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ABSTRACT Identified genetic mutations cause 20% of frontotemporal dementia (FTD) and 5-10% of amyotrophic lateral sclerosis (ALS) cases: however, for the remainder of patients the origin of

disease is uncertain. The overlap in genetic, clinical and pathological presentation of FTD and ALS suggests these two diseases are related. Post-mortem, ~ 95% of ALS and ~ 50% of FTD

patients show redistribution of the nuclear protein TDP-43 to the cytoplasm within affected neurons, while ~ 5% ALS and ~ 10% FTD show mislocalisation of FUS protein. We exploited these

neuropathological features to develop an unbiased method for the in vitro quantification of cytoplasmic TDP-43 and FUS. Utilising fluorescently-tagged cDNA constructs and

immunocytochemistry, the fluorescence intensity of TDP-43 or FUS was measured in the nucleus and cytoplasm of cells, using the freely available software CellProfiler. Significant increases

in the amount of cytoplasmic TDP-43 and FUS were detectable in cells expressing known FTD/ALS-causative _TARDBP_ and _FUS_ gene mutations. Pharmacological intervention with the apoptosis

inducer staurosporine and mutation in a secondary gene (_CYLD_) also induced measurable cytoplasmic mislocalisation of endogenous FUS and TDP-43, respectively. These findings validate this

methodology as a novel in vitro technique for the quantification of TDP-43 or FUS mislocalisation that can be used for initial prioritisation of predicted FTD/ALS-causative mutations.

SIMILAR CONTENT BEING VIEWED BY OTHERS P62 OVEREXPRESSION INDUCES TDP-43 CYTOPLASMIC MISLOCALISATION, AGGREGATION AND CLEAVAGE AND NEURONAL DEATH Article Open access 01 June 2021 C9ORF72

DIPEPTIDES DISRUPT THE NUCLEOCYTOPLASMIC TRANSPORT MACHINERY AND CAUSE TDP-43 MISLOCALISATION TO THE CYTOPLASM Article Open access 21 March 2022 EARLY ACTIVATION OF CELLULAR STRESS AND DEATH

PATHWAYS CAUSED BY CYTOPLASMIC TDP-43 IN THE RNLS8 MOUSE MODEL OF ALS AND FTD Article Open access 03 April 2023 INTRODUCTION Frontotemporal dementia (FTD) is one of the most common forms of

presenile dementia and involves the progressive degeneration of the frontal and temporal lobes of the brain. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder

that affects the upper and lower motor neurons leading to muscle weakness and paralysis1. Increasing genetic evidence, neuropathological and clinical observations have identified a

substantial overlap between these two disorders2. Several genes have been identified where mutations can cause either FTD or ALS (FTD-ALS genes) including _C9orf72_, _VCP_, _OPTN_, _SQSTM1_,

_TBK1_3 and, most recently, _CYLD_4. In addition, FTD and ALS share neuropathological similarities: ~ 95% of ALS and ~ 50% of FTD patients show cytoplasmic inclusions of TAR DNA-binding

protein 43 (TDP-43) in the brain5,6, while ~ 5% of ALS and ~ 10% of FTD patients show cytoplasmic inclusions of fused in sarcoma protein (FUS)6,7,8. In healthy cells, TDP-43 and FUS are

largely expressed within the nucleus. In FTD and ALS, affected neurons can display a redistribution of TDP-43 or FUS to the cytoplasm, as well as insoluble TDP-43 or FUS aggregates6,9,10.

