Characterization of monoamine oxidase-B (MAO-B) as a biomarker of reactive astrogliosis in Alzheimer’s disease and related dementias

8-μm-thick formalin-fixed paraffin-embedded (FFPE) tissue sections from the temporal [including Brodmann area (BA) 38], frontal (BA8/9), and occipital cortex (BA17/18), and cerebellum of n = 52 donors spanning the AD neuropathological continuum from normal aging brain (CTRL, “not AD/low ADNC burden”) to severe AD (high ADNC burden) were obtained from the Massachusetts Alzheimer’s Disease Research Center (MADRC) Neuropathology Core Brain Bank. FFPE sections from the hippocampus of selected donors were also used for illustration purposes. Table 1 depicts the demographic (age, sex) and neuropathological (Thal amyloid phase, CERAD neuritic plaque score, and Braak neurofibrillary tangle stage) characteristics, and the APOE genotype of the n = 52 study donors split by ADNC burden [32]. Frozen samples from the cerebellum were also available for n = 50 donors for DNA purification and MAOB SNP genotyping. We validated the main findings of this study in a separate sample of n = 51 donors with intermediate or high ADNC burden, for which quantitative neuropathological data was available [44]. Specifically, we used Aβ+ area fraction, cortical thickness, and stereology-based count data of pTau+ (PHF1) neurofibrillary tangles, GFAP+ astrocytes, and CD68+ microglia from the temporal association neocortex of this validation sample, which were obtained following procedures detailed elsewhere [44].

In addition, 8-μm-thick FFPE sections from the frontal association cortex (BA8/9) of n = 70 donors with a primary neuropathological diagnosis of ADRD—comprising n = 30 LBD (including five with brainstem/limbic and 25 with neocortical Lewy body stage), n = 30 FTLD-Tau (including ten of each PiD, PSP, and CBD), and n = 10 FTLD-TDP—were also obtained from the MADRC Neuropathology Core Brain Bank (Table 2). Donors or their next-of-kin gave a written informed consent for the brain donation and this study was conducted under the MADRC Neuropathology Core Institutional Review Board.

To determine the cell type expressing MAO-B, we performed double fluorescent immunohistochemistry with antibodies for MAO-B and cell-type-specific markers on FFPE sections from the temporal association cortex of selected CTRL and AD donors followed by laser confocal microscopy. Briefly, FFPE sections were dewaxed in xylenes and rehydrated in decreasing concentrations of ethanol. Next, sections were microwaved in boiling citrate buffer (0.01 M, pH 6.0 with 0.05% Tween20) for 20 min at 95 °C for antigen retrieval. Subsequently, sections were blocked with 10% normal donkey serum (NDS) in Tris-buffered saline (TBS) for 1 h at room temperature (RT) prior to incubation with primary antibodies in 5%NDS/TBS at 4 °C overnight. On the following day, sections were washed with TBS before incubation with secondary antibodies at 1:200 in 5%NDS/TBS for 2 h at RT, washed with TBS, and counterstained with Thioflavin-S (ThioS, Sigma, T1892) by immersion in 0.05% ThioS in 50% ethanol for 8 min, followed by differentiation with 80% ethanol for 10 s. Finally, sections were incubated with Autofluorescence Eliminator Reagent (Millipore, 2160) for 5 min at RT to quench endogenous tissue autofluorescence, cleared with serial 70% ethanol washes of 20 s each, rehydrated in TBS, and coverslipped with Fluoromount-G mounting media with DAPI (Southern Biotech, 0100-01). The following primary antibodies were used: rabbit anti-MAO-B monoclonal antibody (raised against amino acids 1-100 of human MAO-B, clone EPR7102, Abcam, ab133270, 1:500), mouse anti-Aβ monoclonal antibody (clone 6E10, BioLegend, 803003, 1:100), mouse anti-ALDH1L1 monoclonal antibody (clone N103/39, Millipore, MABN495, 1:500), mouse anti-CD31 monoclonal antibody (clone 89C2, Cell Signaling Technology, #3528, 1:100), mouse anti-GFAP monoclonal antibody (clone G-A-5, Sigma, G3893, 1:1000), goat anti-IBA1 polyclonal antibody (Abcam, ab107159, 1:100), mouse anti-microtubule-associated protein 2 (MAP2) monoclonal antibody (clone SMI-52, Biolegend, 801801, 1:250), mouse anti-myelin basic protein (MBP) monoclonal antibody (clone 1, Millipore, MAB382, 1:500), mouse anti-platelet derived growth factor receptor-β (PDGFRβ) monoclonal antibody (clone D-6, Santa Cruz Biotechnology, sc-374573, 1:200). Secondary antibodies included a donkey anti-rabbit Cy3-conjugated antibody (Jackson ImmunoResearch) for MAO-B and species-appropriate donkey antibodies conjugated with Cy5 (ThermoFisher Scientific) for all other primary antibodies.

