Expression of Fused in sarcoma mutations in mice recapitulates the neuropathology of FUS proteinopathies and provides insight into disease pathogenesis

We utilized SBT to express wild type human FUS and two FUS mutations associated with ALS in the brains of mice to investigate the role of FUS in neurodegeneration using an in vivo model. In this experimental paradigm, newly born (P0) litters of mice were administered recombinant AAV1 encoding FUS_WT, FUS_R521C, or FUS_Δ14, through bilateral intracerebroventricular injection. The FUS R521C mutation, which has been identified in 16 ALS families to date, occurs within the PY nuclear localization signal (PY-NLS) region, and results in an average age of onset of 40 years [1, 14, 15]. The third model, FUS_Δ14,was based on a de novo mutation found in a patient with sporadic ALS that we reported previously [16]. Briefly, a mutation in intron 13 of the FUS gene (g.10747A>G) causes skipping of exon 14, a frame shift, and premature termination in exon 15, leading to a truncated FUS protein of 478 amino acids that lacks the C-terminal PY-NLS (Figure 1A). This mutation (FUS_Δ14) is associated with early disease onset (20 years) and a rapid disease progression (22 months).

Three months after viral injection, mice were killed and one brain hemisphere was fixed for neuropathologic characterization, while the other hemisphere was flash frozen for biochemical fractionation. Mice appeared healthy at the time of death and did not display obvious motor impairment or an abnormal grasping phenotype (data not shown). FUS constructs contained a V5 epitope tag on the amino-terminus to aid visualization of protein expression and do not interfere with protein function or cellular localization [16]. Based on V5 immunohistochemistry FUS_WT, FUS_R521C, and FUS_Δ14 mice had widespread FUS protein expression, throughout the brain, with the highest levels in the cerebral cortex and the hippocampus (Figure 1B-G). Using the SBT paradigm transgene expression is neuronal with no detectable glial expression, as assessed by double immunofluorescence (Additional file 1: Figure S1). Neurons expressing FUS_WT showed predominantly nuclear localization, with low, but detectable, levels of cytoplasmic protein based on immunohistochemistry and subcellular fractionation (Figure 1 and 2). FUS_R521C mice had marked increases in FUS immunoreactivity in the neuronal cytoplasm. The presence of nuclear FUS_R521C was a consistent feature; however, FUS_R521C was also detected in the soma, dendrites, and axons of neurons in mice, especially in the hippocampus (Figure 1C and F). Despite increased cytoplasmic levels of FUS_R521C, no obvious inclusions or aggregates of FUS were observed in mice injected with FUS_WT or FUS_R521C. FUS_Δ14 mice showed the greatest cytoplasmic redistribution, with some neurons showing no nuclear FUS reactivity but strong labelling of the cell body and processes in cortex. A portion of neurons in FUS_Δ14 mice contained FUS-positive neuronal cytoplasmic inclusions (NCIs), which bared striking resemblance to the NCIs that are a characteristic pathologic feature of ALS and FTD-FUS (Figure 1D and 1G). In cortex, the percentage of transduced neurons with cytoplasmic distribution of FUS significantly increased in FUS_R521C and FUS_Δ14 mice (Figure 1H). The FUS_Δ14 mice were the only group that had NCI, reaching ~20% of neurons expressing FUS (Figure 1H). Mutation-dependent FUS redistribution also was confirmed by double labelling with V5 and a neuronal marker (Additional file 2: Figure S2). Despite the presence of NCI, we did not observe any obvious neuronal loss or degeneration when examining haematoxylin and eosin (H&E) stained sections. Activated caspase-3 and TUNEL assays were also negative (data not shown), suggesting that apoptosis is not occurring in FUS mice at this age. Further, we did not observe marked astrocytosis or microglial activation at this age (Additional file 3: Figure S3).

We next examined the biochemical solubility of the different FUS proteins expressed in the SBT mice. The cytoplasm and intact nuclei were isolated from the frozen brain hemisphere of the FUS mice analysed above using immunohistochemistry (Figure 1). Nuclei were lysed in detergent to isolate the soluble nuclear fraction. The cytoplasmic and nuclear extracts were centrifuged at 20,000 x g to pellet insoluble proteins. Samples were separated by SDS/PAGE, analysed on immunoblots, and quantified using densitometry (Figure 2A). Comparison of the ratio of soluble cytoplasmic to nuclear FUS protein using densitometry confirmed that the steady state levels of the FUS mutants are higher in the cytoplasm, with FUS_Δ14 showing the strongest shift (Figure. 2A and B). Intriguingly, the levels of FUS_Δ14 detected in the cytoplasmic and nuclear lysates did not appear to match the immunohistochemistry (Figure 1B-H) that suggested robust expression of all FUS constructs. We then analysed the pellets resulting from the protocol used biochemical isolation of the cytoplasm and nucleus, and discovered that the majority of FUS_Δ14 protein partitions into the insoluble fraction (Figure 2C). No FUS_WT, but a portion of FUS_R521C protein was also detected in the insoluble fraction.

