Disease and Region Specificity of Granulin Immunopositivities in Alzheimer Disease and Frontotemporal Lobar Degeneration

Abstract

Heterozygous loss-of-function mutations in GRN, the progranulin gene, which result in progranulin (PGRN) protein haploinsufficiency, are a major cause of frontotemporal lobar degeneration with TDP-43 proteinopathy (FTLD-TDP). PGRN is composed of seven and a half repeats of a highly conserved granulin motif that is cleaved to produce the granulin peptides A-G and paragranulin. To better understand the role of PGRN and granulin (Grn) peptides in the pathogenesis of neurodegeneration, we evaluated PGRN/Grn in brains of patients with Alzheimer disease, FTLD-TDP type A with or without GRN mutations, and normal individuals, using a panel of monoclonal antibodies against Grn peptides A-G. In the neocortex, Grn peptide-specific immunostains were observed, for example, membranous Grn E immunopositivity in pyramidal neurons, and Grn C immunopositivity in ramified microglia. In the hippocampus, Grn immunopositivity in the CA1 and CA2 regions showed disease-specific changes in both neurons and microglia. Most interestingly, in FTLD-TDP type A with GRN mutations, there is a 60% decrease in the density of Grn-positive microglia in the hippocampal CA1, suggesting that haploinsufficiency of the GRN mutations also extends to PGRN expression in microglia. This study provides important insights into future studies of the pathogenesis and treatment of FTLD-TDP.

Keywords: Alzheimer disease; Frontotemporal lobar degeneration; Granulin; Hippocampal sclerosis; Microglia; Neuroinflammation; Progranulin.

FIGURE 1

Cellular immunostaining patterns of anti-Grn mAbs. All of the anti-Grn mAbs revealed diffuse cytoplasmic positivity in neocortical neurons, which were stronger in the pyramidal neurons (A, illustrated by anti-Grn A antibody). The anti-Grn E mAb also showed a membranous staining in scattered neurons (B). Except for a ramified microglial population revealed by the anti-Grn C mAb (C), all of the anti-Grn mAbs showed granular cytoplasmic positivity of microglia and macrophages (D, E, illustrated by anti-Grn D antibody). Scale bar: 100 µm.

FIGURE 2

Immunostains with anti-Grn mAbs of normal neocortex. The labeling of anti-Grn A and anti-Grn B mAbs was stronger in neurons; that of anti-Grn D, F, and G mAbs were stronger in microglial cells; and that of anti-Grn C was strong in both neuronal and microglial cells. Arrow shows a Grn C-immunopositive, ramified microglial cell with abundant processes. Anti-Grn E shows membranous immunopositivity in the pyramidal neurons. Scale bar: 100 µm.

FIGURE 3

Immunohistochemical evaluation with anti-Grn mAbs of middle frontal cortices from patients with different diseases. Representative images (the upper 4 panels) of immunostains by the anti-Grn C mAb showed decreased immunopositivity in neurons, and increased numbers of microglia cells in the middle frontal cortices of patients with AD, and FTLD-TDP type A with or without GRN mutation, when compared with the controls. Similar results were obtained from all other mAbs. In the cortex from AD, and FTLD-TDP type A with GRN mutation, anti-Grn C mAb also labeled a group of ramified microglia cells with abundant processes (arrows). The bottom 2 panels showed that Grn E-positive neurons were markedly increased in number in FTLD-TDP type A with GRN mutation, when compared with controls and the other diseases. Scale bar: 100 µm. CON, control; AD, Alzheimer disease; FTLD-TDP type A, FTLD-TDP type A without GRN mutation; GRN, FTLD-TDP type A with GRN mutation; Grn C, anti-Grn C mAb; Grn E, anti-Grn E mAb.

FIGURE 4

Grn C-immunopositive ramified microglia had different distribution patterns in the frontal cortices of patients with different diseases. In the frontal cortices of AD patients and normal aging controls, Grn C-positive ramified microglia had a transcortical distribution that may correlate to amyloid/tau deposits, as revealed by Abeta and AT8 immunostaining. In some FTLD-TDP type A with GRN mutation cases, Grn C-immunopositive ramified microglia cells were localized in layer 3 of the frontal cortex, while TDP-43-positive inclusions were mainly in layer 2. Inset in the right lower panel, TDP-43 neuronal intranuclear inclusion. Bar, 100 µm. GRN, FTLD-TDP type A with the GRN mutation; AD, Alzheimer disease.

FIGURE 5

Neuronal PGRN expression in the hippocampi of the different diseases, evaluated by the anti-Grn B mAb. Anti-Grn B mAb reveals weak immunopositivity in hippocampal CA1, and moderate immunopositivity in hippocampal CA2, 3, and 4 of the control (not shown) or AD. Compared with the control (not shown) or to AD, the intensity of the immunostains was lower in the CA1 regions of ADHS, FTLD-TDP type A without GRN mutation (not shown), and FTLD-TDP type A with GRN mutation samples due to neuronal loss and gliosis in this region. Compared with the control (not shown) and to AD, the immunostaining intensity was higher in the CA2 regions in FTLD-TDP type A without GRN mutation (not shown) and FTLD-TDP type A with GRN mutation samples, while no significant difference was found between ADHS and AD or the controls. CA3 and CA4 showed no significant differences in immunostaining intensity between the different diseases. Bar, 100 µm. AD, Alzheimer disease; ADHS, Alzheimer disease with hippocampal sclerosis; GRN, FTLD-TDP type A with GRN mutation.

FIGURE 6

IBA-1- and Grn D-positive microglia in the hippocampi of FTLD-TDP with the GRN mutation. Representative photos from IBA-1- and Grn D-positive microglia in CA1 and CA4. Inset in the lower right panel shows higher power image of Grn D-positive microglia. Scale bar: 100 µm.

FIGURE 7

Density of IBA-1- and Grn D-positive microglia in hippocampal CA1 regions of patients with the different diseases. The full height of each bar (both dark and light gray) represents the average number of IBA-1-positive microglia per mm² in CA1, while the light gray bar represents that of Grn D-positive microglia. Values are mean ± SD. The densities of IBA-1-positive microglia were compared with the control value by ANOVA with Dunnett's post hoc test (*p < 0.05). The density of Grn D-positive microglia was compared between FTLD-TDP type A with and without GRN mutation, and statistical significance was determined by Student’s t-test (#p < 0.05). CON, normal control; AD, Alzheimer disease; ADHS, Alzheimer disease with hippocampal sclerosis; FTLD-TDP type A, FTLD-TDP type A without GRN mutation; GRN, FTLD-TDP type A with GRN mutation; Grn D, anti-Grn D mAb.