The importance of TDP-43 and FUS is further demonstrated by the fact that mutation of their encoding genes _TARDBP_ and _FUS_ is sufficient to cause ALS7,11,12 or, rarely, FTD13. Identified

genetic mutations cause 20% of FTD and 5-10% of ALS cases14,15; however, for the remainder of patients the origin of the disease is uncertain. Despite TDP-43 being a prominent feature in the

majority of FTD and ALS cases, including all those carrying FTD-ALS gene mutations13, this fact has not been applied to the screening of FTD and ALS candidate gene variants on a large

scale. In previous studies, TDP-43 and FUS mislocalisation has largely been assessed by subcellular fractionation, immunohistochemistry and manual analysis of confocal

microscopy7,16,17,18,19,20,21,22,23,24 or high-content imaging25,26,27. Whilst studies utilising these techniques have been informative, these assays are labour intensive and low-throughput 

or, expensive and in some cases produce qualitative data. In addition, some microscopy-based techniques could be subject to bias due to manual selection of the cells to be analysed. Here we

report the development of a rapid, cheap and unbiased technique for the in vitro study of TDP-43 and FUS cytoplasmic mislocalisation. TDP-43 or FUS staining in nuclear and cytoplasmic

subcellular compartments was measured in thousands of cells from confocal microscope images, using the freely available analysis software CellProfiler28. We validated this technique using

known genetic drivers of TDP-43 or FUS mislocalisation. We were able to detect increased cytoplasmic localisation of exogenously expressed _FUS_ and _TARDBP_ mutations relative to wild-type

(WT) sequence. We also observed mislocalisation of endogenous FUS upon treatment with the apoptosis inducer staurosporine. Importantly, we could also detect more subtle changes in the

cellular distribution of endogenous TDP-43, arising from mutation in a secondary gene (_CYLD_ p.M719V). This validated methodology can now be utilised for rapidly prioritising newly

discovered FTD and ALS gene variants by quantifying their effect on TDP-43 and/or FUS localisation. RESULTS DETECTION OF FUS CYTOPLASMIC MISLOCALISATION WITH EXOGENOUS EXPRESSION OF FUS

MUTATIONS In order to validate our method for the unbiased quantification of FUS and TDP-43 mislocalisation we initially utilised known pathogenic _FUS_ mutations that drastically alter FUS

subcellular localisation7,29. Expression of green fluorescent protein (GFP)-tagged FUS protein with missense mutation FUSR521C or truncation mutant FUSR495X in the neuronal-like

neuroblastoma cell line, SH-SY5Y, led to a significant increase in the fluorescence intensity of cytoplasmic FUS (Fig. 1e–k) when compared to FUSWT (Fig. 1a–c,h). This increase in

cytoplasmic FUS was accompanied by a corresponding decrease in the fluorescence intensity of nuclear FUS (Fig. 1d). Together, these resulted in a 9.5- and 12.5-fold increase in the FUS

cytoplasmic/nuclear ratio for cells expressing FUSR521C (0.97 ± 0.04; p < 0.0001) and FUSR495X (1.28 ± 0.04; p < 0.0001), respectively, relative to FUSWT (0.10 ± 0.00; Fig. 1l).

Similar results were found when overexpressing the FUSR521C (1.04 ± 0.05; p < 0.0001) and FUSR495X (1.81 ± 0.10; p < 0.0001) mutants in human embryonic kidney (HEK293) cells

(Supplementary Fig. S1), although the decrease in nuclear fluorescence intensity of FUS was not as prominent (Supplementary Fig. S1). DETECTION OF ENDOGENOUS FUS CYTOPLASMIC MISLOCALISATION

FOLLOWING STAUROSPORINE TREATMENT To test our ability to quantify the subcellular distribution of endogenous FUS we utilised the apoptosis inducer staurosporine, which has been previously

reported to cause cytoplasmic mislocalisation of FUS16. Application of increasing doses of staurosporine showed an increase in the fluorescence intensity of cytoplasmic FUS in cells treated

with the two highest doses of staurosporine (1 and 10 μM; Fig. 2a–d,f). A corresponding decrease in the nuclear intensity of FUS was also seen in SH-SY5Y cells (Fig. 2e). There was a

significant increase of cytoplasmic/nuclear FUS ratio for all treatment groups (0.1 μM: 0.13 ± 0.01; p = 0.0405; 1 μM: 0.33 ± 0.05; p = 0.0152; 10 μM: 1.53 ± 0.12; p = 0.0002) relative to

vehicle-treated cells (0.09 ± 0.00; Fig. 2g). HEK293 cells treated with staurosporine also exhibited a dose–response effect on FUS subcellular localisation. In general, HEK293 cells were

more tolerant of the staurosporine treatment (Supplementary Fig. S2) as shown by the lower cytoplasmic/nuclear ratios observed when compared to the SH-SY5Y cells (Fig. 2g and Supplementary