We next examined FUS mice for the presence of neuropathologic markers found in ALS or FTD. Sections from eGFP, FUS_WT and FUS_R521C mice had diffuse ubiquitin staining with no detectable inclusions (Figure 3A, E and I). In contrast, FUS_Δ14 mice had frequent ubiquitin-positive NCIs (Figure 3M and Additional file 4: Figure S4). NCIs varied in size from small puncta to large, round inclusions (Additional file 5: Figure S5). Double-label immunofluorescence confirmed that FUS_Δ14 and ubiquitin co-localize to the same inclusion (Figure 4). We did not observe an increase in high molecular weight smearing of FUS, an indicator of poly-ubiquitination, on immunoblots of brain tissue of FUS_Δ14 compared to FUS_WT (data not shown). Furthermore, immunoblots directly for poly-ubiquitin did not detect a difference between FUS_WT, FUS_R521C or FUS_Δ14 mice, suggesting that FUS is not robustly ubiquitinated (data not shown).

Based on the robust formation of NCIs in the FUS_Δ14 mice, we examined a panel of protein markers that were previously described in the NCIs of FUS proteinopathies. Many of the NCI in FUS_Δ14 mice were basophilic by H&E staining, similar to the NCI found in BIBD, aFTLD-U, and NIFID cases (Figure 3N) [17‐19]. Many of the basophilic inclusions were cytoplasmic, adjacent to the nucleus, and had a rounded, “Pick-body” like structure (Figure 3N insert). Eosinophilic staining was also noted on the periphery of inclusions, distinct spots within basophilic NCIs, or as distinct inclusions. We then stained for the neuronal intermediate filament protein α-internexin, which is one of the most abundant markers for NIFID [20]. A portion of the NCIs found in the FUS_Δ14 model were positive for α-internexin (Figure 3O), but they were less frequent than NCIs labelled with ubiquitin (Figure 3M). NCI in FUS_Δ14 were also immunoreactive for PABP-1, which was previously reported to stain NCI in BIBD and NIFID cases (Figure 3P) [21]. There was diffuse cytoplasmic staining of PABP-1 in eGFP, FUS_WT or FUS_R521C mice, but no obvious NCI (Figure 3D, H and L). Double-label immunofluorescence confirmed that α-internexin and PABP-1 co-localized with FUS inclusions in the FUS_Δ14 model (Figure 5E-L).

We also examined the staining patterns of the p62/ sequestosome-1 (p62/SQSTM1) and optineurin (OPTN) proteins in FUS mice, which have been reported to be mutated in some familial and sporadic ALS cases [22, 23]. P62 robustly co-localized with NCI in FUS_Δ14 mice (Figure 5A-D). OPTN immunostaining was diffuse and widespread in the neuronal cytoplasm of FUS_WT, FUS_R521C, as well as FUS_Δ14 mice. We found occasional increased OPTN staining in the neuronal cytoplasm in brain regions of FUS_Δ14 mice with ubiquitin positive NCI, but no definite labelling of NCIs (Additional file 6: Figure S6).

Because FUS and TDP-43 have such striking structural and functional similarities, the relationship between their pathology and disease mechanism is an important unanswered question in the field. We did not find any evidence of TDP-43 redistribution from the nucleus to the cytoplasm or presence of TDP-43 within NCI in FUS_WT, FUS_R521C and FUS_Δ14 mice (Figure 6A, B and C). In contrast, over expression of TDP-43 with a mutated nuclear localization signal in mice using SBT leads to cytoplasmic accumulation of TDP-43 (Additional file 7: Figure S7). Double-label immunofluorescence data also showed TDP-43 predominantly distributed in the nucleus in eGFP, FUS_WT, FUS_{R521C and} FUS_Δ14 mice, even in neurons with well-defined NCIs (Additional file 8: Figure S8 and Figure 6C-J).

The generation of the N-terminally V5 tagged FUS constructs, AAV1-wild type human FUS (FUS_WT), AAV1-human pR521C mutant FUS (FUS_R521C) and AAV1-human p.G466VfsX14 truncated FUS (FUS_Δ14) was previously described [16]. Inserts were cloned into the AAV1-vector using BamHI and XhoI restriction sites. The V5 tag does not alter the normal location or function of FUS and was added to the N-terminus of all constructs to facilitate detection and analysis without interference from endogenous FUS protein [32]. The sequences of all AAV1-FUS expression constructs were confirmed by direct sequencing of the complete cDNA inserts and flanking vector sequences.

