Colocalization of TDP-43 and stress granules at the early stage of TDP-43 aggregation in amyotrophic lateral sclerosis

Abstract

TDP-43 aggregates (skeins and round inclusions [RIs]) are frequent histopathological features of amyotrophic lateral sclerosis (ALS). We have shown that diffuse punctate cytoplasmic staining (DPCS) is the earliest pathologic manifestation of TDP-43 in ALS, corresponding to nonfibrillar TDP-43 located in the rough endoplasmic reticulum. Previous in vitro studies have suggested that TDP-43 inclusions may be derived from stress granules (SGs). Therefore, we investigated the involvement of SGs in the formation of TDP-43 inclusions. Formalin-fixed spinal cords of six ALS patients with a disease duration of less than 1 year (short duration), eight patients with a disease duration of 2-5 years (standard duration), and five normal controls were subjected to histopathological examination using antibodies against an SG marker, HuR. In normal controls, the cytoplasm of anterior horn cells was diffusely HuR-positive. In short-duration and standard-duration ALS, the number of HuR-positive anterior horn cells was significantly decreased relative to the controls. DPCS and RIs were more frequent in short-duration ALS than in standard-duration ALS. The majority of DPCS areas and a small proportion of RIs, but not skeins, were positive for HuR. Immunoelectron microscopy showed that ribosome-like granular structures in DPCS areas and RIs were labeled with anti-HuR, whereas skeins were not. These findings suggest that colocalization of TDP-43 and SGs occurs at the early stage of TDP-43 aggregation.

Keywords: HuR; TDP-43 inclusions; amyotrophic lateral sclerosis; anterior horn cell; stress granule.

FIGURE 1

Immunoreactivity for HuR in the anterior horn of the lumbar spinal cord from normal controls (A) and patients with short‐duration ALS (B) and standard‐duration ALS (C). (A) In controls, the majority of anterior horn cells (AHCs) show moderate immunoreactivity for HuR in the cytoplasm and nucleus. (B) In short‐duration ALS, AHCs occasionally show no HuR immunoreactivity (arrows). (C) In standard‐duration ALS, the immunoreactivity is severely decreased in AHCs (arrows). Bar = 100 μm. The number of HuR‐positive AHCs (D), the somatic area of HuR‐positive AHCs (E), and the proportions of HuR‐positive AHCs in relation to the total number of AHCs (F) in control cases (n = 5), short‐duration ALS (n = 6) and standard‐duration ALS (n = 8). ALS, amyotrophic lateral sclerosis. *p < 0.05.

FIGURE 2

Immunoreactivity for HuR (A, D, G, J), nTDP‐43 (B, E, H, K), and pTDP‐43 (C, F, I, L) in serial sections of AHCs in ALS. (A–C) A normal‐looking AHC showing immunopositivity for HuR (A), but not for nTDP‐43 (B) or pTDP‐43 (C), in the cytoplasm. The nucleus is diffusely immunopositive for HuR (A) and nTDP‐43 (B). (D–F) A slightly atrophic AHC containing diffuse punctate cytoplasmic staining. The cytoplasm and nucleus are immunopositive for HuR (D), but not for nTDP‐43 (E) or pTDP‐43 (F). (G–I) A slightly atrophic AHC containing a round inclusion (RI) (arrows). The cytoplasm and nucleus are immunopositive for HuR (G), but not for nTDP‐43 (H) or pTDP‐43 (I). The RI is also immunopositive for HuR (G). (J–L) A severely atrophic AHC containing skein‐like inclusions (SLIs) (arrowheads). The cytoplasm and nucleus are immunonegative for HuR (J), nTDP‐43 (K), and pTDP‐43 (L). The SLIs are also immunonegative for HuR (J). AHC, anterior horn cell; ALS, amyotrophic lateral sclerosis. Bars = 10 μm.

