV encephalitis

Introduction

HIV-associated neurological abnormalities are seen in approximately 25% of terminally ill AIDS patients (Cinque et al., 1997, Dore et al., 1999). Deficits observed in these individuals range from minor cognitive impairment, motor and psychiatric dysfunctions to frank dementia thought to result from neuronal damage (Nath et al., 2006). Neuronal and synaptic injury is attributed to viral proteins and toxic products derived from HIV-infected and activated infiltrating macrophages (Ellis et al., 2007). These infected and activated macrophages manifest in the brain as microglial nodules, multinucleated giant cells and perivascular macrophage infiltrates, histopathologically referred to as HIVE. SIV infection in macaques has been used to model HIVE with a variable percentage of macaques developing SIVE (Lackner et al., 1991). Since activated and infected macrophages in the brain are central to the pathology of lentiviral encephalitis, our goal is to image these cells in vivo using PET to enable the detection and progression of lentiviral encephalitis in the macaque model and eventually in human subjects.

Brain macrophages can be imaged in vivo using PET by taking advantage of increased expression of the peripheral benzodiazepine receptor (PBR) (Banati, 2002). Unlike the central benzodiazepine receptor, PBR is expressed at relatively low levels in the normal brain on resting brain macrophages and astrocytes (Cagnin et al., 2002). PK11195, the prototype PBR ligand, has been shown to cross the blood brain barrier and achieve specific binding to brain macrophages in both animal models and human subjects with neuroinflammation (Banati, 2002).

We and others have shown that activated brain macrophages can be labeled with [3H](R)-PK11195 in SIVE (Mankowski et al., 2003, Venneti et al., 2004) and that [11C](R)-PK11195 can be used to image activated brain macrophages in vivo using PET in macaques that develop encephalitis (Venneti et al., 2004). However, translation of these studies to human subjects has been less promising. [11C](R)-PK11195 PET retention in the brain did not differ between HIV infected patients on retroviral drugs with and without neurological deficits (Hammoud et al., 2005, Wiley et al., 2006). These findings may be in part due to decreased brain inflammation seen in neurologically impaired HIV infected subjects on antiretroviral therapy (ART) without frank dementia (Gray et al., 2003b, Gray et al., 2003a). It is also possible that [11C](R)-PK11195 shows low sensitivity of in detecting milder forms of neuroinflammation. Ligands that bind to PBR with higher affinity compared to PK11195 may show greater sensitivity in detecting milder forms of neuroinflammation. DAA1106 [N-(2,5-dimethoxybenzyl)-N-(4-uoro-2-phenoxyphenyl)-acetamide] is an aryloxyanilide derivative, that binds with higher affinity to PBR compared to PK11195 (Chaki et al., 1999, Zhang et al., 2003, Maeda et al., 2004). We hypothesized that DAA1106 would label activated brain macrophages in encephalitic brain tissues with higher binding affinity when compared to that of PK11195. We show that [3H]DAA1106 binding is higher in lentiviral encephalitis with greater Bmax/KD ratios compared to [3H](R)-PK11195 correlating with infected and activated macrophages in areas with synaptic damage. These data suggest that DAA1106 possesses binding characteristics that could lead to improved PET imaging of lentiviral encephalitis.

Methods

Archival human and macaque brain tissue

Brain tissue was obtained from the University of Pittsburgh brain bank. Neuropathological microscopic analysis was performed in all human and macaque brain tissues. Only brains that showed no evidence of opportunistic infections were chosen for analysis. Encephalitis was diagnosed on the basis of distribution of macrophage infiltrates, microglial nodules, multinucleated giant cells and abundant macrophages that immunostained for HIV gp41 or SIV gp110 in human and macaque cases respectively (Budka, 1991, Lackner et al., 1991). Frozen tissue from the basal ganglia of patients with HIVE (n=5), HIV infected, non-encephalitis (n=5) and non-infected controls (n=3) and from the frontal cortex of macaques with SIVE (n=3), SIV infected, non-encephalitis (n=4) and non-infected controls (n=3) were used (Table 1).

Table 1.

