A new subtype of frontotemporal lobar degeneration with FUS pathology

Abstract

Frontotemporal dementia (FTD) is a clinical syndrome with a heterogeneous molecular basis. The neuropathology associated with most FTD is characterized by abnormal cellular aggregates of either transactive response DNA-binding protein with Mr 43 kDa (TDP-43) or tau protein. However, we recently described a subgroup of FTD patients, representing around 10%, with an unusual clinical phenotype and pathology characterized by frontotemporal lobar degeneration with neuronal inclusions composed of an unidentified ubiquitinated protein (atypical FTLD-U; aFTLD-U). All cases were sporadic and had early-onset FTD with severe progressive behavioural and personality changes in the absence of aphasia or significant motor features. Mutations in the fused in sarcoma (FUS) gene have recently been identified as a cause of familial amyotrophic lateral sclerosis, with these cases reported to have abnormal cellular accumulations of FUS protein. Because of the recognized clinical, genetic and pathological overlap between FTD and amyotrophic lateral sclerosis, we investigated whether FUS might also be the pathological protein in aFTLD-U. In all our aFTLD-U cases (n = 15), FUS immunohistochemistry labelled all the neuronal inclusions and also demonstrated previously unrecognized glial pathology. Immunoblot analysis of protein extracted from post-mortem aFTLD-U brain tissue demonstrated increased levels of insoluble FUS. No mutations in the FUS gene were identified in any of our patients. These findings suggest that FUS is the pathological protein in a significant subgroup of sporadic FTD and reinforce the concept that FTD and amyotrophic lateral sclerosis are closely related conditions.

Figure 1

FUS IHC performed on sections of post-mortem brain tissue from normal control (A) and aFTLD-U subjects (B–I). The normal physiological staining pattern, consisting of strong immunoreactivity of neuronal nuclei, weaker but consistent staining of neuronal cytoplasm and more variable reactivity of glial nuclei was demonstrated in all cases, including normal controls (A), neurological controls and aFTLD-U subjects (B). In patients with aFTLD-U, neurons with inclusions (arrow) retained at least some of the normal nuclear and cytoplasmic staining (B). Neuronal cytoplasmic inclusions (NCI) were most numerous in the middle and deeper layers of neocortex (C–E) and dentate granule cells of the hippocampus (F) and ranged in morphology from round or oval (D and F) to crescentic (E). Dentate granule cells with vermiform neuronal intranuclear inclusions (arrows) often also had NCI (G). Globular NCI were present in lower motor neurons (H). Flame-shaped glial cytoplasmic inclusions were present in white matter (I). FUS IHC with concentration of primary antibody adjusted to demonstrate normal physiological staining (A and B) or optimize visualization of pathological inclusions (C–I). Scale bar: A and H, 20 μm; B, E, G and I, 10 μm; C, 50 μm; D, 15 μm; F, 30 μm.

Figure 2

Double-label immunofluorescence of inclusions in aFTLD-U and FTLD-TDP. Neuronal cytoplasmic inclusions (A) and neuronal intranuclear inclusions (B and C) in aFTLD-U cases showed colocalization of ubiquitin (green) and FUS (red). Note the presence of a cytoplasmic and intranuclear inclusion (arrow) in a single cell (C). No obvious difference in the intensity of FUS nuclear staining was observed between neurons with and without inclusions (A). In sharp contrast, ubiquitin-positive inclusions in FTLD-TDP were not stained with anti-FUS antibodies (D). Scale bar: A and D, 50 µm; B, 20 µm; C, 16.5 µm.

Figure 3

Biochemical analysis of FUS. Proteins were sequentially extracted from aFTLD-U, FTLD-TDP and control (CO) brains. High salt (lane 1), Triton X-100 (lane 2), RIPA (lane 3), 2% SDS (lane 4) and formic acid (lane 5) fractions were separated by 7.5% SDS–PAGE and immunoblotted with anti-FUS antibody (RP A300-302A). All cases showed a strong ∼73-kDa band in the soluble high-salt fraction (lane 1). Although the amount of SDS–soluble FUS (lane 4) was variable within each group, aFTLD-U cases always showed a strong band that was greater than that seen for most of the controls.

Figure 4

Ratio of insoluble to soluble FUS. Band intensities of FUS in insoluble (SDS fraction) and soluble (high salt and Triton-X100 fractions) were analysed and the ratio calculated. Ratios are depicted as a box and whiskers blot that shows the range of values, with the box being subdivided into the 25 and 75% quartiles by the median; circles represent outliers, filled rhombus represent the mean. Although there is some overlap, the aFTLD-U group showed significantly higher ratios compared to both the FTLD-TDP and control groups (P < 0.05).