Fig. S2). Treatment with 10 μM staurosporine caused the majority of FUS to be present in the cytoplasm rather than the nucleus in SH-SY5Y cells (1.53 ± 0.12; p = 0.0002; Fig. 2d,g) whilst

the same treatment in the HEK293 cells caused only a small proportion of FUS protein to be mislocalised (0.28 ± 0.01; p < 0.0001; Supplementary Fig. S2). Despite these differences in

effect size, we could detect a significant effect of staurosporine treatment on mislocalisation of endogenous FUS in both cell lines. DETECTION OF TDP-43 CYTOPLASMIC MISLOCALISATION WITH

EXOGENOUS EXPRESSION OF _TARDBP_ MUTATIONS We took the same initial approach used for FUS to validate this method for the quantification of TDP-43 cytoplasmic mislocalisation: i.e. exogenous

expression of known pathogenic mutations in _TARDBP_. We examined p.A315T, one of the earliest detected and thus most extensively examined mutations30,31,32, and two of the mutations most

commonly observed in ALS patients: p.M337V and p.A382T11,31,32,33. In SH-SY5Y cells, a significant increase in the cytoplasmic/nuclear ratio of GFP-tagged TDP-43A315T (0.87 ± 0.01; p = 

0.0016; Fig. 3b,f,i–k) and TDP-43A382T (0.86 ± 0.02; p = 0.0032; Fig. 3d,h–k) was observed when compared to TDP-43WT (0.75 ± 0.01; Fig. 3a,e,i–k). In contrast, there was a significant

decrease in the cytoplasmic/nuclear ratio of GFP-tagged TDP-43M337V (0.63 ± 0.01; p = 0.0028; Fig. 3c,g,i–k) relative to TDP-43WT. Similar to the experiments with FUS, expression of TDP-43

mutations had a lesser effect in HEK293 cells (Supplementary Fig. S3). Once again, there was a significant increase in the cytoplasmic/nuclear ratio of TDP-43A315T (0.57 ± 0.02; p = 0.0021;

Supplementary Fig. S3) and TDP-43A382T cells (0.66 ± 0.03; p = 0.0026; Supplementary Fig. S3) when compared to TDP-43WT (0.50 ± 0.02; Supplementary Fig. S3). However, no significant effect

was observed in TDP-43M337V cells (0.52 ± 0.01; Supplementary Fig. S3). DETECTION OF ENDOGENOUS TDP-43 CYTOPLASMIC MISLOCALISATION IN CELLS EXPRESSING A CAUSATIVE FTD/ALS MUTATION To

demonstrate the potential of this methodology for identifying potentially pathogenic variants in other FTD/ALS genes, we expressed a known familial FTD/ALS-causative mutation in the _CYLD_

gene, p.M719V4, and observed the changes in endogenous TDP-43 localisation. We previously determined that overexpression of CYLDM719V increases the proportion of cytoplasmic TDP-43-positive

mouse primary cortical neurons by ~ 20% when compared to CYLDWT4. When compared to GFP-only vector control (0.079 ± 0.001; Fig. 4a–d), expression of CYLDWT-GFP (Fig. 4e–h) in SH-SY5Y cells

led to a significant 1.2-fold increase in the TDP-43 cytoplasmic/nuclear ratio (0.095 ± 0.001; p = 0.0002; Fig. 4q–s). In turn, expression of CYLDM719V-GFP (Fig. 4m–p) caused a further

1.3-fold increase in cytoplasmic/nuclear TDP-43 relative to CYLDWT-GFP (0.121 ± 0.002; p < 0.0001; Fig. 4q–s). The CYLDM719V mutation had the same effect in HEK293 cells, causing a

