Frontotemporal dementia-like disease progression elicited by seeded aggregation and spread of FUS

Abstract

RNA binding proteins have emerged as central players in the mechanisms of many neurodegenerative diseases. In particular, a proteinopathy of fused in sarcoma (FUS) is present in some instances of familial Amyotrophic lateral sclerosis (ALS) and about 10% of sporadic Frontotemporal lobar degeneration (FTLD). Here we establish that focal injection of sonicated human FUS fibrils into brains of mice in which ALS-linked mutant or wild-type human FUS replaces endogenous mouse FUS is sufficient to induce focal cytoplasmic mislocalization and aggregation of mutant and wild-type FUS which with time spreads to distal regions of the brain. Human FUS fibril-induced FUS aggregation in the mouse brain of humanized FUS mice is accelerated by an ALS-causing FUS mutant relative to wild-type human FUS. Injection of sonicated human FUS fibrils does not induce FUS aggregation and subsequent spreading after injection into naïve mouse brains containing only mouse FUS, indicating a species barrier to human FUS aggregation and its prion-like spread. Fibril-induced human FUS aggregates recapitulate pathological features of FTLD including increased detergent insolubility of FUS and TAF15 and amyloid-like, cytoplasmic deposits of FUS that accumulate ubiquitin and p62, but not TDP-43. Finally, injection of sonicated FUS fibrils is shown to exacerbate age-dependent cognitive and behavioral deficits from mutant human FUS expression. Thus, focal seeded aggregation of FUS and further propagation through prion-like spread elicits FUS-proteinopathy and FTLD-like disease progression.

Keywords: Aggregation; Amyotrophic lateral sclerosis (ALS); FUS-proteinopathy; Frontotemporal lobar degeneration (FTLD); Spreading.

Fig. 1

HA-FUS^R495X fibrils induce human FUS mislocalization and aggregation in aged humanized mutantFUS (mFUS^KO/hFUS^R521H) mice. A Coomassie blue staining of recombinant HA-FUS protein. B Electron micrograph of fibrils of HA-FUS^R495X recombinant protein purified from bacteria (Left panel). HA-FUS^R495X fibrils after sonication before inoculating them into mice (Right panel). Scale bars: 1 µm (before sonication), 0.2 µm (after sonication). C Sonicated HA-tagged FUS^R495X fibrils were injected unilaterally into the cortex and hippocampus of 16 months old humanized, mutant FUS mice (mFUS^KO/hFUS^R521H). D, E Immunostaining of FUS (green) and DAPI (blue) of the side of the mouse brain hemisphere (ipsilateral side) injected either with PBS (D) or with HA-FUS^R495X fibrils (E) after 1 month and 6 months post-injection. Scale bars: 10 µm, inset: 5 µm. The top panel illustrates the regions of the brain that were analyzed and the site of injection (pink box). Yellow arrows indicate cytoplasmic FUS aggregates at 1- and 6-months post-injection (p.i.). F Quantification of the percentage of cells containing endogenous cytoplasmic FUS aggregates in the cortex and hippocampus at the injection (ipsilateral) side 1-, 2- and 6-months post-injection. N=3 animals. Kruskal-Wallis test with Dunn’s multiple test post-hoc p-values: cortex p = 0.0429 and hippocampus p = 0.0219. Data is presented as mean ± SEM. G, H Immunostaining of FUS (green) and DAPI (blue) of the opposite side of the mouse hemisphere (contralateral side) that was injected either with PBS (G) or with HA-FUS^R495X fibrils (H) after 1 month and 6 months post-injection. Yellow arrows indicate FUS cytoplasmic inclusions after 1- and 6-months post-injection. Scale bars: 10 µm, inset: 5 µm. I Quantification of the percentage of cells containing endogenous FUS aggregates in the cortex and hippocampus at the contralateral side over 1-, 2- and 6-months post-fibril injection. N=3 animals. Kruskal-Wallis test with Dunn’s multiple test post-hoc p-values: p = 0.0225