Demographics of archival human and macaque post mortem tissue

Neuropathological diagnosis ID # Age (years) Sex PMI (h) ART Cause of Death
HIV encephalitis 1021 31 M 21 None UN
3067 28 M 18 None Kaposi sarcoma lung
3099 36 M 23 None CMV adrenalitis
5087 47 M 24 None Aplastic anemia
9030 UN M 18 UN UN
HIV non-encephalitis 4044 43 M 20 None Hepatic failure due to cirrhosis
4084 39 M 10 None Cardiac failure
4097 33 M 12 AZT- 1year CMV adrenalitis
4144 32 M 20 None Acute peritonitis due to Small bowel perforation
9081 55 M 25 None Diabetic ketoacidosis
Control 3182 69 M <24 NA Acute non lymphocytic leukemia
3186 60 M <24 NA Surgical complications of liver transplant
3188 54 F <24 NA Myocardial infarction
Neuropathological diagnosis ID # Age (mo.) Sex Length of infection (days) Disease at time of sacrifice Clinical symptoms
SIV encephalitis 13901 34 M 56 AIDS Anorexia and diarrhea
14001 46 M 56 AIDS Anorexia and diarrhea
16103 59 M 119 AIDS Ataxia and pneumocystitis pneumonia
SIV non-encephalitis 10797 168 F 722 AIDS Anorexia
14101 22 M 101 AIDS Diarrhea
15201 39 M 30 Asymptomatic Diarrhea
17000 84 F 1062 AIDS Anorexia and diarrhea
Control 40500 UN M NA NA NA
42100 66 F NA NA NA
42200 UN UN NA NA NA
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PMI (h) - Post mortem interval in hours, M - male, F - Female, ART - Antiretroviral therapy, AZT - azidothymidine, CMV- cytomegalovirus, mo. - months, UN - Unknown, NA - Not applicable.

Tissue culture experiments

Primary human macrophage cultures were isolated from peripheral blood mononuclear cells isolated from HIV and Hepatitis B seronegative buffy coats obtained from the blood bank (Central Blood Bank, Pittsburgh, PA) using established protocols (Bergamini et al., 1999) and cultured for 7 days. Human fetal brain tissue was gathered according to the standards of the University of Pittsburgh ethics and biosafety guidelines. Primary human astrocyte and microglial cell were isolated as per established protocols (Lee et al., 1992, Barami et al., 2001). Astrocytes and microglia were treated with 1μg/ml LPS (Sigma, Sr. Louis, MO) for 48hrs. Astrocytes were additionally activated with 100 μM dibutyryl cyclic AMP (dB-CAMP) (Sigma) for 48hrs in parallel cultures. dB-CAMP has been extensively used to model in cell culture the morphological and proliferative changes seen in reactive astrocytosis(Padmanabhan et al., 1999, Abe et al., 2000). Mitochondrial extracts were obtained from primary human macrophages, microglia and astrocytic cultures utilizing a kit obtained from Pierce (Rockford, IL) based on the metrizamide gradient centrifugation method that yields 97.71% pure mitochondrial preparations (Taylor et al., 2003).

Filtration radioligand binding assays

(a) Saturation binding curves

Homogenized brain tissue samples (total protein concentration ranging from 150 to 200 μg) were incubated with 0.2 to 25 nM [3H]-DAA1106 (sp. act., 80 Ci/mmol; American Radiolabeled Chemical, St Louis, MO) or 0.5-100 nM [3H](R)-PK11195 (sp. act., 89.9 Ci/mmol; NEN Life Sciences Products, Boston, MA) at 4°C for 2 hr in a final volume of 250 μl of HEPES (4°C, pH 7.4). This was defined as total binding. Nonspecific binding was excluded by the inclusion of 10 μM DAA11106 or 10 μM PK11195 respectively.

(b) Specific binding analyses in mitochondrial extracts

Mitochondrial extracts derived from primary human astrocytes, macrophages and microglia were incubated with 1 nM [3H]DAA1106 and non-specific binding was excluded by the inclusion of 1 μM DAA1106.