1.3-fold increase in the cytoplasmic/nuclear ratio relative to CYLDWT (0.145 ± 0.007; p = 0.0115; Supplementary Fig. S4). We also transfected cells with the CYLDD681G mutation (Fig. 4i–l and

Supplementary Fig. S4) which is catalytically inactive and known to cause CYLD cutaneous syndrome, a skin tumour disorder34,35. Our data corroborated previous results4 that the CYLDD681G

mutation had no effect on TDP-43 localisation when compared to the GFP-only vector control in either cell type (Fig. 4s and Supplementary Fig. S4). DISCUSSION In this study we have validated

a novel methodology for the unbiased quantification of cytoplasmic TDP-43 and FUS in human cell lines under three different experimental paradigms: determining localisation of exogenously

expressed mutant TDP-43 or FUS; detecting the effect of a chemical modulator on endogenous FUS localisation; and detecting the effect of a secondary gene on endogenous TDP-43 localisation.

This rapid, cheap, quantitative assay can now be utilised for rapidly assessing drug treatments and the potential pathogenicity of newly discovered FTD and ALS gene variants by quantifying

their effect on TDP-43 and/or FUS localisation. Previous studies examining TDP-43 and FUS have quantified cytoplasmic mislocalisation in different ways. Many studies have reported the

proportion of the cell population that display cytoplasmic TDP-43 or FUS expression12,18,19,21,24. This may present a problem with reproducibility, since fluorescence detection thresholds

for considering a cell as ‘positive' for cytoplasmic protein likely differ between labs and will vary according to the microscopy equipment used. We selected the ratio of cytoplasmic to

nuclear expression as our primary measure, having observed reduced variability between experimental replicates in comparison to individual nuclear and cytoplasmic measurements. Some studies

have reported similar measurements using techniques such as high-content screening confocal microscopy to study TDP-43 or FUS mislocalisation under different conditions25,26. Whilst

high-content screening can allow for a more high-throughput study design than our methodology, the extensive infrastructure required is expensive, not readily available and can require

substantial optimisation. We note that while we selected the CellProfiler program for post-imaging analysis, other software platforms are capable of the same type of analysis e.g. Imaris

(RRID:SCR_007370), ImageJ36 and Huygens (RRID:SCR_014237). We chose CellProfiler based on its ease of use, the ability to perform batch analysis of images, cost and accessibility. Another

general observation in our experiments was that SH-SY5Y cells often displayed a greater degree of cytoplasmic mislocalisation than that in HEK293 cells under the same conditions. This may be

due to SH-SY5Y cells being a neuronal cell line and thus more disease relevant, or due to the longer transfection time required to achieve optimal transfection efficiency in SH-SY5Y cells

(48 h, versus 24 h in HEK293), causing additional stress on the cells. It should also be noted that transfection efficiency also dictated the number of images required to reach the desired

cell number per replicate (300 cells). SH-SY5Y cells, which had a lower transfection efficiency, required more images to achieve the same number of cells for quantification. The cytoplasmic

mislocalisation and/or aggregation of FUS in neurons and glia of post-mortem tissue from patients with ALS caused by _FUS_ mutations has been widely established7,12. This pathology has been

recapitulated by expression of FUS mutations in mammalian cells7,12,29. In this study, exogenous expression of the severe truncation mutation FUSR495X caused the majority of FUS to be

mislocalised to the cytoplasm as previously demonstrated by Bosco et al.29 using live-cell confocal microscopy and 3D images (11–28 cells). Our quick and simple methodology was able to

duplicate these results using 2D images and a larger number of cells (1800 cells total). While we recognise the merits of quantifying the entire cell volume, measuring such a small number of

selected cells is not representative of the population and may not be able to detect more subtle or variable effects in protein localisation. Expression of the FUSR521C missense mutation

and treatment with staurosporine (1–10 μM) also reproduced previous qualitative microscopy and subcellular fractionation results7,16,37 showing a significant increase in the FUS

cytoplasmic/nuclear ratio. Confirmation of these results demonstrates the effectiveness of this new methodology in providing quantitative data where previously qualitative or

