Our study is the first to use SBT to model FUS gene mutations in the mammalian central nervous system. The SBT paradigm was chosen because 1) mice can be generated quickly (a few months) compared to traditional transgenic techniques (a few years), 2) gene expression reaches a maximum ~3 weeks after birth, potentially avoiding toxicity during development, as has been recently observed for TDP-43
, and 3) recombinant AAV vectors can be rapidly generated to test different constructs in vivo, such as alternative promoters or putative disease associated mutations.
A key question in the field is how mutations in FUS cause neurodegeneration in ALS or FTD. Different pathogenic mechanisms for FUS mutants including toxic gain-of-function, loss-of-function, or a combination of effects have been hypothesized
[10, 25, 26]. The SBT FUS mice we have described provide additional insight into this issue. Over expression of either FUSWT, FUSR521C, or FUSΔ14 was not overtly toxic to mice on an organismal level after 3 months. Similarly, transgenic rats expressing wild-type human FUS do not have acute neuronal degeneration or behavioural impairment up to the first year of life; although transgenic lines expressing FUSR521C have rapid motor impairment and neuronal degeneration
. Despite this ALS-like phenotype, FUS R521C rat lines did not have classic neuropathology associated with FUS proteinopathies. Intriguingly, both FUS WT and R521C rats accumulated ubiquitin; however FUS did not co-localize with ubiquitin and there was no formation of distinct NCI
. Similar to this result we did not detect NCI in our FUSR521C mice. In contrast, SBT generated FUSΔ14 mice have FUS and ubiquitin positive NCI, suggesting that we observed a much greater accumulation of neuropathology due to the use of this mutation, which causes a dramatic redistribution of FUS into the cytoplasm
. One deficiency of the SBT FUSR521C or FUSΔ14 mice we have described is the lack of a motor phenotype or neurodegeneration. A simple explanation is that neuronal death is not present at the three-month time point we have examined. Larger cohorts of SBT FUS mice are being generated and aged to answer this question.
To date 46 mutations in FUS that are associated with ALS or FTD have been discovered, but the mechanism of their toxicity is still being deciphered
[26, 28, 29]. A majority of these mutations cluster in or near the C-terminal PY-NLS signal, and a number of groups have now reported that in cell culture these mutations inhibit nuclear import of FUS to varying levels and increase cytoplasmic levels of FUS
[27, 30–32]. Our data provide the first in vivo evidence in mouse neurons that both ALS mutations studied, FUSR521C and FUSΔ14, translocate to the cytoplasm at higher levels compared to control. FUSΔ14, which lacks the entire PY-NLS domain, had the highest levels of FUS in the neuronal cytoplasm, lowest levels in the nucleus, and was the only mutation that developed robust inclusions and insoluble FUS. The degree of FUS re-localization caused by a mutation and age of disease onset has been interpreted to mean that cytoplasmic accumulation of FUS is a primary event that drives neurodegneration
. Experiments in yeast
[33, 34], Drosophila[35–38], and C. elegans[39, 40] support the concept that cytoplasmic accumulation of FUS is toxic. In contrast, Xia et al. have reported that FUS toxicity in Drosophila requires nuclear localization
. Our observation that FUSΔ14, which produces the earliest disease onset in humans, accumulates at the highest levels in the cytoplasm and rapidly induces multiple pathological features of FUS proteinopathies, broadly supports the hypothesis that cytoplasmic FUS is toxic . Further experiments will be necessary to dissect whether chronic cytoplasmic accumulation of FUSΔ14 in our mice leads to neurodegeneration and if so by what molecular mechanism.
Many neurodegenerative diseases have NCI or glial inclusions and the identity of the aggregated molecule(s) has proven to be a useful tool to characterize disease sub-types and help define disease pathogenesis
. Despite the lack of an obvious motor or behavioural phenotype, the SBT FUSΔ14 mice recapitulate many key features of FUS proteinopathies
[10, 19]. The most striking feature in FUSΔ14 mice is the robust formation of NCIs, which are immunopositive for FUS, ubiquitin, PABP1and p62/SQSTM1. NCIs containing ubiquitin and p62 are common to all sub-types of FTD and ALS-FUS. More informative is the frequent presence of basophilic NCI in FUSΔ14 mice, which are numerous in BIBD cases, but also present in aFTLD-U and NIFID to a lesser extent
. Basophilic staining of NCIs has recently been linked to the presence of RNA and RNA-binding proteins, which is logical based on the function of FUS
. In contrast, we only detected infrequent α-internexin staining of NCI. This may indicate that FUSΔ14 pathology more closely resembles BIBID and aFTLD-U. Alternatively, NCI formation may start with FUS aggregation and accumulation of α-internexin is a downstream event. Further, when FUSΔ14 NCIs do stain with α-internexin, it is only a portion of the total inclusion (see Figure
5). We also asked if OPTN occurred in FUS NCI based on recent reports that OPTN is a prominent marker of NCI in a subset of ALS and FTLD
[43, 44]. In our FUSΔ14 mouse model, only a small percentage of neurons had small extra-nuclear aggregates of OPTN and these did not robustly overlap with NCI detected by FUS and ubiquitin immunohistochemistry (Additional file
5: Figure S5). α-internexin inclusions were more frequent and distinct than OPTN, but still only labelled a fraction of the total FUS positive NCIs (Figure
3 and Figure
5). This observation is reminiscent of recent pathological studies of NIFID cases which found that many NCI were immunoreactive for FUS and in some cases FUS-immunoreactive NCI were more numerous than α-internexin immunoreactive NCI
[17, 45]. The lack of α-internexin or OPTN positive NCI in FUSWT or FUSR521C mice implies that inclusion formation is a requirement for the development of this pathology. Based on these findings, and the referenced pathological findings in human cases, we suggest that α-internexin and OPTN pathology are downstream events and are not a major driver of pathology and neurodegeneration in most FUS proteinopathies. Electron microscopy of NIFID tissue supports the idea that neuronal intermediate filament accumulates following FUS aggregation in the cytoplasm
. On-going experiments with FUSΔ14 mice will address whether aging increases the amount of α-internexin staining.