In both the above experimental conditions the reaction was terminated by filtration through glass fiber filters (Brandel, Gaithersburg, MD) by the addition of HEPES (4°C, pH 7.4) in a vacuum cell harvester (Brandel). Filter bound radioactivity was counted in a liquid scintillation spectrometer (Perkin Elmer Life Sciences, Wellesley, MA). Specific binding at each concentration of 3H-ligand was defined as the difference between total binding and nonspecific binding. Specific binding in brain tissues ranged from 80-90% of total binding (nonspecific binding values at 1nM [3H]-ligand were ∼ 8% in macaque and ∼ 10% of total binding in human brain tissues respectively). All samples were run in duplicate. Bmax (fmols/mg protein) and KD (nM) were determined using PRISM software (Graphpad, San Diego, CA). Bmax/KD ratios (representing a measure of the binding potential of each ligand) were obtained to compare both ligands.

Immunohistochemistry

Immunostaining and laser confocal microscopic imaging was performed as described previously (Venneti et al., 2004). Paraffin sections containing the frontal cortex in the same macaques from which frozen tissue was obtained were used. Sections were stained with mouse monoclonal antibodies against CD68 (marker for activated macrophages, 1:50,000, DAKO, Carpinteria, CA) or MAP-2 (1:1500, Sternberger Inc., Lutherville, MD), or synaptophysin (1:100, DAKO), or GFAP (1:500, DAKO) or rabbit polyclonal antibody against the SIV envelope gp110 (5 μg/ml, gift from Dr. Kelly Stefano Cole, University of Pittsburgh, Pittsburgh, PA) with secondary Cy5-conjugated goat anti-mouse IgG and Cy3-conjugated goat anti-rabbit IgG (1:200, Jackson ImmunoResearch Laboratories, Inc.). Immunostained sections were scanned and quantified on a laser confocal microscope equipped with an argon laser with 458, 477, 488, and 514 nm primary emission lines (LSM 150; Carl Zeiss GmbH, Heidelberg, Germany). Each section was scanned along the z-axis to define the middle optical plane used in quantification (262,144 pixels/plane; 1 pixel = 0.25 mm2). Scanning parameters such as laser power aperture, gain, and photomultiplier tube settings were kept constant for each wavelength. An individual blinded to the experimental design imaged ten areas (x40) encompassing 106,100 μm2. For each cell phenotype scanned, contribution to signal intensity from autofluorescence was minimized using a threshold that was kept constant. In each area the average pixel fluorescence along with the pixel counts for a given cell phenotype marker that exceeded the threshold were enumerated. The average pixel fluorescence was multiplied by the total number of pixels to measure the total fluorescence for that cell phenotype marker in that area. The total fluorescence values determined from the ten scanned areas in one brain region were averaged to represent a measure of the cell phenotype in that brain region.

Autoradiography

Autoradiography was performed as described(Venneti et al., 2004). Briefly, 15 μm thick frozen brain sections were placed on SuperfrostTm glass slides (Sigma) and incubated in 50 mM HEPES containing 1 nM [3H]DAA1106 for 30 min. Specificity of binding was ensured by the inclusion of 1 μM DAA1106 in parallel sections. The sections were mounted with a layer of autoradiographic LM-1 emulsion (Amersham, UK), were developed after 4 weeks. We combined immunostaining with autoradiography to evaluate the cellular localization of [3H]DAA1106. Sections were first immunostained and then processed for autoradiography, following which they were imaged on the confocal microscope.

Statistical analysis

Data were analyzed using PRISM software (Graphpad, San Diego, CA). Students t test or one-way ANOVA tests with post-test Bonferroni correction with 95% confidence intervals were used to analyze data. Non parametric correlations using 95% confidence intervals were performed to quantify the relationship between [3H]DAA1106 binding and various immunohistochemical markers represented by r, the Spearman’s coefficient.

