FUS-immunoreactive inclusions are a common feature in sporadic and non-SOD1 familial amyotrophic lateral sclerosis

Abstract

Objective: Amyotrophic lateral sclerosis (ALS) is a fatal disorder of motor neuron degeneration. Most cases of ALS are sporadic (SALS), but about 5 to 10% of ALS cases are familial (FALS). Recent studies have shown that mutations in FUS are causal in approximately 4 to 5% of FALS and some apparent SALS cases. The pathogenic mechanism of the mutant FUS-mediated ALS and potential roles of FUS in non-FUS ALS remain to be investigated.

Methods: Immunostaining was performed on postmortem spinal cords from 78 ALS cases, including SALS (n = 52), ALS with dementia (ALS/dementia, n = 10), and FALS (n = 16). In addition, postmortem brains or spinal cords from 22 cases with or without frontotemporal lobar degeneration were also studied. In total, 100 cases were studied.

Results: FUS-immunoreactive inclusions were observed in spinal anterior horn neurons in all SALS and FALS cases, except for those with SOD1 mutations. The FUS-containing inclusions were also immunoreactive with antibodies to TDP43, p62, and ubiquitin. A fraction of tested FUS antibodies recognized FUS inclusions, and specific antigen retrieval protocol appeared to be important for detection of the skein-like FUS inclusions.

Interpretation: Although mutations in FUS account for only a small fraction of FALS and SALS, our data suggest that FUS protein may be a common component of the cellular inclusions in non-SOD1 ALS and some other neurodegenerative conditions, implying a shared pathogenic pathway underlying SALS, non-SOD1 FALS, ALS/dementia, and related disorders. Our data also indicate that SOD1-linked ALS may have a pathogenic pathway distinct from SALS and other types of FALS.

Figures

Figure 1

FUS-immunoreactive aggregates/inclusions in spinal cord of SALS patients. (A, C) Representative FUS-immunoreactive inclusions detected with antibody 11570-1-AP in several spinal anterior horn neurons of SALS cases. (B) Higher magnification image of the boxed area in panel A. (D) Representative FUS-positive inclusions detected with antibody HPA008784 in SALS cases. Small arrows in (A) and (C) indicate representative neurons with FUS-positive inclusions. Large arrows in (B) and (D) indicate neuronal cytoplasmic FUS-positive inclusions. Arrowheads in (B) and (D) indicate neuritic FUS-positive inclusions. Bar: A, C=100μm; D=25 μm.

Figure 2

Representative FUS-immunoreactive aggregates/inclusions in SALS, ALS/dementia and non-SOD1 FALS. Human spinal cord sections were analyzed with immunohistochemistry using an affinity purified anti-FUS antibody (11570-1-AP). Representative FUS-immunoreactive aggregates/inclusions are shown here in anterior horn neurons in patients with SALS (A), FALS without known genetic mutations in SOD1, FUS or TDP43 (B), ALS/dementia (C), FALS with FUSR521L mutation (D), and FALS with TDP43G298S mutation (E). Most of the inclusions were skein-like (A-E), but some of them appeared compact (F). The FUS inclusions were predominantly localized in the large neurons of the anterior horn (A-F). Some glial cells exhibited significantly increased FUS staining (G). No FUS inclusions were found in primary sensory neurons of the dorsal root ganglia (H) of a patient exhibiting FUS inclusions in spinal neurons (A). Representative TDP43-immunoreactive inclusions in spinal cord neurons of SALS patients are shown in (I). Bar, 50μm.

Figure 3

Co-localization of TDP43, p62 and ubiquitin in FUS-positive inclusions. FUS-positive inclusions (green) were also immunoreactive to TDP43, p62 and ubiquitin (red) antibodies (A-I). In a patient with the TDP43G298S mutation, TDP43-positive inclusions (red) were also immunoreactive to the FUS antibody (green) (J-L).

Figure 4

Confocal analysis of the FUS pathology with different FUS antibodies. The representative FUS pathology is shown with FUS antibodies HPA008784 (A-C) and sc-47711 (D-F) in spinal neurons of SALS cases. The ubiquitin-positive inclusions in aFTLD-U were immunoreactive to FUS antibody (G-I), but not to TDP43 antibody (J-L).

Figure 5

Western blots with antibodies to FUS and TDP43. Twenty micrograms of protein from spinal cord homogenates of ALS patients were loaded and blotted using affinity-purified rabbit anti-human FUS polyclonal antibody 11570-1-AP (A) and anti-TDP43 antibody (B). (A) Lane 1, lysate of THP-1, a human monocytic leukemia cell line (sc-2238, Santa Cruz Biotechnology, Santa Cruz, CA, USA); (B) lane 1, lysate of transiently transfected HEK293 cells overexpressing TDP43; (A and B) lane 2, blank; lanes 3-5, spinal cord homogenates from patients with SALS; lane 6, spinal cord homogenates from a patient with FUSR521L mutation. A single FUS band of the expected size (A) and multiple TDP43 bands (B) detected with each antibody are indicated with arrows. The sizes of molecular weight (MW) are indicated on the left (kd). Lower panel, actin control.

Figure 6

Sensitivity of FUS antibodies for detection of the FUS inclusions in aFTLD-U. Confocal microscopy of the hippocampal sections from two aFTLD-U cases stained with antibodies against ubiquitin and FUS.

Fig. 7

Effect of the antigen retrieval protocols on detection of the inclusions. The representative ubiquitin- (A-F) and p62- (G-L) positive inclusions in spinal neurons of SALS cases were not apparently FUS-positive when antigen was retrieved using either boiling (A-C and G-I) or microwave (D-F and J-L) protocol. (M-O) Representative TDP43- and ubiquitin-positive inclusions are shown with microwave antigen retrieval protocol.