All experiments with mice were approved by the Emory University and Mayo Clinic Institutional Animal Care and Use Committees and performed according to the guidelines for the care and use of laboratory animals. Animals were housed under circadian conditions and had free access to food and water. Recombinant adeno-associated virus serotype 1 (AAV1) generation and neonatal injection procedures were previously described [60]. Briefly, through viral transduction of the neuron, the protein of interest is expressed under the control of the cytomegalovirus enhancer/chicken β-actin promoter. P0 mouse pups (0–12 hours old) were cryoanesthetized on ice and bilaterally injected with 2 μl of virus (10¹³ particles/ml) per cerebral ventricle. After injection, the pups were wrapped in cage bedding, recovered on a heating pad then returned to their mother. Three groups of wild type B6C3F1 mice were injected with virus encoding FUS_WT (n=9), FUS_R521C (n=16) and FUS_Δ14 (n=11).

Brains were harvested at 3 months of age, except the hTDP43_M337V mice, which were sacrificed at 3 weeks of age. Half of the brain was immersion fixed in 4% paraformaldehyde for 24 hours and washed in Tris Buffered Saline (TBS). Sections were embedded in paraffin, sectioned in the sagittal plain (5 μm thick) and mounted on glass slides. The other half of the brain was frozen on dry ice for biochemistry.

Sections were deparaffinized in xylene and rehydrated in a graded series of alcohol followed by dH₂O. Antigen retrieval was performed in a dH₂O steam bath for 30 minutes. Immunohistochemistry was performed on an automated stainer (DAKO Auto Machine Corporation) and the DAKO EnVision+ HRP system. All sections were briefly counterstained with hematoxylin. For Double labelling immunofluorescence.

After deparaffinised and rehydrated, sections were incubated in retrieval solution (DAKO) for 30 min at 95 degree. Tissues were immunostained with the following primary antibodies at the indicated concentrations: V5 (monoclonal, Invitrogen, 1:1,000 and polyclonal, Bethyl Labs, 1:1000), FUS (polyclonal, Sigma, 1:2,500), TDP-43 (monoclonal human specific, Novus Biologicals, 1:3,000 and polyclonal, Proteintech, 1:5,000), PABP1 (polyclonal, Cell signaling, 1:100), α-internexin (monoclonal IgG; from Gerry Shaw, University of Florida, 1:50), OPTN (polyclonal, Abcam, 1:100) and ubiquitin (monoclonal, Millipore,1:60K and polyconal, DAKO, 1:200), TAF15(polyclonal, Bethyl Lab, 1:250), P62(monoclonal, BD Biosciences, 1:100), NeuN(polyclonal, Millipore, 1:200), IBA1(polyclonal, WAKO, 1:1000), EWS(polyclonal, Epitomics, 1:250), GFAP(polyclonal, Millipore, 1:1000). For Double labelling immunofluorescence, the sections were incubated in the secondary antibodies conjugated to Cy3 and fluorescein(1:250, Invitrogen). Hematoxylin-eosin (H&E) staining was performed on paraffin sections. Stained sections were captured using the ScanScope XT image scanner (Aperio, Vista, CA, USA) and processed with ImageScope software. Ubiquitin accumulation was quantified using the ImageScope positive pixel count algorithm for DAB staining (Aperio). Other photomicrographs were captured on an Olympus BX50 microscope with DP12 digital camera (Olympus, PA, USA). Confocal images were collected with a Zeiss LSM 510 NLO META system.

Nuclear and cytoplasmic enriched protein fractions were isolated from the tissue using the ProteoJET Cytoplasmic and Nuclear Extraction Kit (Fermentas, Ontario, Canada) following the manufacturer’s protocol. Protein separation and immunoblot were performed as previously described [61, 62]. Briefly, proteins were separated on 4-12% Bis-Tris XT gels (Bio-Rad, CA, USA) with XT-MES running buffer and transferred to a 0.2μm nitrocellulose membrane. After overnight blocking at 4°C in a 0.5% casein block solution, blots were probed with anti-V5 (monoclonal, Invitrogen, 1:1K) and anti-histone 3 (polyclonal, Cell Signaling, 1:1,000) primary antibody, followed by horseradish peroxidase (HRP)-conjugated secondary antibody against mouse and rabbit IgG (Jackson Immuno Research, 1:5K). Relative band intensity was quantified using ImageJ software (NIH).