Hortobágyi T, Troakes C, Nishimura AL, et al.: OPTN in ALS and FTLD-TDP

ELECTRONIC Supplementary Material

Legends

Supplementary Figures

Figure S1: OPTN immunohistochemistry in the brain. Diffuse cytoplasmic labelling in controls and FTLD-TDP with ALS – comparison of different antibodies.

There is diffuse cytoplasmic labelling in control (a,c) and FTLD-TDP with ALS(b,d) with OPTI-HPA (a, b) and OPTI-C (c,d) antibodies in the temporal lobe neocortex. There is no obvious difference in staining intensity. OPTI-C antibody gives a cleaner background and appears more suitable for visualisation of cytoplasmic staining with DAB as a chromogen. In contrast, to detect inclusions, OPTN Sigma HPA 3360 is more sensitive (See Figures 1, 2) (Scale bar: 100 m (a, c); 200 m (b, d)).

Figure S2: OPTN in Alzheimer’s disease.

In the hippocampus OPTN positive neuronal cytoplasmic inclusions, dystrophic neurites and glial pathology is shown (a) in Alzheimer’s disease (AD). In the granule cell layer of the dentate fascia (b) the tau-positive pathology (red) is far more numerous then the occasional OPTN positivity (green) which does not co-localise with tau.

(OPTN immunohistochemistry with DAB as chromogen) (a). Double labelling immunofluorescence with tau (red)(b), p-TDP-43(red)(c), respectively, and OPTN-C (green)(b,c) antibodies. DAPI nuclear counterstain is blue.(Scale bar: 120 m (a);20 m (b); 15m (c); 7 m (c, inset)).

Figure S3: OPTN in Huntington’s disease.

In the striatum neuronal cytoplasmic granular staining is detectable. Intranuclear inclusions (a characteristic feature of HD) are not OPTN positive. Immunohistochemistry with OPTN (Sigma HPA 3360) antibody(Scale bar: 80 m).

Figure S4: Correlation analyses between levels of OPTN and -3tubulin.

OPTN and neuronal marker -3tubulin were normalized by levels of general cell marker Histone 3 (H3) to study the effect of neuronal loss on OPTN levels. There was a positive correlation in all groups with statistical significance in FTLD-TDP and AD.

Spinal cord samples from ALS patients (Pearson’s correlation coefficient r=0.39, n.s.)(a);brain samples from FTLD-TDP (r=0.98, p=0.0001)(b); brain samples fromAD (r=0.82, p=0.001)(c);brain samples from FTLD-tau (r=0.54, ns)(d).(n.s. – not significant, p>0.05)

Supplementary Tables

Table S1: Patient and tissue data of cases used for immunohistochemistry.

List of post-mortem brain samples obtained from control subjects and patients with sporadic ALS (SALS) with TDP-43 positive inclusions; familial ALS (FALS) with SOD-1 and FUS mutation, respectively; frontotemporal lobar degeneration (FTLD) with TDP-43 positive inclusions (FTLD-TDP); FTLD-TDP with ALS; FTLD with hyperphosphorylated tau inclusions (FTLD-tau): FTLD-tau with MAPT mutation, Pick’s disease (PiD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD); FTLD with FUS inclusions (FTLD-FUS), Alzheimer’s disease (AD) with and without TDP-43 pathology; dementia with Lewy bodies (DLB); Huntington’s disease (HD); and spinocerebellar ataxia type 3 (SCA).

Table S2: Patient and tissue data of cases for tissue microarray (TMA).

List of post-mortem brain samples obtained from controls and patients with sporadic ALS (SALS) and dementia with Lewy bodies (DLB), respectively.

Table S3: Patient and tissue data of cases for Western blotting on brain samples. Post-mortem brain samples were obtained from controls and patients with frontotemporal lobar degeneration (FTLD) with TDP-43 positive ubiquitinated inclusions (FTLD-TDP), and FTLD-TDP with ALS.

Table S4: Patient and tissue data of cases for Western blotting on spinal cord samples.

Post-mortem spinal cord samples were obtained from controls and patients with TDP-43 positive sporadic ALS (SALS).

Table S5: Comparison of clinical phenotype and tissue data in cases with and without OPTN positive pathological inclusions. There is no significant (p0.05) difference between any of the variables (Mann-Whitney test), although mean disease duration is longer in cases without OPTN pathology (p=0.08).

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