For quantitative analyses, we immunostained FFPE sections from all donors with the peroxidase-3,3′-diaminobenzidine (DAB) method using the BOND Polymer Refine Detection kit (Leica Biosystems, DS9800) in a Leica BOND RX Fully Automated Research Stainer (Leica Biosystems). Specifically, the following primary antibodies were used: rabbit anti-MAO-B monoclonal antibody (clone EPR7102, Abcam, ab133270, 1:1000), rabbit anti-Aβ monoclonal antibody (clone D52D4, Cell Signaling Technology, 1:2000), mouse anti-CD68 monoclonal antibody (clone KP-1, Dako, M0814, 1:500), mouse anti-GFAP monoclonal antibody (clone G-A-5, Sigma-Aldrich, G3893, 1:20,000), and mouse anti-pTau^Ser396/404 monoclonal antibody (clone PHF1, kind gift from Dr. Peter Davies, 1:500). MAO-B immunohistochemistry was performed in all four regions (temporal, frontal, occipital, and cerebellum) of all donors in Table 1 except for one donor for which cerebellum was not available, in the hippocampus of selected donors in Table 1, as well as in frontal sections from all donors in Table 2. Aβ, CD68, GFAP, and pTau immunostains were only performed in temporal cortex sections from all donors in Table 1. The automated immunohistochemistry protocol consists of: (1) baking at 80 °C for 60 min; (2) dewaxing using BOND Dewax solution (Leica Biosystems) followed by rehydration; (3) heat-induced epitope retrieval (HIER) with citrate (ER1) at 100℃ for either 20 min (MAO-B, Aβ, and pTau) or 30 min (CD68) except for GFAP, which did not require this step; (4) blocking of endogenous peroxidase activity with a 4% (v/v) hydrogen peroxide solution in methanol for 20 min; (5) primary antibody incubation at 37℃ for 35 min except for anti-CD68 antibody, which was incubated for 60 min; (6) secondary antibody incubation with post-primary solution containing 10 g/mL Poly-HRP IgG in 0.01% 2-methylisothiazol-3(2H) for 10 min followed by polymer solution containing 25 g/mL Poly-HRP IgG in 0.01% 2-methylisothiazol-3(2H) for 8 min; (7) DAB substrate-peroxidase reaction for 1 min; (8) counterstaining with 0.1% hematoxylin for 15 min; and (9) washes with wash buffer and double-distilled water. Immunostained sections were dehydrated in increasing concentrations of ethanol and coverslipped with Permount mounting media (Fisher Scientific, SP15-500).