FIGURE 3

Cytoplasmic HuR‐immunoreactivity of AHCs without inclusions and with DPCS, RI, and SLI in short‐duration (A) and standard‐duration ALS (B). (A) In short‐duration ALS, the proportion of HuR‐positive AHCs without inclusions relative to the total number of AHCs was 73.0%. The proportion of HuR‐positive AHCs with DPCS or RIs relative to the total number of AHCs with each inclusion was 54.6% or 64.2%, respectively. All of the SLI‐containing AHCs are immunonegative for HuR. (B) In standard‐duration ALS, the proportion of HuR‐positive AHCs without inclusions relative to the total number of AHCs was 45.2%. The proportion of HuR‐positive AHCs with DPCS or RIs relative to the total number of AHCs with each inclusion was 59.8% or 43.3%, respectively. All of the SLI‐containing AHCs were immunonegative for HuR. AHC, anterior horn cell; ALS, amyotrophic lateral sclerosis; DPCS, diffuse punctate cytoplasmic staining; RI, round inclusion; SLI, skein‐like inclusion. *p < 0.05; **p < 0.01.

FIGURE 4

Double‐labeling immunofluorescence for HuR (A, D, G, J), nTDP‐43 (B), or pTDP‐43 (E, H, K) in AHCs in ALS. HuR appears green, nTDP‐43 appears red, and overlap of HuR and nTDP‐43 or pTDP‐43 (C, F, I, L) appears yellow. (A–C) The cytoplasm and nuclei of AHCs without inclusions are diffusely immunopositive for HuR and nTDP‐43. (D–E) DPCS is immunopositive for HuR and pTDP‐43. (G–I) An RI (arrows) is immunopositive for HuR and pTDP‐43. (J–L) SLIs (arrowheads) are immunonegative for HuR. AHC, anterior horn cell; ALS, amyotrophic lateral sclerosis; DPCS, diffuse punctate cytoplasmic staining; Lf, lipofuscin granules; Nu, nucleus; RI, round inclusion. Bars = 10 μm.

FIGURE 5

HuR immunoreactivity of DPCS, RI, and SLI in short‐duration (A) and standard‐duration ALS (B). (A) In short‐duration ALS, the proportion of AHCs with HuR‐positive DPCS relative to the total number of AHCs with DPCS (54.6%) was significantly higher than that of AHCs with HuR‐positive RIs (21.1%) and SLIs (0%). (B) In standard‐duration ALS, the proportion of AHCs with HuR‐positive DPCS or RIs relative to the total number of AHCs with each inclusion was 59.8% or 10.0%, respectively. All of the SLIs were immunonegative for HuR. AHC, anterior horn cell; ALS, amyotrophic lateral sclerosis; DPCS, diffuse punctate cytoplasmic staining; RI, round inclusion; SLI, skein‐like inclusion. *p < 0.05; **p < 0.01.

FIGURE 6

Immunoelectron microscopy using anti‐HuR antibody in ALS. (A) An AHC containing an RI (arrow). (B) A higher‐magnification view of the area indicated by the arrow in (A) showing gold particles in an accumulation of ribosome‐like structures containing autolysosomes (white arrowheads). (C) A higher‐magnification view of the area indicated by the asterisk in (B) showing accumulated ribosome‐like structures containing early stage Bunina bodies (white arrows). (D) A higher‐magnification view of the area indicated by the star in (C) showing ribosome‐like granular structures immunolabeled with gold (black arrowheads). Filamentous structures are not evident in this RI. AHC, anterior horn cell; ALS, amyotrophic lateral sclerosis; RI, round inclusion. Bars: A = 5 μm; B, C = 1 μm; D = 0.2 μm.

FIGURE 7

Immunoelectron microscopy using anti‐HuR antibody in ALS. (A) An AHC containing diffuse punctate cytoplasmic staining (DPCS) and skein‐like inclusions (SLIs, black arrows). (B) A higher‐magnification view of the area indicated by the asterisk in (A) showing DPCS (fragmented Nissl bodies) and SLIs (black arrows). (C) A higher‐magnification view of the area indicated by the black star in (B) showing fragmented Nissl bodies labeled by HuR immunoproducts (white arrows). (D) A higher‐magnification view of the area indicated by the white star in (C) showing ribosome‐like granular structures labeled with HuR immunoproducts (black arrowheads). HuR immunoproducts are not evident in SLIs (black arrow). AHC, anterior horn cell; ALS, amyotrophic lateral sclerosis; RI, round inclusion. Bars: A = 5 μm; B, C = 1 μm; D = 0.2 μm.