Glia Maturation Factor and Mitochondrial Uncoupling Proteins 2 and 4 Expression in the Temporal Cortex of Alzheimer's Disease Brain

Abstract

Alzheimer's disease (AD) is characterized by the presence of neuropathological lesions containing amyloid plaques (APs) and neurofibrillary tangles (NFTs). AD is associated with mitochondrial dysfunctions, neuroinflammation and neurodegeneration in the brain. We have previously demonstrated enhanced expression of the proinflammatory protein glia maturation factor (GMF) in glial cells near APs and NFTs in the AD brains. Parahippocampal gyrus consisting of entorhinal and perirhinal subdivisions of temporal cortex is the first brain region affected during AD pathogenesis. Current paradigm implicates oxidative stress-mediated neuronal damage contributing to the early pathology in AD with mitochondrial membrane potential regulating reactive oxygen species (ROS) production. The inner mitochondrial membrane anion transporters called the uncoupling proteins (UCPs), function as regulators of cellular homeostasis by mitigating oxidative stress. In the present study, we have analyzed the expression of GMF and mitochondrial UCP2 and UCP4 in the parahippocampal gyrus of AD and non-AD brains by immunostaining techniques. APs were detected by thioflavin-S fluorescence staining or immunohistochemistry (IHC) with 6E10 antibody. Our current results suggest that upregulation of GMF expression is associated with down-regulation of UCP2 as well as UCP4 in the parahippocampal gyrus of AD brains as compared to non-AD brains. Further, GMF expression is associated with up-regulation of inducible nitric oxide synthase (iNOS), the enzyme that induces the production of nitric oxide (NO), as well as nuclear factor kB p65 (NF-κB p65) expression. Also, GMF appeared to localize to the mitochondria in AD brains. Based on our current observations, we propose that enhanced expression of GMF down-regulates mitochondrial UCP2 and UCP4 thereby exacerbating AD pathophysiology and this effect is potentially mediated by iNOS and NF-κB. Thus, GMF functions as an activator protein that interferes with the cytoprotective mechanisms in AD brains.

Keywords: Alzheimer’s disease; NF-κB; UCP2; UCP4; glia maturation factor; inducible nitric oxide; mitochondrial uncoupling protein; para hippocampal gyrus.

Figure 1

Immunohistochemistry (IHC) detection of uncoupling proteins 2 (UCP2) or UCP4 with amyloid plaques (APs) and neurofibrillary tangles (NFTs) in Alzheimer’s disease (AD) and non-AD brains. (A) Representative photomicrographs show UCP2 or UCP4 IHC staining followed by thioflavin-S fluorescence staining in the parahippocampal gyrus of AD (n = 10) and non-AD brains (n = 10). Both UCP2 and UCP4 expression (brown color, arrows) were decreased in AD brains when compared to non-AD brains. Thioflavin-S staining (green color, arrowheads) showed APs and NFTs in the AD brain (magnifications = 400×). (B) We also counted UCP2 and UCP4-positive cells in AD (n = 10) and non-AD (n = 10) brains using the IHC slides. The counting was performed under the microscope using high magnification objectives at five different fields in each section and then averaged. The data were presented as mean ± SEM of the number of UCP2 or UCP4-positive cells/95 mm², *p < 0.05, t test.

Figure 2

Double immunofluorescence detection of glia maturation factor (GMF) with UCP2 or UCP4 in parahippocampal gyrus of AD brain and compared with non-AD brain (n = 7–10). GMF monoclonal antibody and UCP2 or UCP4 polyclonal antibodies were mixed and the sections were incubated with this mixture. GMF was visualized using goat anti-mouse IgG conjugated with green fluorescent dye Alexa Fluor 488. (A) UCP2 and (B) UCP4 proteins were visualized using goat anti-rabbit IgG conjugated with red fluorescent dye Alexa Fluor 568. UCP2 and UCP4 expressions are reduced in AD brains when compare to non-AD brains. We have observed increased expression of GMF (green color, arrows) in AD brain when compare to non-AD brain. UCP2 and UCP4 expressions are observed at the same location where GMF is expressed (magnification = 400×). To quantitate the immunostaining, we counted (C) GMF and UCP2 or (D) GMF and UCP4-positive cells under the microscope at five different fields in each section and then averaged (n = 7–10). The data were presented as mean ± SEM of the number of GMF or UCP2 or UCP4-positive cells/95 mm², *p < 0.05, t test. (E,F) Confocal microscopic double immunofluorescence labeling of AD brain sections. Sections were incubated with GMF monoclonal antibody and UCP2/UCP4 polyclonal antibodies. UCP2/UCP4 (green) were visualized with goat anti-rabbit IgG conjugated with green fluorescent dye Alexa Fluor 488. GMF (red) labeling visualized with goat anti-mouse IgG conjugated with green fluorescent dye Alexa Fluor 568. Boxed area show colocalization of GMF and UCP2 or UCP4 in the merged image (magnification 63× oil immersion objective).