To genotype the MAOB rs1799836 SNP, we used a commercially available Taqman PCR assay on genomic DNA extracted from frozen cerebellar samples. Briefly, we purified genomic DNA from ≈ 25 mg of frozen cerebellum using the PureLink Genomic DNA Extraction Mini Kit (ThermoFisher Scientific, K182002) following manufacturer’s instructions. We measured the DNA concentration in a DS-11 spectrophotometer (DeNovix Inc) and prepared 1.8 ng/μL working dilutions for the Taqman PCR assay. The latter reaction volume was 25 μL comprising 1.25 μL of 20 × TaqMan MAOB rs1799836 genotyping assay (ThermoFisher Scientific, Assay ID C—8878790_10), 12.50 μL of 2 × TaqMan Fast Universal PCR Master Mix, no AmpErase UNG (Thermo Scientific, 4352042), and 11.25 μL of DNA sample (20 ng). DNA samples were run in duplicates in 96-well plates (Bio-Rad) in a Bio-Rad CFX96 Touch Real-Time PCR Detection System with the following protocol: 95 °C × 10 min (ramp 1 °C/s), 95 °C × 15 s, and 60 °C × 1 min, for 45 cycles. Principal component analysis of VIC versus FAM fluorescence [corresponding to base A (major allele) vs. G (minor allele), respectively] enabled allele discrimination and genotype assignment (AA, AG, or GG).

To validate the specificity of the anti-MAO-B antibody used in the immunohistochemical studies, we performed western blot in MAOB-overexpressing and knock-down human cell line lysates and human recombinant MAO-A and MAO-B proteins. The human recombinant MAO-A protein was purchased from LS-Bio (LS-G3233), whereas the human recombinant MAO-B protein was purchased from Abcam (ab82944), both purified from Escherichia coli with a 6xHis-tag. The human cell line lysates were kindly provided by Dr. Boyang (Jason) Wu (Washington State University, Spokane, WA) and consisted of lysates from PrSC human prostate stroma cells stably expressing either a control plasmid or a MAOB plasmid, and lysates from HepG2 human liver carcinoma cells stably expressing either a control shRNA or one of two anti-MAOB shRNAs [50]. Briefly, sample protein concentration was measured with the Pierce BCA Protein Assay Kit (ThermoFisher Scientific, 23225). After boiling at 95 °C for 5 min with 10X sample reducing agent (NuPAGE, ThermoFisher Scientific, NP0009) and 4X protein sample loading buffer (LI-COR Biosciences, P/N: 928-40004) for protein denaturalization, samples (4 μg for recombinant proteins, 10 μg for cell lysates) were loaded onto a 4–12% Bis–Tris gradient gel (NuPAGE, ThermoFisher Scientific, NP0343BOX) together with Precision Plus Dual Color Protein Standards (Bio-Rad, 1610374). SDS-PAGE was run in MOPS SDS running buffer (NuPAGE, ThermoFisher Scientific, NP0001) at 130 V for ~ 2 h. The gel proteins were transferred to a nitrocellulose membrane using a wet method at 310 mA for 2 h in ice. The membrane was blocked with Intercept Blocking Buffer (TBS) (LI-COR Biosciences, P/N 927-50000) for 1 h at RT, followed by incubation with primary antibodies in the Intercept Blocking Buffer (TBS) overnight at 4 °C. We used a mouse monoclonal anti-β-actin clone AC-15 (Sigma-Aldrich, A5541, 1:3000) as housekeeping loading control. Next day, the membrane was washed with TBS-Tween20 and incubated with the appropriate near-infrared fluorescent secondary antibodies (LI-COR Biosciences, 1:5000) for 1 h at RT. Finally, the membrane was thoroughly washed with TBS-Tween20 followed by TBS and scanned in an Odyssey CLx Imager (LI-COR Biosciences).