Results

[3H]DAA1106 binding is higher in lentiviral encephalitis compared to controls

Bmax and KD values reflective of the total number of binding sites per mg protein and binding affinity of [3H]DAA1106 to PBR respectively were determined in post mortem brain tissue derived from the basal ganglia from HIVE, HIV infected non-encephalitis and age-matched non-infected cases. Bmax with [3H]DAA1106 was significantly higher in HIVE compared to controls (Figure 1A-C & table 3, p=0.0003). KD, the affinity of binding of [3H]DAA1106 did not differ in all these conditions (Figure 1D & table 3, p=0.0712). Similar results were obtained in SIVE compared to SIV infected non-encephalitis and control uninfected tissues obtained from the frontal cortex (Figure 2C & table 3, Bmax, p=0.0038 and 2D, KD, p=0.2492). These results were confirmed by performing autoradiography on brain tissue obtained from the frontal cortex of SIVE macaques (Figure 2A and B).

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

Figure 1

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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 Bmax, and slope represents KD) with [3H]DAA1106 from HIVE (black squares), HIV no-encephalitis (grey squares) and an age-matched control (clear squares).

C, The Bmax (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 KD (nM), reflective of the ligand binding affinity was not significantly different in all three conditions, p=0.0712. Data was analyzed using ANOVA.

Table 3.

Comparison of [3H](R)-PK11195 and [3H]DAA1106 Bmax and KD values in human and macaque postmortem tissues

Bmax (fmols/mg) KD (nM)
Condition [3H]DAA1106 [3H](R)-PK11195 p value [3H]DAA1106 [3H](R)-PK11195 p value
Control 1775 ± 46 2030 ± 74 0.0526 3.5 ± 0.5 17 ± 0.2 0.0004
SIV 1856 ± 165 2299 ± 149 0.1290 4.8 ± 0.9 14 ± 1.8 0.0080
SIVE 5631 ± 946** 4815 ± 626** 0.5238 6.4 ± 1.3 18 ± 4.3 0.0320
Control 536 ± 34 649 ± 275 0.6280 3.4 ± 0.6 24 ± 1.0 0.0002
HIV 448 ± 69 848 ± 139 0.1010 3.3 ± 0.5 32 ± 2.6 0.0004
HIVE 1158 ± 104*** 1660 ± 105*** 0.2074 4.8 ± 1.3 30 ± 4.1 0.0037
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Bmax (reflective of the total number of binding sites) and KD (inversely proportional to the binding affinity) were compared in macaque and human brain tissues using [3H]DAA1106 and [3H](R)-PK11195. With each ligand, Bmax values were significantly higher in encephalitis compared to controls (***p<0.01, **p<0.01 data was analyzed using ANOVA). Bmax values were not significantly different between [3H]DAA1106 and [3H](R)-PK11195 in any of the conditions. KD values were significantly lower with [3H]DAA1106 compared to [3H](R)-PK11195 in all the conditions (data was analyzed using students t test).

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

Figure 2

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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, Bmax (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 KD (nM) was not significantly different in all three conditions, p=0.2492, (D). Data was analyzed using ANOVA.

[3H]DAA1106 binding is higher in activated macrophages compared to controls in cell culture

We compared changes in [3H]DAA1106 binding in primary human macrophages, microglia and astrocytes with and without activation. Primary human microglia and macrophages were activated with LPS (Figure 3B and C, untreated controls shown in insets). Astrocytic cultures were activated with dB-cAMP (Figure 3A, untreated control shown in inset). Astrocytes treated with LPS served as additional controls. As PBR is a mitochondrial receptor (Casellas et al., 2002), we assessed changes in [3H]DAA1106 binding in mitochondrial extracts obtained from each of these conditions. [3H]DAA1106 binding did not differ significantly in astrocytes activated with dB-cAMP or LPS from untreated cultures (Figure 3D). In contrast, [3H]DAA1106 binding was significantly higher in both LPS-activated macrophage and microglial cultures compared to untreated controls and all the astrocytic conditions (Figure 3D).

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

Figure 3

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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.