An interesting question raised by our data is the identity of the ubiquitinated protein(s) in FUSΔ14 inclusions. Ubiquitin is the most enriched marker, besides FUS, in the NCI of FUSΔ14, but we do not detect mono or poly-ubiquitination of FUS. This data is in agreement with multiple reports that aggregated FUS isolated from human brain is not modified by post-translational modifications, such as ubiquitin or phosphorylation
[2, 46–48]. Taken together, we hypothesize that accumulation of FUSΔ14 into NCI recruits other protein(s) that are ubiquitinated. The identity of these proteins remains to be determined and may reveal additional insights into FUS pathogenesis.
PABP-1 was another protein frequently detected in FUSΔ14 NCI. PABP-1 binds the poly(A) tail of mRNA and is involved in multiple steps of mRNA metabolism, including pre-mRNA splicing and regulation of translation. PABP-1 has recently gained attention in the neurodegeneration field due to its involvement in the formation of stress granules. Stress granules are dense cytoplasmic foci composed of non-translated messenger RNA, ribonucleoproteins, and other proteins that vary depending on the cell type and stress inducer
. Stress granules are thought to protect mRNA from harmful conditions or serve as a mechanism to rapidly modulate the types and quantities of mRNA in response to changes in the environment
. PABP-1 is one of the more common RNA-binding proteins that reliably associates with the various types of stress granules and is therefore commonly used as a specific marker
. PABP-1 labels NCI in ALS-FUS with a R521C mutation, as well as NCI in FTLD-FUS, BIBD and NIFID
. In cell culture, mutation of the PY-NLS can efficiently redistribute FUS into the cytoplasm, but an additional stressor appears necessary to induce localization to stress granules
. This finding lead the authors to speculate that two hits may be necessary to induce abnormal accumulation of FUS into stress granules and eventually end-stage NCIs
. This does not appear to be the case in the FUSΔ14 mice, because we observe numerous NCIs that co-localize with ubiquitin, p62, and PABP-1. However, milder mutations such as FUS R521C or sporadic cases may indeed require additional genetic or environmental factors to induce abnormal FUS pathology.
A major difference between the FUS positive NCI found in ALS-FUS or FTLD-FUS is that they are much larger and more insoluble than the stress granules observed in cell culture. More detailed examination of the spectrum of FUSΔ14 transduced neurons reveals a spectrum of aggregates ranging from multiple small foci in a neuron to a single large inclusion filling the cell body (Additional file
5: Figure S5). We hypothesize that FUS-immunoreactive inclusions evolve in stages, and may represent a transition from stress granules, which are reversible and can rapidly be dissolve, to the large, insoluble, basophilic inclusions found in end-stage FUS pathology.
Mutations in FUS were first identified in ALS cases because sequencing of the FUS gene was prioritized based on its functional similarity to TDP-43, another RNA-binding protein that had been discovered to harbour causative mutations in ALS patients. Abnormal function of FUS, TDP-43, and other RNA-binding proteins has been recently proposed to be part of a common pathway linking defects in RNA quality control to neurodegeneration in ALS and FTLD
. Therefore it is imperative to determine if FUS and TDP-43 share pathogenic mechanisms or interact in some way. To date, most ALS cases with FUS mutations or FTLD cases with FUS pathologies do not show abnormal TDP-43 redistribution or pathology, although one group has reported co-deposition of both proteins in NCIs
[18, 52]. Experiments in Drosophila imply that both proteins share a common pathway, with FUS acting downstream of TDP-43
. Other model systems suggest that FUS and TDP-43 act through distinct pathways and cause disease through independent mechanisms, but a consensus has not yet been reached in the field
[28, 53, 54]. We find no evidence of TDP-43 redistribution into the cytoplasm or co-aggregation into NCI in any of the FUS mice examined, even in the presence of NCIs (Figure
5). Thus in our mouse model, FUS and TDP-43 aggregation appear distinct, and lead us to speculate that despite their many similarities
, FUS and TDP-43 have unique biological functions and their dysfunction may cause neurodegeneration through RNA dysfunction, but the precise targets and pathways are distinct.