We classified donors in three groups corresponding to their ADNC burden [32]. Normality of all datasets was determined by D’Agostino-Pearson Omnibus test. We ran one-way ANOVA with Tukey’s multiple comparison test if all datasets were normally distributed and Kruskal–Wallis ANOVA with Dunn’s multiple comparison test if one or more datasets were non-normally distributed. For correlation analyses between MAO-B area fraction and other neuropathological measures, we used Spearman’s rank correlation tests (since MAO-B area fraction was non-normally distributed) and simple linear regression. To investigate the effects of sex and MAOB rs1799836 SNP genotype on MAO-B area fraction, we used two-way ANOVA with ADNC burden and either sex or MAOB rs1799836 SNP genotype as factors and the sex × ADNC burden or genotype × ADNC burden interaction terms. To examine the independent associations between MAO-B expression and all other neuropathological measures, we built multiple linear regression models with temporal cortex MAO-B area fraction as outcome variable; temporal cortex Aβ, pTau, GFAP, and CD68 measures, and cortical thickness as independent variables; and age at death as co-variate (to account for possible influences of aging on cortical thickness, GFAP immunoreactivity, and diffuse Aβ deposits). Lastly, for the plaque-centered analyses, we used mixed effects models with MAO-B or GFAP area fraction as outcome variable, diagnosis (CTRL vs. AD) and location (sham or ThioS+ plaque, ≤ 50 μm peri-plaque halo, or > 50 μm), with or without a diagnosis × location interaction term, as fixed effects, and controlling for donor ID (random effect). The significance level was set at a two-sided p < 0.05 in all statistical analyses. All statistical analyses and graphs were performed with GraphPad Prism version 9.4.1 (GraphPad Inc., La Jolla, CA) except mixed-effect models, which were run in STATA version 15.0 (StataCorp, LLC., College Station, TX).

We first characterized the ADNC and reactive gliosis of CTRL and AD donors via quantitative immunohistochemistry for Aβ, pTau (PHF1, pTau^Ser396/404), reactive astrocytes (GFAP), and reactive microglia (CD68), and also measured the cortical thickness in the temporal association neocortex. Figure 1 shows representative images of these immunostainings and the results of these neuropathological quantitative analyses by ADNC burden. As expected, high ADNC burden donors exhibited significantly higher levels of Aβ plaques and pTau relative to CTRL (i.e., not AD/low ADNC burden), with intermediate ADNC burden donors displaying intermediate levels between CTRL and definite AD donors. High ADNC burden donors had significantly more severe atrophy (i.e., lower cortical thickness) than intermediate ADNC burden and not AD/low ADNC burden donors, who did not differ in cortical thickness. Lastly, we found a marginally significant (p = 0.0590) increase in GFAP+ area fraction and a significant increase in CD68+ area fraction in high ADNC burden versus not AD/low ADNC burden donors, with intermediate ADNC burden donors exhibiting intermediate levels of reactive gliosis. Thus, we concluded that this sample is representative of the normal aging-severe AD neuropathological continuum.

To validate the specificity of the MAO-B antibody used for immunohistochemistry, we performed western blot on lysates from human cell lines stably overexpressing or downregulating MAOB as well as on recombinant MAO-B and MAO-A proteins. Western blot revealed a single band of ~ 59 kDa which increased upon MAOB overexpression and virtually disappeared upon MAOB silencing with specific shRNAs in human cell lines. Moreover, recombinant MAO-B produced a higher band (~ 62 kDa) due to its His-tag, whereas no band was detectable with recombinant MAO-A, thus confirming the specificity of the antibody for MAO-B (Supplemental Fig. S1).

Next, to determine the cell type(s) expressing MAO-B, we performed double fluorescence immunohistochemistry and confocal microscopy for MAO-B and various cell type-specific markers on FFPE sections from the temporal association cortex. Co-immunostaining of MAO-B with GFAP and ALDH1L1 revealed colocalization of MAO-B with astrocytes in both CTRL (Fig. 2 and Supplemental Fig. S2) and AD donors (Fig. 3 and Supplemental Fig. S3). In CTRL brains, MAO-B was predominantly expressed by both subpial and perivascular astrocytes in the cortex (Fig. 2) as well as by fibrous astrocytes, particularly those surrounding blood vessels, in the white matter (Supplemental Fig. S2). In the AD cortex, MAO-B was highly expressed by many GFAP+ reactive astrocytes surrounding Aβ plaques (Fig. 3), whereas in the AD white matter it was apparent in both perivascular and non-perivascular astrocytes (Supplemental Fig. S3). In hippocampal and cerebellar sections immunostained with the peroxidase-DAB method we had noted that only rare pyramidal neurons were immunoreactive for MAO-B in the hippocampal CA1 region from either CTRL or AD donors, whereas many granular cells in the dentate gyrus did express MAO-B (Supplemental Fig. S4a) and cerebellar Purkinje cells exhibited very limited or no immunostaining in both CTRL and AD donors (Supplemental Fig. S4b). Confocal microscopy confirmed very low MAO-B expression, when detectable, in cortical pyramidal MAP2+ neurons, and no expression at all in IBA1+ microglia, MBP+ oligodendrocytes, or CD31+ endothelial cells in either cortex (Figs. 2 and 3 and Supplemental Fig. S5) or white matter (Supplemental Figs. S2 and S3). Similarly, cortical PDGFRβ+ pericytes (Supplemental Fig. S6) did not appear to express MAO-B. Negative control images of sections immunostained only for MAO-B ruled out “bleeding through” as the explanation for the colocalization with astrocyte markers (not shown).