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

We wanted to determine the relative contributions of astrocytes and activated brain macrophages to [3H]DAA1106 binding. Since macrophages are the predominant infected cells in the brain in lentiviral encephailitis (Ellis et al., 2007), we also determined the association between infected macrophages and [3H]DAA1106 binding in the brain. We combined immunostaining for astrocytes (GFAP), activated brain macrophages (CD68) and the SIV envelope protein gp110 with [3H]DAA1106 autoradiography on frozen brain sections obtained from the frontal cortex of SIVE macaques. [3H]DAA1106 binding overlapped with CD68 labeled activated macrophages (Figure 4A) and SIV-infected macrophages (Figure 4C), but not with GFAP labeled astrocytes (Figure 4B). Similar results including colocalization of HIV infected cells with [3H]DAA1106 autoradiography were seen in HIVE brain tissues (Figure 4D).

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

Figure 4

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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.

Next, we tested whether [3H]DAA1106 binding in homogenized brain tissue correlated with the abundance of astrocytes or activated macrophages or SIV-infected macrophages labeled with GFAP, CD68 and SIV gp110 respectively. Each cell-type was quantified using confocal microscopy and correlated with [3H]DAA1106 Bmax values obtained from the same brain regions in the same macaques. [3H]DAA1106 binding correlated significantly with the abundance of SIV-infected macrophages (r=0.9646; p=0.0084) and activated macrophages (r=0.8308; p=0.0160) but weakly with the abundance of GFAP-stained astrocytes (r=0.6755; p=0.0958) (Table 2).

Table 2.

[3H]DAA1106 binding correlates with activated and infected brain macrophages and areas of synaptic damage

Parameter Marker r value p value
Activated macrophages CD68 0.8308 0.0106
Infected macrophages SIV gp110 0.9646 0.0084
Reactive astrocytosis GFAP 0.6755 0.0958
Presynaptic protein SYN -0.7806 0.0383
Postsynaptic protein MAP-2 -0.7868 0.0205
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[3H]DAA1106 Bmax values in SIVE and SIV brain tissues were correlated with changes in the abundance of activated (CD68) and infected macrophages (SIV gp110) or (GFAP) or with the synaptic markers (SYN and MAP-2) in the frontal cortical of the same cases. Increases in [3H]DAA1106 binding correlated best with activated and infected macrophages. Increase in [3H]DAA1106 binding correlated significantly with decreases in both presynaptic and postsynaptic markers.

Decreases in the presynaptic protein SYN and the postsynaptic protein MAP-2 correlate with increases in [3H]DAA1106 binding

The presynaptic protein SYN and the postsynaptic protein MAP-2 were quantified in macaque brain tissues. Both proteins were decreased in SIVE compared to controls (Figure 5). Decreases in SYN (r= - 0.7806, p=0.0383) and MAP-2 (r= - 0.7868, p=0.0205) correlated with increases in [3H]DAA1106 Bmax values (Table 2)

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

Figure 5

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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.

Binding affinities (reflected by low KD values) and Bmax/KD ratios with [3H]DAA1106 are higher than that with [3H](R)-PK1195 in lentiviral encephalitis

Bmax and KD values with [3H]DAA1106 and [3H](R)-PK1195 were compared in macaque and human tissues. Bmax values did not differ between [3H]DAA1106 and [3H](R)-PK1195 in all conditions (table 3). KD values with [3H]DAA1106 were significantly lower compared to [3H](R)-PK1195 in all conditions assessed, suggesting higher affinity with DAA1106 compared to PK11195 (table 3). Bmax/KD ratios representing the binding potential of either ligand, was significantly higher with both [3H]DAA1106 and [3H](R)-PK11195 in HIVE compared to HIV non-encephalitis and age-matched non-infected controls (Figure 6A). Within each group Bmax/KD ratio with [3H]DAA1106 was significantly higher compared to [3H](R)-PK11195 with the greatest differences obsereved in HIVE. Similar results were observed in SIVE compared to controls (Figure 6B).

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

Figure 6

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A & B, The ratio of Bmax/KD (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 Bmax/ KD 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.

