TREM2 Protein Expression Changes Correlate with Alzheimer's Disease Neurodegenerative Pathologies in Post-Mortem Temporal Cortices - PubMed (original) (raw)

TREM2 Protein Expression Changes Correlate with Alzheimer's Disease Neurodegenerative Pathologies in Post-Mortem Temporal Cortices

Lih-Fen Lue et al. Brain Pathol. 2015 Jul.

Abstract

Triggering receptor expressed by myeloid cells 2 (TREM2), a member of the immunoglobulin superfamily, has anti-inflammatory phagocytic function in myeloid cells. Several studies have shown that TREM2 gene variant rs75932628-T increased the risks for Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia and amyotrophic lateral sclerosis. It has been suggested that the risks could be resulted from the loss of TREM2 function caused by the mutation. Indeed, new evidence showed that several mutations in the immunoglobulin-like V-region led to low cell surface expression of TREM2 and reduced phagocytic function. Because of the emerging importance in understanding TREM2 expression and functions in human neurodegenerative diseases, we conducted biochemical and morphological studies of TREM2 expression in human post-mortem temporal cortical samples from AD and normal cases. Increased expression of TREM2 protein was found to significantly correlate with increases of phosphorylated-tau and active caspase 3, a marker of apoptosis, and also loss of the presynaptic protein SNAP25. Strong intensities of TREM2 immunoreactivity were observed in the microglia associated with amyloid plaques and in neuritic pathology-enriched areas. Based on the findings that TREM2 expression correlated with neurodegenerative markers, further investigation on whether there is abnormality of TREM2 functions in AD brains with nonmutated TREM2 is needed.

Keywords: TREM2; amyloid; brain; human; microglia; tau.

© 2014 The Authors. Brain Pathology published by John Wiley & Sons Ltd on behalf of International Society of Neuropathology.

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Figures

Figure 1

Figure 1

Identification of

TREM

2 (triggering receptor expressed by myeloid cells 2) rs75932628‐

T

.

DNA

samples from cerebellum were amplified by polymerase chain reaction (

PCR

) followed by restriction enzyme digestion using primer pairs that allow the identification of rs75932628 genotypes. The gel image of amplified

PCR

products is shown here. Lane 1 is the

DNA

ladder. In lane 2, the band at 172 bp indicated the presence of rs75932628‐

T

in this case caused by the loss of a

H

ha

I

restriction enzyme site. The other lanes were from

DNA

samples without this polymorphism. For the purpose of this study, we only used autopsy cases that did not have rs75932628‐

T

genotype.

Figure 2

Figure 2

Antibody characterization in the

HEK

cells and human brain homogenates. A. Lysate samples of

HEK

cells transfected with human

TREM

2 (triggering receptor expressed by myeloid cells 2) and

DAP

12 (DNAX‐activation protein 12) plasmids were used to characterize

TREM

2 antibodies by immunoblotting.

TREM

2 protein bands were detected in the range of molecular weights of 35–40 kDa with three antibodies, only in the samples with

TREM

2 transfection. Among these antibodies, the goat polyclonal antibody from

R

&

D

System performed more robustly in

TREM

2‐transfected cells.

DAP

12 protein bands were detected in

HEK

cells transfected with human

DAP

12 plasmids at 12 kDa, but not in the

TREM

2‐transfected samples.

Figure 3

Figure 3

The expression levels of

TREM

2 (triggering receptor expressed by myeloid cells 2),

DAP

12 (DNAX‐activation protein 12),

IBA

1, cleaved caspase 3,

SNAP

25 and

PSD

95 by disease groups. The expression levels of microglial proteins

TREM

2,

DAP

12 and

IBA

1, apoptotic marker active caspase 3, and synaptic proteins

SANP

25 and

PSD

95 were analyzed in the brain samples using protein‐specific antibodies by our standard immunoblotting procedure. The intensities of the specific band in each sample were normalized by β‐actin followed by statistical analysis by disease groups: nondemented controls (