Once established that MAO-B is mainly expressed by cortical and white matter astrocytes, we sought to quantify MAO-B expression via automated immunohistochemistry with the peroxidase-DAB method, and measure MAO-B+ area fraction in both cortex and white matter of temporal, frontal, occipital, and cerebellar regions. We found significantly higher MAO-B expression in AD (high ADNC burden) than in CTRL (not AD/low ADNC burden) in temporal cortex and white matter, frontal cortex and white matter, and occipital cortex, whereas MAO-B+ area fraction did not significantly differ across ADNC groups in occipital white matter, cerebellar cortex, and cerebellar white matter (Fig. 4). Of note, the cerebellar cortex displayed the lowest MAO-B expression of all regions-of-interest in all donors, very close to zero. Also of note, MAO-B+ area fraction did not differ by sex (Supplemental Fig. S7) and did not significantly correlate with postmortem interval (Supplemental Fig. S8) or age at death (data not shown).

Next, to determine the possible drivers of MAO-B expression level, we investigated correlations between MAO-B+ area fraction and ADNC burden (Aβ+ and pTau+ area fractions), cortical thickness, and reactive gliosis (GFAP+ and CD68+ area fractions for reactive astrocytes and microglia, respectively) in the temporal neocortex. Univariate analyses (Spearman’s rank test) revealed statistically significant positive correlations between MAO-B+ area fraction and Aβ +, pTau +, GFAP +, and CD68+ area fractions as well as a statistically significant negative correlation between MAO-B+ area fraction and cortical thickness (Fig. 5 and Table 3). A multivariable linear regression analysis with MAO-B+ area fraction as dependent variable; Aβ +, pTau +, GFAP +, and CD68+ area fractions, and cortical thickness as independent variables, and adjusted by age at death confirmed that MAO-B expression level is independently associated with pTau+ and CD68+ area fractions and showed trends toward a positive independent association with GFAP+ area fraction (p = 0.0997) and a negative association with cortical thickness (p = 0.0627), but no significant association with Aβ+ area fraction (Table 4).

It should be noted that the Aβ+ area fraction encompasses both diffuse and compact Aβ plaques, and that compact, Thioflavin-S+ (ThioS +) dense-core Aβ plaques are known to trigger more prominent reactive gliosis around them than diffuse deposits [25, 39, 45]. Indeed, MAO-B+ astrocytes appeared to localize around ThioS+ dense-core plaques (Supplemental Fig. S9), whereas diffuse ThioS-Aβ plaques present in some CTRL donors (not AD/low ADNC in Fig. 1b) did not seem to trigger MAO-B upregulation by astrocytes (Supplemental Fig. S10). To unequivocally determine whether MAO-B expression is increased in reactive astrocytes around dense-core plaques, we performed double fluorescent immunohistochemistry for MAO-B and GFAP with ThioS counterstaining in temporal cortex sections from n = 10 AD (high ADNC) and n = 10 CTRL (not AD/low ADNC) donors. We then quantified MAO-B+ and GFAP+ area fraction within 50 randomly selected ThioS+ plaques, the 50 µm peri-plaque halo, and areas distant (> 50 µm) from the nearest ThioS+ plaque of each AD donor. We did the same in sham plaques randomly placed throughout the temporal cortex of the CTRL donors for comparison. Mixed effect models revealed a significant effect of diagnosis and location on both MAO-B and GFAP immunoreactivity, demonstrating that MAO-B expression is higher in AD versus CTRL, especially in the GFAP+ reactive astrocytes surrounding and penetrating the dense-core Aβ plaques of AD donors (Fig. 6). Indeed, a highly significant (p < 0.0001) diagnosis × location interaction (i.e., the closer to ThioS+ plaques from AD donors the higher the MAO-B area fraction versus no change in MAO-B area fraction with distance to sham plaques in CTRL donors) supports this interpretation.

