Increased MANF Expression in the Inferior Temporal Gyrus in Patients With Alzheimer Disease

Abstract

Alzheimer disease (AD) is an aging-related disorder linked to endoplasmic reticulum (ER) stress. The main pathologic feature of AD is the presence of extracellular senile plaques and intraneuronal neurofibrillary tangles (NFTs) in the brain. In neurodegenerative diseases, the unfolded protein response (UPR) induced by ER stress ensures cell survival. Mesencephalic astrocyte-derived neurotrophic factor (MANF) protects against ER stress and has been implicated in the pathogenesis of AD. MANF is expressed in neurons of the brain and spinal cord. However, there have been no investigations on MANF expression in the brain of AD patients. This was addressed in the present study by immunohistochemistry, western blotting, and quantitative analyses of postmortem brain specimens. We examined the localization and expression levels of MANF in the inferior temporal gyrus of the cortex (ITGC) in AD patients (n = 5), preclinical (pre-)AD patients (n = 5), and age-matched non-dementia controls (n = 5) by double immunofluorescence labeling with antibodies against the neuron-specific nuclear protein neuronal nuclei (NeuN), ER chaperone protein 78-kDa glucose-regulated protein (GRP78), and MANF. The results showed that MANF was mainly expressed in neurons of the ITGC in all 3 groups; However, the number of MANF-positive neurons was significantly higher in pre-AD (Braak stage III/IV) and AD (Braak stage V/VI) patients than that in the control group. Thus, MANF is overexpressed in AD and pre-AD, suggesting that it can serve as a diagnostic marker for early stage disease.

Keywords: Alzheimer disease; MANF; cerebral cortex; endoplasmic reticulum stress; hyperphosphorylated tau; senile plaque.

FIGURE 1

p-tau and Aβ_17–24 expressions in the ITGC detected by immunohistochemistry. (A–H) p-tau and Aβ_17–24 immunoreactivity in control subjects (left) and AD patients (right). Patients with AD showed extensive accumulation of Aβ plaques [arrowheads in panels (F,H)] and NFTs [arrowheads in panels (B,D)] in the ITGC. Scale bar, 50 μm (A,B,E,F), 20 μm (C,D,G,H).

FIGURE 2

Mesencephalic astrocyte-derived neurotrophic factor levels detected by western blotting with a polyclonal antibody. Cell lysis solutions and human brain temporal cortex protein extracts were probed with a polyclonal anti-MANF antibody to evaluate specificity. The immunoblot shows a clear single band of ∼18 kDa in all protein extracts and cell lysis solutions.

FIGURE 3

Mesencephalic astrocyte-derived neurotrophic factor levels in the HKO control mice and WT control mice detected by immunohistochemistry with polyclonal antibodies. No positive staining was detected in the hepatocellular tissues of the HKO control mice (A,B). MANF was detected in the hepatocellular tissues of the WT control mice (C,D). Scale bar: 50 μm (A,C) and 20 μm (B,D).

FIGURE 4

Cytoarchitecture of the ITGC. (A–C) Organization of cortical laminae visualized in tissue sections immunolabeled with an antibody against MANF. Layers are indicated by roman numerals. Scale bar, 200 μm (A) and 50 μm (B,C).

FIGURE 5

Representative images of the expression of MANF and GRP78 in the ITGC. (A–H) The pictures above are representative images of immunofluorescence labeling for MANF (red) and GRP78 (green). The nuclei were stained with DAPI (blue). Picture D is a merged image of panels (A–C). The magnified images show the expression and cellular distribution of MANF and GRP78, respectively. Scale bar: 50 μm (A–D) and 10 μm (E–H).

FIGURE 6

Representative images of MANF and NeuN expression in the ITGC. (A–F) Representative micrographs of double immunolabeling with antibodies against MANF (red) and NeuN (green). MANF was mainly detected in neurons. Scale bar: 100 μm (A–F).

FIGURE 7

Mesencephalic astrocyte-derived neurotrophic factor expression in the ITGC of AD, pre-AD, and control cases detected by immunohistochemistry. (A–F) MANF expression in non-dementia control (A,B), pre-AD (C,D), and AD (E,F) samples shown at low (20×) (A,C,E) and high (40×) (B,D,F) magnification.

FIGURE 8

Increased MANF expression in the ITGC of pre-AD and AD groups. Overall expression in both groups was higher than in the control group, while the expression level was lower in the AD group than in the pre-AD group, although the difference was non-significant. **P < 0.05 vs. control group.