ND

); possible

AD

(

P

oss

AD

); and

A

lzheimer's disease (

AD

). Significant changes (P < 0.05) were detected between

AD

and

ND

groups in

TREM

2 (A),

DAP

12 (B),

IBA

1 (C), and active caspase 3 (D),

SNAP

25 (E) and

PSD

95 (F). However, the significant differences between

AD

and

P

oss

AD

groups were only detected in

TREM

2,

DAP

12 and

IBA

1. Expression levels of the apoptotic marker active caspase 3, presynaptic protein

SNAP

25 and post‐synaptic protein

PSD

95 were not significantly different between

P

oss

AD

and

ND

groups. There were no significant differences between

ND

and

P

oss

AD

in all of the markers. The horizontal lines in the dot plots represent the values of mean and standard errors. Representative immunoblot images for the proteins

TREM

2,

DAP

12,

IBA

1,

PSD

95,

SNAP

25 and cleaved caspase 3 were compiled in G. The disease groups for the samples in each lane were shown above gel images:

AD

for

A

lzheimer's disease;

PA

for possible

A

lzheimer's disease,

ND

for nondemented normal controls. The blank lane was the lane that molecular weight ladders were loaded. The molecular weights corresponding to each protein were indicated at the right side of the gel images. The brain

TREM

2 has molecular weights of 35–40 kDa.

Figure 4

Figure 4

Immunohistochemical characterization of

TREM

2 (triggering receptor expressed by myeloid cells 2) expressions in postmortem human brain tissues. Representative images of double immunohistochemistry of

TREM

2 (dark blue) and

MHCII

(major histocompatibility complex II) (brown) are shown in A–D;

TREM

2 immunoreactive microglia in association with neurons in E–F;

TREM

2 (dark blue) and p‐tau (brown) in G–I; and

TREM

2 and Aβ in J–N. A–D.

TREM

2‐immunoreactive microglia are present in

MHCII

‐negative (arrows) cells and also in

MHCII

‐positive cells (arrow heads). E–I.

TREM

2 immunoreactive microglia are in close contact with neuronal cell bodies (neutral red stained; E, F) in the tissues with minimal

A

lzheimer's disease (

AD

) pathology. Increased

TREM

2 immunoreactivity (dark blue, arrows) could be seen in the areas enriched with p‐tau immunoreactive neurites (brown; G–I). J–N.

TREM

2 immunoreactive microglia (dark blue, arrows; J–N) are observed in the vicinity of amyloid plaques (J–K) and inside amyloid plaques with different compactness of the amyloid aggregates (L–N). All of the micrograph images were taken with 40× objective. The 10‐μm magnification calibration bars were shown in the first picture in each row (A, E, G, J). The brain tissues for the morphological study were

ND

cases in A, B, E; PossAD cases in C, D, F, J, K;

AD

cases in G–I and L–N.

Figure 5

Figure 5

Double immunohistochemistry of

TREM

2 (triggering receptor expressed by myeloid cells 2) with astrocyte and oligodendrocyte markers. Double immunohistochemistry of

TREM

2 with astrocyte marker [glial acidic filament protein (

GFAP

)] or oligodendrocyte‐specific protein shown in this figure were performed in the brain tissues of nondemented controls (

ND

) (A, C), possible

AD

(

P

oss

AD

) (D) and

A

lzheimer's disease (

AD

) (B) cases.

TREM

2 was demonstrated with nickel enhanced

DAB

(DNAX‐activation protein) substrate (dark blue, indicated by black arrows), whereas

GFAP

and oligodendrocyte‐specific protein were demonstrated with

DAB

without enhancement (light brown, indicated by white arrows).

TREM

2 immunoreactivities were not observed in the same cells marked by

GFAP

or oligodendrocytes‐specific protein. Images were taken at 40× objective in A, B, and at 100× with oil immersion lens in C, D. The calibration bars represent 10‐μm length.

Figure 6

Figure 6

Double immunofluorescence labeling of

TREM

2 (triggering receptor expressed by myeloid cells 2) and

DAP

12 (DNAX‐activation protein 12) in human brain tissues. To illustrate the relationship of

TREM

2 and

DAP

12 expressions in brain cells, results of double immunofluorescence labeling are shown here: A, D:

TREM

2 expression in green; B, E:

DAP

12 expression in red; C, F: overlay of

TREM

2 and

DAP

12 images. In both overlay images, the co‐localization of

TREM

2 and

DAP

12 immunoreactivities were shown (arrow heads). However, some of the

DAP

12 immunoreactive profiles are not associated with

TREM

2 immunoreactivity (indicated by arrows). Magnification: calibration bars: 10 μm.

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