It should also be noted that the pTau+ area fraction is mainly driven by pTau+ neuropil threads and plaque-associated neuritic dystrophies, whereas neurofibrillary tangles only account for 10% or less of the total pTau+ area fraction [7, 31]. To determine whether the number of pTau+ neurofibrillary tangles indeed correlates with MAO-B expression level, we measured the MAO-B+ area fraction in the temporal cortex of a validation sample of intermediate and high ADNC donors (n = 51) for which FFPE sections were available and stereology-based counts of PHF1+ neurofibrillary tangles, GFAP+ reactive astrocytes, and CD68+ microglia as well as Aβ+ area fraction and cortical thickness measures had been obtained for a previous study [44]. Univariate correlations revealed statistically significant positive correlations between MAO-B+ area fraction and the number of GFAP+ reactive astrocytes and CD68+ reactive microglia as well as a statistically significant negative correlation between MAO-B+ area fraction and cortical thickness, but no significant correlation between MAO-B+ area fraction and either Aβ plaque burden or the number of PHF1+ neurofibrillary tangles (Fig. 7 and Table 5). A multivariate regression analysis with MAO-B area fraction as outcome variable, Aβ+ area fraction, number of pTau+ neurofibrillary tangles, GFAP+ reactive astrocytes, and CD68+ reactive microglia, and cortical thickness as independent variables, and adjusted by age at death, confirmed that MAO-B expression level is independently associated with cortical thickness and number of CD68+ reactive microglia and showed a trend (p = 0.0546) toward a positive independent association with the number of GFAP+ reactive astrocytes, but no significant association with either total Aβ plaque burden or the number of neurofibrillary tangles (Table 6).

We also investigated the possible impact of the MAOB rs1799836 SNP genotype on MAO-B expression level, since its G (minor) allele has been associated with lower MAO-B enzymatic activity [3], which could potentially affect radiotracer binding. Of the 50 donors in whom this SNP genotyping could be assayed, 20 were AA, 17 AG, and 13 GG. MAO-B+ area fraction increased with increasing ADNC burden in all brain regions regardless of the MAOB rs1799836 SNP genotype, indicating no effect of this SNP on MAO-B expression level (Fig. 8).

Lastly, we asked if this MAO-B upregulation by reactive astrocytes is specific of AD or also occurs in ADRD. To address this question, we performed quantitative immunohistochemistry with the peroxidase-DAB method followed by the same analytical pipeline in FFPE sections from the frontal association cortex (BA8/9) of n = 30 LBD and n = 10 of each PiD, PSP, CBD, and FTLD-TDP donors. We chose this brain region because it is known to be significantly affected in all these ADRD [2, 8]. We found that, in these conditions, astrocytes are also the main source of MAO-B expression in both cortex and white matter (Fig. 9a). Surprisingly, quantitative analyses of MAO-B-immunoreactive area fraction revealed that CBD, PiD, and FTLD-TDP donors exhibit an even higher MAO-B expression than donors with high ADNC burden and much higher than CTRL donors (Not AD/Low ADNC burden). By contrast, MAO-B area fraction in LBD and PSP donors did not differ from that of CTRL donors (Fig. 9b, c).