Figure 3

Confocal microscopy image of double immunofluorescence labeling of GMF (red) and voltagedependent anion-selective channel 1 (VDAC1; green) in the brain section of AD. Merged image showed the colocalization of GMF and VDAC1. Boxed area show the enlarged view of co-localization of GMF and VDAC1 (magnification 63× oil immersion objective).

Figure 4

Double immunofluorescence and double IHC detection of GMF and inducible nitric oxide synthase (iNOS) or GMF and nuclear factor kB p65 (NF-κB p65) in AD brains. (A) Parahippocampal gyrus sections from AD patient brain were incubated with a mixture of GMF monoclonal antibody and polyclonal iNOS or NF-κB antibodies. GMF was visualized with goat anti-mouse IgG conjugated with green fluorescent dye Alexa Fluor 488, and iNOS or NF-κB were visualized with goat anti-rabbit IgG conjugated with red fluorescent dye Alexa Fluor 568 to iNOS or NF-κB. (A) iNOS (red color, arrowheads) and NF-κB (red color, arrowheads) were localized at the vicinity of GMF (green color, arrows) expression. (A) Merged image show the co-localization of GMF with iNOS or NF-κB (magnification = 200×). (B) We have also co-localized GMF with iNOS and GMF with NF-κB by double IHC staining in AD brains (n = 3). The brain sections were first incubated with GMF monoclonal antibody and 3,3’-diaminobenzidine (DAB) substrate solution (brown color) followed by incubation for iNOS or NF-κB detection. Then the sections were incubated with Vector SG substrate solution (blue color). Double IHC staining also revealed the co-localization of GMF (brown color, arrows) with iNOS (blue color, arrowheads) or GMF with NF-κB (blue color, arrowhead). Glial cells show the expression of GMF as well as iNOS or NF-κB (magnification = 400×). Positive cells were counted from the images and presented in bar graphs. (C) NF-κB immunofluorescence labeling with DAPI counter staining showing NF-κB cytoplasmic expression. Boxed area show enlarged view of NF-κB and DAPI co-localization (magnification 63× oil immersion objective).

Figure 5

Double IHC detection of APs with GMF or iNOS or NF-κB in parahippocampal gyrus of AD brains (n = 3). We have performed co-localization of APs (6E10 antibody) with GMF or iNOS or NF-κB subunit p65. We have first performed IHC for APs with 6E10 antibody (brown color, arrowheads). After the visualization of immunoreactivity for APs with DAB substrate (brown color), the sections were then incubated with antibodies for GMF or iNOS or NF-κB at 4°C for overnight. The second immunoperoxidase reaction was developed with Vector SG substrate (blue color). GMF, iNOS and NF-κB (blue color, arrows) were co-localized at the vicinity of 6E10 labeled APs (brown color) in the AD brains (magnification = 200×). Bar graph show positive cells in these images.

Figure 6

IHC of fatty acid synthase (FASN), and double labeling of FASN with AT8, and FASN with 6E10. (A) Immunohistochemical staining of FASN in AD and non-AD brain sections. Note the presence of strong immunoreactive cells (arrows) in the plaque area. (B) Double immunostaining for FASN (DAB, brown color) and tau (AT8 antibody; blue-gray color, SG Vector) showing the association of these (magnification = 400×). Positive cells were counted from these images and presented in graphs. (C) Confocal microscopy double immunofluorescence labeling of 6E10 (red) and FASN (green) in the AD brain showing the colocalization (yellow color; white arrows) in the merged image (magnification 63× oil immersion objective). *Significantly increased (p < 0.05) as compared with Non-AD.