Discussion

Despite the absence of productive infection of neurons lentiviral encephalitis is characterized by progressive neurodegeneration. Neuronal and synaptic damage has been hypothesized to be mediated by neurotoxins derived from activated and infected macrophages that infiltrate the brain (reviewed in (Ellis et al., 2007)). Since brain macrophages are key to the pathogenesis of lentiviral encephalitis, imaging these cells in vivo will enable detection and progression of encephalitis. DAA1106 is a ligand that binds to PBR enriched in activated macrophages, but present in low levels in the normal brain on astrocytes and resting brain macrophages ((Banati, 2002, Venneti et al., 2006)). Our data show that both HIVE and SIVE brain tissues have significantly higher [3H]DAA1106 binding compared to controls, which in macaque tissues correlated with the abundance of activated and SIV-infected macrophages, but not with the abundance of reactive astrocytes. Further, decreases in both the presynaptic protein SYN and the postsynaptic protein MAP-2 correlated with increases in [3H]DAA1106 binding in SIVE, suggesting that regions showing increased [3H]DAA1106 binding labeled areas with synaptic damage. Finally, the binding potential of [3H]DAA1106, reflected by the Bmax/KD ratio was significantly higher when compared with that of [3H](R)-PK11195 in the same brain region in the same cases in the both HIVE and SIVE suggesting that DAA1106 possesses better binding characteristics over PK11195 for labeling activated and infected macrophages in lentiviral encephalitis. The choice of brain tissues as the basal ganglia in human subjects and the frontal cortex was based on tissue availability. Histopathology in both human subjects and macaques suggest that lentiviral encephalitis does not target a specific area of the brain. However, prominent involvement of basal ganglia has been suggested in human subjects (Brew et al., 1995), while in macaques a more diffuse pattern of disease has been noted (Lackner et al., 1991). Despite these differences, the pathology of disease is both humans and macaques is similar, suggesting that our data obtained from the basal ganglia of human subjects and from the frontal cortex of macaques are comparable.

We have shown that [11C](R)-PK11195 can image activated brain macrophages in macaques with terminal AIDS that develop SIVE using PET (Venneti et al., 2004). However, no differences were seen in [11C](R)-PK11195 brain retention in HIV infected individuals with or without neurological deficits (Hammoud et al., 2005, Wiley et al., 2006). Although, subjects in these two studies showed neurological impairments, none of the patients had frank dementia. Therefore, none of these subjects would be expected to have HIVE. Further, most of the patients in both these studies were on ART. Indeed, the incidence of HIVD has been reported to decrease in HIV infected patients on ART. However, the prevalence of HIVD may increase due to the longer life spans of HIV infected patients on ART (Geraci et al., 2001, Valcour et al., 2004). The neuropathology seen in these patients is also different from frank encephalitis: subtle degeneration of dendritic arbors and interneuron populations (Langford et al., 2003), suggesting that activation of brain macrophages in these subjects may be less severe until progression to encephalitis and frank dementia. Analogous to these findings, [11C](R)-PK11195 retention in the brain does not differ in normal subjects when compared with patients with minor cognitive impairment, a condition thought to progress to Alzheimer’s disease in approximately 50% of subjects (Schuitemaker et al., 2004, Schuitemaker et al., 2006). These data suggest that [11C](R)-PK11195 may not be able detect milder forms of neuroinflammation highlighting the significance of developing newer ligands that show high specificity and sensitivity for PET imaging of activated brain macrophages in HIVE. The Bmax/KD ratio (reflective of the binding potential of a PET ligand (Mintun et al., 1984)) with [3H]DAA1106 was significantly higher when compared with that of [3H](R)-PK11195 in both SIVE and HIVE, suggesting that DAA1106 may be able to address some of these issues. However, It is also possible that the lack of increase of [11C](R)-PK11195 binding in HIV-infected patients with neurological signs could be due to the effects of ART in potentially decreasing brain inflammation, in which case either ligand would be insensitive. Future studies in SIV-infected macaques and HIV-infected patients will be able to address these issues.