labour-intensive experiments were required. Our ability to quantify FUS cytoplasmic mislocalisation following staurosporine treatment also demonstrates the possibility of this technique for

use in testing the ability of novel FTD and ALS drug treatments to modulate FUS or TDP-43 mislocalisation. The majority (~ 95%) of ALS and ~ 50% of FTD cases are characterised by the

abnormal accumulation of TDP-43 in the cytoplasm of neurons and glia, even though in most cases mutations in _TARDBP_ are absent6. Mutations in _TARDBP_ have been associated with both FTD

and ALS and lead to TDP-43 cytoplasmic mislocalisation11,13. Unlike FUS, the expression of _TARDBP_ mutations in primary cells and cell lines does not reproduce the dramatic cytoplasmic

mislocalisation seen in post-mortem tissue38,39,40. In addition, overexpression of exogenous TDP-43 in vitro is sufficient to induce mislocalisation even for WT sequence19,23. Any

differences between WT and mutant TDP-43 for this characteristic are therefore harder to discern and represent a significant limitation for all TDP-43 exogenous expression assays, including

the one described here, and may be responsible for differing results being reported for the same mutations. Our technique was able to detect a significant increase in the TDP-43

cytoplasmic/nuclear ratio in TDP-43A315T and TDP-43A382T cells when compared to TDP-43WT. This confirms previous results for TDP-43A315T18,19 and TDP-43A382T41,42. The TDP-43M337V variant,

which displayed a significant decrease in cytoplasmic TDP-43 in SH-SY5Y cells, has previously been shown to increase cytoplasmic TDP-43 in some19,21,24, but not all studies22. We note that

TDP-43M337V has previously been shown to aggregate into nuclear puncta in SH-SY5Y cells21, which we observed in our experiments. As these puncta fluoresce at a higher intensity than diffuse

TDP-43 expression, this may lead to an overall decrease in cytoplasmic/nuclear fluorescence ratio relative to TDP-43WT. Thus, in some cases quantification of nuclear and cytoplasmic puncta

may be a useful adjunct to assess the pathogenicity of a given mutation. However, the disease relevance of nuclear inclusions in in vitro studies of TDP-43M337V is unclear: although both

intranuclear and cytoplasmic inclusions were reported in a transgenic TDP-43M337V mouse model43 and it is reminiscent of intranuclear inclusions observed in post-mortem tissue from sporadic

FTD patients9, such inclusions are rare in human disease. In the only neuropathological report to our knowledge that concerns a patient with a _TARDBP_ M337V mutation, no mention was made of

intranuclear inclusions44. We also note that mislocalisation of TDP-43 is not the only mechanism by which _TARDBP_ mutations lead to disease: for example, changes in protein stability or

interaction partners have also been described31. However, we envisage that the mislocalisation assay as implemented here would be a useful tool in a battery of assays to screen novel

_TARDBP_ variants. Lastly, we were able to recapitulate the effect of WT CYLD, the FTD-ALS-causative mutation p.M719V and the catalytically inactive mutation p.D681G on subcellular

localisation of endogenous TDP-43, which we previously observed in mouse cortical neurons4. In comparison to the other experiments used to validate this quantification method, the degree of

change in the endogenous TDP-43 cytoplasmic/nuclear ratio between the empty vector, the CYLDWT and the CYLD mutations was very small: e.g., absolute increase in ratio of ~ 0.03–0.04 between

CYLDWT and CYLDM719V (Fig. 4s and Fig. S4s). Our ability to detect significant differences for such a subtle change demonstrates the sensitivity of this technique for detecting changes in