Fig. 2

HA-FUS^R495X fibrils induce aggregation and spreading of human FUS WT but not of mouse FUS. A Electron micrograph of FUS monomeric protein (scale bar: 1 µm) which was injected into 16-month old mFUS^KO/hFUS^R521H mice. B Immunostaining of FUS (green) and DAPI (blue) of the side of the mouse brain in which FUS monomers were injected after 2 months post-injection. Scale bars: 10 µm. C Coomassie blue staining of recombinant His-SOD1 protein. D Electron micrograph of His-SOD1 fibrils obtained from recombinant protein purified from bacteria (left panel) and sonicated His-SOD1 fibrils before inoculating them into 16 months old humanized  mFUS^KO/hFUS^R521H mice. Scale bar: 200 nm. E Immunostaining of FUS (green) and DAPI (blue) of the side of the mouse brain in which FUS monomers were injected after 2 months post-injection. Scale bars: 10 µm. F Sonicated HA-tagged FUS^R495X fibrils were injected unilaterally into the cortex and hippocampus of 16 months old humanized, FUS wild-type mice (mFUS^KO/hFUS^WT). G Immunostaining of a PBS-injected mFUS^KO/hFUS^WT mouse brain, 8 months post-injection using a FUS (green) antibody. H Immunostaining of FUS (green) and DAPI (blue) of the side of the mouse brain hemisphere (ipsilateral side) injected with HA-FUS^R495X fibrils. Yellow arrows indicate FUS cytoplasmic aggregates after 1-, 3- and 8-months post-injection. Scale bars: 10 µm, inset: 5 µm. I Quantification of the percentage of cells with endogenous human FUS aggregates in the cortex and hippocampus at the ipsilateral and contralateral side, 1-, 3- and 8-months post-injection. N=3 animals. Kruskal-Wallis test with Dunn’s multiple test post-hoc p-values: cortex ipsilateral p = 0.0190, cortex contralateral p = 0.0312, hippocampus ipsilateral p = 0.0299 and hippocampus contralateral p = 0.0190. Data is presented as mean ± SEM. J, K Immunostaining of FUS (green) and DAPI (blue) of the side of injection (ipsilateral side) in non-transgenic C57BL/6J mice (mouse FUS) brains injected with HA-FUS^R495X after 6- and 9-months post-injection. Scale bars: 10 µm, inset: 5 µm

Fig. 3

Human FUS aggregates are insoluble, display pre-amyloid properties and recapitulate features of human FUS pathology. A Schematic overview of the timepoints at which FUS aggregation was analyzed using immunofluorescence-based assays and biochemical insolubility assays at 3 hours, 1 months and 6 months post-injection. B, C Representative confocal images of mFUS^KO/hFUS^R521H mouse brains injected either with HA-FUS^R495X fibrils at 3 hours, 1 months and 6 months post-injection (B) or with PBS at 6 months post-injection (C) immunolabelled using antibodies against the pre-amyloid oligomer marker A11 (red), HA after 3 hours post-injection (green) and FUS after 1 month and 6 months post-injection (green). Yellow arrows indicate co-localization between A11 and FUS cytoplasmic inclusions detected in fibril-injected mice. DAPI (blue) as nuclear counterstaining. Scale bars: 10 µm, inset: 5 µm. D, E Representative confocal micrographs of mFUS^KO/hFUS^R521H mouse brains injected with HA-FUS^R495X fibrils using ubiquitin/p62/TDP-43 (red) and FUS (green) antibodies after 1 month (D) and 6 months (E) post-injection. Yellow arrows indicate co-localization between either ubiquitin/p62/TDP-43 and FUS cytoplasmic aggregates. Scale bar: 10 µm, inset: 5 µm. F Experimental outline of the serial fractionation of brain homogenates derived from mFUS^KO/hFUS^R521H mice either PBS- or HA-FUS^R495X fibril-injected and R6/2 Huntington’s model mice as a positive control for FUS insolubility [58]. G Immunoblotting of the sequential biochemical fractions from mouse brains using anti-FUS, anti-TAF15 and anti-TDP-43 antibodies. Anti-GAPDH was used as loading control

Fig. 4

Human FUS aggregates exacerbate cognitive impairments and provoke behavioral deficits and neurodegeneration in ALS-FUS mice. A Novel object recognition test was performed in 16 months (before injection) and 22 months old HA-FUS^R495X fibril-injected mFUS^KO/hFUS^R521H animals (6 months post-injection) compared to PBS-injected controls and non-transgenic HA-FUS^R495X fibrils or PBS injected controls. N=5–12 animals per group. Unpaired t-test p-value = 0.0293. Data is presented as mean ± SEM. B Open field test was performed in 22 months old HA-FUS^R495X fibril injected mFUS^KO/hFUS^R521H animals (6 months post-injection) compared to PBS-injected controls and non-transgenic HA-FUS^R495X fibrils or PBS injected controls. N=5–12 animals per group. Unpaired t-test p-value = 0.0498. Data is presented as mean ± SEM. C Representative immunofluorescence labelling for the neuronal marker NeuN (green) and DAPI in the hippocampus (upper panel) and cortex (lower panel) of humanized mutant mice mFUS^KO/hFUS^R521H non-injected, and injected either with PBS or HA-FUS^R495X fibrils. Scale bar: 25 µm. D,E Quantification of neurons in hippocampus (D) and cortex (E) in HA-FUS^R495X fibril-injected mice compared to PBS and non-injected controls. N=4 animals per condition. Data is presented as mean ± SEM. Kruskal-Wallis test with Dunn’s multiple test post-hoc p-values: hippocampus (D) p* = 0.0112 and cortex (E) p* = 0.0455 and p** = 0.0053