The major cell type that DAA1106 binds to in neuroinflammatory conditions is not known. We show that [3H]DAA1106 binds mainly to activated and infected brain macrophages in SIVE brain tissue. This is further substantiated by significant correlations of [3H]DAA1106 binding with areas of synaptic damage, since activated and infected macrophages are thought to mediate synaptic injury in lentiviral encephalitis (Masliah et al., 1997, Ellis et al., 2007). Some studies report significant contributions from astrocytes to [3H](R)-PK11195 binding in animal treated with neurotoxins and tissue culture systems (Itzhak et al., 1994, Kuhlmann et al., 2000, Chen et al., 2004). However, we found that the majority of [3H]DAA1106 binding correlated with activated and infected macrophages in SIVE brain tissues correlating weakly with the abundance of astrocytosis.

PBR is an outer mitochondrial membrane protein in a hetero-oligomeric complex comprised of the voltage-dependent anion channel and the adenine nucleotide carrier (Casellas et al., 2002). We found increases in [3H]DAA1106 binding in mitochondrial fractions derived from LPS activated macrophages and microglia compared to controls and activated and control astrocytes. Further, no differences were observed in [3H]DAA1106 binding between LPS activated primary macrophages and microglia suggesting that PBR binding sites do not differ between activated resident microglia and activated infiltrating macrophages in the brain.

PK11195 has been extensively used to image activated brain macrophages in several neurological disorders. However, the specific binding signal with [11C](R)-PK11195 is generally low and challenging to quantitative with typical PET image noise levels (Petit-Taboue et al., 1991, Groom et al., 1995, Banati et al., 2000, Pappata et al., 2000). Another consideration is that [11C](R)-PK11195 shows increased binding in regions traditionally not associated with activated brain macrophages in regions such as the thalamus in Alzheimer’s disease (Cagnin et al., 2001), Parkinson’s disease (Ouchi et al., 2005) and Huntington’s disease (Pavese et al., 2006), and the occipital cortex in amyotrophic lateral sclerosis (Turner et al., 2004). Since it is not possible to confirm the histopathological presence of activated brain macrophages in these regions in human PET studies, it is possible that these findings reflect activated brain macrophages in regions connected by synapses to areas of pathology. Alternatively, they may represent regional variations in the constitutive PBR or some degree of non-uniform non-specific binding in the CNS. These concerns along with the possibility of low sensitivity in milder forms of neuroinflammation highlight the importance of developing newer ligands that show more specificity and sensitivity to activated brain macrophages for PET imaging in diseases such as HIVE. [3H]DAA1106 binds with higher Bmax/KD ratios in comparison to PK11195 to activated and infected macrophages in lentiviral encephalitis in areas of synaptic damage suggesting that DAA1106 possesses better binding characteristics over PK11195 for labeling activated and infected macrophages in lentiviral encephalitis. It is possible that filtration binding experiments in brain tissues may not be entirely reflective of in vivo properties of the ligand. Various parameters such as the ability of a given ligand to cross the blood brain barrier, in vivo binding kinetics to PBR, issues of in vivo non specific binding and the rate of metabolism of the ligand influence the utility of the ligand in question as a PET tracer. We are currently conducting experiments in macaques infected with SIV using [11C]DAA1106 to address these points. Nevertheless, the assertion that a PET radioligand with potential clinical utility is binding to the intended cellular component; activated and infected brain macrophages, and that changes in measured Bmax values determined using a standardized assay are observed in a manner that is consistent with the known disease pathology is an important validation that is often overlooked or bypassed in the progression to human investigational studies. Overall, these results suggest that DAA1106 has binding characteristics that might offer improved sensitivity to image activated and infected macrophages in lentiviral encephalitis in vivo using PET. Further studies in HIV infected human subjects with and without neurological impairments are required to fully characterize the potential enhancements of DAA1106.

Acknowledgements

We thank James Kasenchak, Jason Nugyen and Susan Slagel for technical help, Brian Lopresti for helpful comments, Dr. Stephanie J. Bissel for banking macaque brain tissues, Dr. Ronald L. Hamilton, Jonette Werley and the University of Pittsburgh brain bank for human post mortem tissues. This work was supported by the National Institutes of Health: RO1 MH64921 (CAW), K24 MH01717 (CAW).

Footnotes

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