TDP-43 and FUS localisation in vitro. We note that this assay relies on overexpression of the secondary gene and it would therefore be judicious to perform follow-up studies to examine

whether the effect is still observed when the gene is expressed at physiological levels (e.g. by CRISPR/Cas9-editing to introduce candidate gene mutations) and/or in more biologically

relevant cells (e.g. induced pluripotent stem cell-derived neurons). However, the utilisation of our quantification method in this format demonstrates what we believe to be its primary use,

prioritising potentially pathogenic newly identified FTD- or ALS-associated variants in genes other than _FUS_ and _TARDBP_ for more in-depth investigation. An important parameter to

consider when visualising mislocalisation of TDP-43 and FUS in exogenous expression assays is the positioning of the fluorescent tag in the TDP-43 or FUS fusion proteins. In this study we

utilised a N-terminal GFP tag for FUS constructs, due to the location of its nuclear localisation signal (NLS) at the C-terminus (aa497-526)45. Conversely, we used a C-terminal GFP tag for

TDP-43, as its NLS is located near the N-terminus of the protein (aa82-98)46, and TDP-43 forms homodimers via its N-terminus47,48. By tagging the protein at the opposite end to its NLS or

dimer-binding region we hoped to minimise any effect on the normal localisation pattern of TDP-43 and FUS. Additionally, aggregates of TDP-43 contain C-terminal fragments49, which may have

been undetectable had we used a N-terminal GFP tag. We note that the cytoplasmic/nuclear ratio of exogenous GFP-FUSWT (0.10 ± 0.00; Fig. 1l) was almost identical to that of endogenous FUS in

vehicle-treated cells (0.09 ± 0.00; Fig. 2g), implying that this tag does not affect FUS localisation. For TDP-43, the cytoplasmic/nuclear ratio of the GFP-tagged protein (0.75 ± 0.01; Fig.

 3k) was much higher than that of endogenous TDP-43 (0.079 ± 0.001; Fig. 4s). This is seen for exogenous expression of TDP-43 even with small peptide tags19,23. Nevertheless, we acknowledge

that the presence of the larger GFP tag may further affect TDP-43 localisation and represents a limitation of this assay. A parameter of equivalent importance for detection of endogenous FUS

and TDP-43 mislocalisation is selection of an appropriate detection antibody. This is particularly important for TDP-43, where C-terminal TDP-43 fragments observed in FTD and ALS patients9

contribute to its mislocalisation and aggregation49. Antibodies raised against the extreme N-terminus of TDP-43 would therefore not be suitable for a mislocalisation assay, as they would not

detect these cleavage products. In contrast, the anti-TDP-43 antibody we used (Proteintech; #10782-2-AP) recognises all major truncated TDP-43 forms9. In summary, this study demonstrates a

simple methodology for unbiased quantification of TDP-43 and FUS cytoplasmic mislocalisation in vitro. This technique can easily be added to studies utilising qualitative data to strengthen

clearly noticeable phenotypes in an unbiased manner, as well as to highlight subtle changes that may have not been previously identified. Utilisation of this fast and cost-effective

technique to aid in assessing pathogenicity of gene variants observed in patients with FTD or ALS will help to prioritise these variants for more intensive research efforts into how they

cause disease. METHODS DNA CONSTRUCTS _CYLD, TARDBP_ and _FUS_ mutations were introduced into the pCMV6-CYLD, pCMV6-TARDBP and pCMV6-FUS constructs (Origene) by site-directed mutagenesis

using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent). Mutated _CYLD_ and _TARDBP_ cDNA sequences were then subcloned, using AsiSI and MluI restriction sites, into the

pCMV6-AC-GFP vector (Origene) for expression of C-terminal GFP-tagged CYLD and TDP-43 proteins, respectively. Mutated _FUS_ cDNA sequences were subcloned similarly into the pCMV6-AN-GFP

vector (Origene) for expression of N-terminal GFP-tagged FUS protein, to avoid interference of the GFP tag with the C-terminal NLS of the FUS protein45. All clones were verified by

restriction digestion and sequence analysis. CELL CULTURE HEK293 and SH-SY5Y were purchased from ATCC and authenticated by STR analysis prior to use in this study. HEK293 cells were

maintained in Eagle’s Minimum Essential Medium (EMEM; Gibco) and SH-SY5Y cells in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F12; 1:1 mixture; Gibco) each containing 10%

