Accumulation of transactive response DNA binding protein 43 in mild cognitive impairment and Alzheimer disease

Abstract

Transactive response DNA binding protein 43 (TDP-43) plays a central role in the neuropathology of frontotemporal lobar degeneration and amyotrophic lateral sclerosis, but the relationship between TDP-43 abnormalities and Alzheimer disease (AD) remains unclear. To determine whether TDP-43 can serve as a neuropathologic marker of AD, we performed biochemical characterization and quantification of TDP-43 in homogenates from parietal neocortex of subjects with aclinical diagnosis of no cognitive impairment (NCI, n = 12), mild cognitive impairment (MCI, n = 12), or AD (n = 12). Immunoblots revealed increased detergent-insoluble TDP-43 in the cortex of 0, 3, and 6 of the 12 individuals with NCI, MCI, or AD, respectively. Detergent-insoluble TDP-43 was positively correlated with the accumulation of soluble Aβ42, amyloid plaques, and paired helical filamenttau. In contrast, phospho-TDP-43 was decreased in the cytosolic fraction and detergent-soluble membrane/nuclear fraction from AD patients and correlated with antemortem cognitive function.Immunofluorescence analysis confirmed that the frequencies of individuals with TDP-43 or phospho-TDP-43 cytoplasmic inclusions were higher in AD than in NCI, with MCI at an intermediate level. These data indicate that abnormalities of TDP-43 occur in an important subset of MCI and AD patients and that they correlate with the clinical and neuropathologic features of AD.

Figure 1

Accumulation of total TDP-43 in detergent-insoluble fractions from the parietal cortex of individuals with mild cognitive impairment (MCI) or Alzheimer disease (AD). (A-C) TDP-43 was measured by Western imunoblotting in the detergent-insoluble fraction (formic acid extracts) using a C-terminal polyclonal antibody (pAb-CT) (A), an N-terminal polyclonal antibody (pAB-NT) (B), and a total TDP-43 monoclonal antibody (mAb [2E2-D3]) (C). Statistical comparisons in panels A-C were performed using Kruskal-Wallis tests. Each point represents an individual and the horizontal bar is the average (n = 11-12 per group). (D) Examples of immunoblots. (E, F) The proportion of subjects with high levels of TDP-43 deposition in the formic acid extracts was higher in AD patients (E) as determined with a contingency analysis followed by a Pearson's chi-square test (F).

Figure 2

Unchanged concentrations of total TDP-43 in soluble fractions from the parietal cortex of individuals with mild cognitive impairment (MCI) or Alzheimer disease (AD). (A-E) TDP-43 was measured by Western immunoblotting in the TBS-soluble (cytosol) (A-C) and detergent-soluble (membrane/nucleus) fractions (D-E) using a C-terminal polyclonal antibody or a monoclonal antibody (2E2-D3). Each point represents an individual and the horizontal bar is the average (n = 11-12 per group). Statistical comparisons between groups were performed with Kruskal-Wallis tests.

Figure 3

Concentrations of phosphorylated TDP-43 in insoluble fractions from the parietal cortex of individuals with mild cognitive impairment (MCI) or Alzheimer disease (AD). (A-C) TDP-43 was measured by Western immunoblotting in the detergent-insoluble fraction (formic acid extracts) using a polyclonal antibody targeting TDP-43 phosphorylated at serine 409/410. The signal was relatively weak and bands at 43 and 25 kDa were quantified. Each point represents an individual and the horizontal bar is the average (n = 11-12 per group). No statistical difference was detected using Kruskal-Wallis tests.

Figure 4

Alteration of phosphorylated TDP-43 in soluble fractions from the parietal cortex of individuals with mild cognitive impairment (MCI) or Alzheimer disease (AD). (A-C) TDP-43 was measured by Western immunoblotting in the TBS-soluble (cytosol) (A, B) and detergent-soluble (membrane/nucleus) fractions (C), using a polyclonal antibody, targeting TDP-43 phosphorylated at serine 403/404. Each point represents an individual; the horizontal bar is the average (n = 11-12 per group). Statistical comparisons were performed using a Mann-Whitney U test (A) or Welch-ANOVA followed by Dunnett's post hoc test (B, C). NC = no cognitive impairment.

Figure 5

TDP-43 immunofluorescence in the cytoplasm of neuron-like cells from the parietal cortex of individuals with mild cognitive impairment (MCI) and Alzheimer disease (AD). (A-F) Cytoplasmic TDP-43 labeling was more frequent in neuron-like cells from individuals with MCI and AD that -in controls (Ctrl) with no cognitive impairment. TDP-43 immunostaining (using anti-N-TDP-43) is shown in green; nuclei are identified with DAPI and shown in blue. All sections were from the parietal cortex. Qualitative assessment showed a trend toward a higher frequency of subjects with high levels of TDP-43 cytoplasmic labeling in AD patients (contingency analysis followed by a Pearson's chi-square test, n = 11-12 per group).

Figure 6

Phospho-TDP-43 immunofluorescence in the cytoplasm of neuron-like cells from the parietal cortex of individuals with mild cognitive impairment (MCI) and Alzheimer disease (AD). (A-F) Cytoplasmic phospho-TDP-43 signal was more frequent in neuron-like cells from individuals with MCI and AD that in controls (Ctrl) with no cognitive impairment. pTDP-43 immunostaining (anti-TDP-43 pS409/S410, green); nuclei are identified with DAPI (blue). All sections are from the parietal cortex. Qualitative evaluation showed that cytoplasmic TDP-43 was more frequently observed in AD patients (contingency analysis followed by a Pearson's chi-square test, n = 11-12 per group).