heat-inactivated fetal calf serum (Sigma-Aldrich). For immunocytochemistry, 8-well chamber slides (Ibidi) were coated with 0.005% poly-L-lysine (Sigma-Aldrich) for 1 h and then washed twice

with Dulbecco’s phosphate-buffered saline (DPBS; Gibco). SH-SY5Y cells were seeded at 5–7.5 × 104 cells/well and HEK293 cells were seeded at 8 × 104 cells/well. After 24 h, cells were

transfected with GFP constructs (250 ng/well) for 24 (HEK293) or 48 (SH-SY5Y) hours using Lipofectamine 3000 (0.75 μL/well; Invitrogen) as per the manufacturer’s protocol. For the

staurosporine experiment, cells were incubated for 48 h after seeding, then treated for 5 h with 0.1, 1 or 10 μM staurosporine, to induce apoptosis. Staurosporine dilutions were prepared

from a 1 mM staurosporine stock solution dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich). All vehicle and staurosporine treatment groups were adjusted to a final concentration of 1%

DMSO. IMMUNOCYTOCHEMISTRY Transfected or drug-treated cells were fixed in 4% paraformaldehyde (PFA) in PBS for 20 min in the dark. For visualisation of exogenous TDP-43 and FUS, cells were

then washed twice with DPBS and mounted using DAKO fluorescent mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; Agilent). For visualisation of endogenous TDP-43 and FUS,

non-specific background staining was blocked after cell fixation using 1% bovine serum albumin (BSA) in PBS for 30 min. Cells were then incubated overnight at 4 °C with rabbit anti-TDP-43

(1:500; Proteintech #10782-2-AP) or rabbit anti-FUS (1:500, Proteintech #11570-1-AP) antibodies. The next day, cells underwent 2 × 5-min washes with DPBS, incubation with rabbit Alexa

Fluor-647 secondary antibody (1:500; Invitrogen #A-21244) for 1 h, followed by 2 × 5-min washes and mounting as above. Cells were imaged for fluorescence intensity quantification with a 40 ×

 objective on a Nikon A1R confocal microscope. Images of representative cells were obtained with a 100 × objective for figure preparation. Laser intensity, gain and offset settings were kept

constant and at least five random fields of view were imaged for each experimental replicate. FLUORESCENCE INTENSITY QUANTIFICATION Quantification of changes in the cellular localisation of

TDP-43 or FUS between the nucleus and the cytoplasm was done by utilising CellProfiler 3.1.9 software (Broad Institute, Cambridge, MA)28. To prepare images for analysis, ND2 microscope

files were converted to TIFFs, blinded and split into individual wavelength channel images using ImageJ (National Institutes of Health, Bethesda MD)36. In CellProfiler, the DAPI channel was

analysed first using the _Identify Primary Objects_ module and a global, three-class Otsu thresholding method. This allowed for identification and separation of each nucleus within an image.

Strict filtering for cell diameter (30–60 pixels) eliminated any irregular or overlapped nuclei that were present. TDP-43- or FUS-positive cells were also identified using the _Identify

Primary Objects_ module. Cell diameter filtering for this step had a much larger range and was dependent on experimental conditions and cell type (20–150 pixels). The _Relate_ and _Filter_

modules were used to link filtered nuclei to TDP-43- or FUS-positive cells. Centroid distance filtering, which limits the distance between the centre of the nucleus and the centre of the

cytoplasm (maximum 40 pixels) was also applied to remove any debris or abnormal cells that may be fluorescing in the TDP-43/FUS channel. For the experiments quantifying endogenous TDP-43 in

cells expressing GFP-tagged CYLD constructs, additional steps in the analysis were required. GFP-positive CYLD cells were identified in the same way as TDP-43- or FUS-positive cells using

the _Identify Primary Objects_ module, described above. However, prior to this, TDP-43-positive cells were identified by association with their corresponding nuclei, using the _Identify

Secondary Objects_ module. The watershed-image method was used with global, three-class Otsu thresholding. Size filtering was also applied (maximum 3000 pixels) to remove any clumped cells.

The _Relate_ and _Filter_ modules were used to select only TDP-43-positive cells that were also positive for GFP. TDP-43- and GFP-positive cells were then linked to their corresponding

nuclei using the _Relate_ and _Filter_ modules described above. For all experiments, in each TDP-43/FUS-positive cell, the nucleus and cytoplasm were separated into individual objects using

the _Identify Tertiary Objects_ module. Integrated (total) fluorescence intensity for both cellular compartments were then measured from the TDP-43/FUS channel image using the _Measure

Object Intensity_ module. Using these measurements, the cytoplasmic to nuclear ratio of TDP-43 or FUS was calculated for 300 cells per group, for each of the six replicates of the

experiment. All filtering and thresholding steps in each experimental pipeline were kept consistent throughout all experimental replicates. Filtering values were specific to each experiment

and require adjustment for different cell types and microscope parameters. STATISTICAL ANALYSIS Data are presented as mean ± standard error of the mean (SEM) from at least six independent

experiments of n = 300 cells. Mean fluorescent intensity of TDP-43 or FUS across six biological replicates was in most cases compared by repeated measures one-way ANOVA and Dunnett’s

multiple comparisons test. The effect of CYLD on endogenous TDP-43 was analysed using repeated measures one-way ANOVA and Sidak’s multiple comparisons test. All statistical analyses were

performed using GraphPad Prism 9. Significance for all tests was set at p < 0.05. DATA AVAILABILITY The datasets and materials generated during the current study are available from the

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the facilities and technical assistance of Microscopy Australia at the Australian Centre for Microscopy and Microanalysis at the University of Sydney. This research was funded by the

National Health and Medical Research Council of Australia (NHMRC) Project Grant 1140708 (to C.D.-S. and J.B.K.), by NHMRC Boosting Dementia Research Leadership Fellowship 1138223 (to

C.D.-S.) and the University of Sydney. J.B.K. is supported by NHMRC Project Grant 1163249, NHMRC-JPND Grant 1151854 and NHMRC Dementia Research Team Grant 1095127. AUTHOR INFORMATION AUTHORS

AND AFFILIATIONS * Brain and Mind Centre and School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Camperdown, NSW, 2006, Australia Lisa J. Oyston, Stephanie

Ubiparipovic, Lauren Fitzpatrick, Marianne Hallupp, Lauren M. Boccanfuso, John B. Kwok & Carol Dobson-Stone * Garvan Institute of Medical Research, Darlinghurst, NSW, 2010, Australia

Stephanie Ubiparipovic Authors * Lisa J. Oyston View author publications You can also search for this author inPubMed Google Scholar * Stephanie Ubiparipovic View author publications You can

also search for this author inPubMed Google Scholar * Lauren Fitzpatrick View author publications You can also search for this author inPubMed Google Scholar * Marianne Hallupp View author

publications You can also search for this author inPubMed Google Scholar * Lauren M. Boccanfuso View author publications You can also search for this author inPubMed Google Scholar * John B.

Kwok View author publications You can also search for this author inPubMed Google Scholar * Carol Dobson-Stone View author publications You can also search for this author inPubMed Google

Scholar CONTRIBUTIONS L.J.O., J.B.K. and C.D.-S. conceived the study. S.U., L.F., M.H. and L.M.B. generated and sequence-verified mutant cDNA constructs. L.J.O. carried out TDP-43 and FUS 

localisation experiments. L.J.O. and C.D.-S. participated in data analysis. L.J.O. and C.D.-S. drafted the manuscript. All authors read and approved the final manuscript. CORRESPONDING

AUTHOR Correspondence to Carol Dobson-Stone. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER'S NOTE Springer Nature

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S., Fitzpatrick, L. _et al._ Rapid in vitro quantification of TDP-43 and FUS mislocalisation for screening of gene variants implicated in frontotemporal dementia and amyotrophic lateral

sclerosis. _Sci Rep_ 11, 14881 (2021). https://doi.org/10.1038/s41598-021-94225-1 Download citation * Received: 06 April 2021 * Accepted: 06 July 2021 * Published: 21 July 2021 * DOI:

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