Case identification and cognitive profiling

Tissue was obtained from the Medical Research Council (MRC) Edinburgh Brain Bank (Table 1). All post-mortem tissue was collected with ethics approval from East of Scotland Research Ethics Service (16/ES/0084) in line with the Human Tissue (Scotland) Act (2006). Use of post-mortem tissue for studies was reviewed and approved by the Edinburgh Brain Bank ethics committee and the Academic and Clinical Central Office for Research and Development (ACCORD) medical research ethics committee (AMREC). Clinical data were collected as part of the Scottish Motor Neurone Disease Register (SMNDR) and Care Audit Research and Evaluation for Motor Neurone Disease (CARE-MND) platform, with ethics approval from Scotland A Research Ethics Committee (10/MRE00/78 and 15/SS/0216) and have been published previously [3, 10, 11]. Donors underwent neuropsychological testing during life with the Edinburgh Cognitive and Behavioural ALS Screen (ECAS) and all patients consented to the use of their data.

Table 1 Clinically stratified cohort of ALS cases and controlsThe clinically diverse patient cohort

To build a suitable patient cohort for our study, we set out to establish numerically balanced groups of four “case types” for comparison: These were (1) “Concordant” cases, where cognitive deficits had been identified along with pTDP-43 brain pathology, (2) “Discordant” cases, where pTDP-43 brain pathology had been identified but not cognitive deficits, (3) “Disease control” cases, constituting ALS patients with identified ALS-associated mutations (SOD1) but where no brain pTDP-43 pathology or cognitive deficit was identified, and (4) “Non-disease control” cases, representing controls with no diagnosis of ALS, no proteinopathy at post-mortem and no cognitive deficits in life (Table 1). For each of two cognitive brain regions (BA44 associated with language and fluency, and BA46 associated with executive function) for which TDP-43 brain pathology had been investigated, we identified three individuals for each of the four case types above. For both cognitive brain regions, cognition measured by the Edinburgh Cognitive ALS Screening tool and brain pTDP-43 pathology information was available and published previously [10].

Immunohistochemistry and BaseScope™ in situ hybridisation

Formalin-fixed, paraffin-embedded (FFPE) tissue was cut on a Leica microtome into 4 μm thick serial sections that were collected on Superfrost (ThermoFisher Scientific) microscope slides. Sections were baked overnight at 40 °C before staining. Sections were dewaxed using successive xylene washes, followed by alcohol hydration and treatment with picric acid to minimise formalin pigment. For pTDP-43 protein staining, antigen retrieval was carried out in citric acid buffer (pH 6) in a pressure cooker for 30 min, after which immunostaining was performed using the Novolink Polymer detection system (Leica Biosystems, Newcastle, UK) with a 2B Scientific (Oxfordshire, UK) anti‐phospho(409–410)‐TDP-43 antibody at a 1 in 4000 dilution and a Novus stathmin-2 antibody (St. Louis, USA) at a 1:500 dilution with no antigen retrieval step.

For validation of STMN-2 expression, two BaseScope™ probes were designed, one that targets STMN-2 downstream of exon 2 to detect normal STMN-2 (STMN-2(N); Catalogue number 1048241-C1), and one that targets the STMN-2 cryptic exon (STMN-2(CE); Catalogue number 1048231-C1). The BaseScope™ protocol was performed as we have published previously with no additional modifications [3, 11]. Slides were counterstained using haematoxylin and blued with lithium carbonate. STMN-2 expression was quantified manually by a pathologist blinded to clinical and phenotypic data. Manual grading was performed by counting the number of transcripts per cell in 20 cells in each of three 40 × fields of view per section. Whole tissue sections were scanned with brightfield illumination at 40 × magnification using a Hamamatsu NanoZoomer XR. Using NDP.view2 viewing software (Hamamatsu), regions of interest (ROIs) were taken from key regions for quantification as described below.

Immunohistochemistry modifications for RNA aptamer staining

Tissue was prepared in the same way for IHC as listed above. Following deparaffinisation, rehydration, and antigen retrieval, slides were incubated with peroxidase block for 30 min followed by 5-min wash step with TBS. Avidin and biotin blocking steps were then carried out using a biotin blocking kit (ab64212) as per the manufacturer’s guidelines followed by a 5-min TBS wash step and a 5-min wash step with milli-Q water. 156nM of aptamer (TDP-43Apt CGGUGUUGCU with a 3' Biotin-TEG modification, ATDBio, Southampton, UK) prepared in Milli-Q water was applied to the tissue and incubated for 3 h at 4 °C followed by incubation with 4% PFA overnight at 4 °C. A 5-min wash with Milli-Q water then preceded incubation with anti-biotin HRP antibody (ab6651) diluted 1 in 200 in milli-Q water for 30 min followed by a 5-min wash step with Milli-Q water and incubation with DAB for 5 min. Slides were then counterstained, dehydrated and cleared as detailed above. For double staining, the entire BaseScope™ staining protocol was implemented and following the application of red chromogen, slides were washed with TBS for 5 min and then taken straight to the avidin/biotin blocking steps of the aptamer staining protocol https://doi.org/10.17504/protocols.io.eq2lyjo4mlx9/v1.

Immunofluorescence staining

Tissue was prepared in the same way for IHC as listed above. Following deparaffinisation, rehydration, and antigen retrieval, slides were permeabilised in PBS + 0.3% Triton-X for 15min and then rinsed with PBS. Autofluorescence quenching was then performed by incubating slides for 2min in 70% ethanol, followed by 0.1% Sudan Black in 70% ethanol (20min) then a further 2 min in 70% ethanol. Slides were then washed in PBS followed by a 5 min incubation with TrueView (prepared as 1:1:1 components A, B and C). Slides were then washed for 5 min in PBS and blocked for 1h in blocking buffer (PBS supplemented with 1% goat serum and 0.1mg/ml salmon sperm DNA). Slides were then incubated overnight at 4°C with 1.2 μg/mL pTDP-43 primary antibody (Proteintech, 22309–1-AP) prepared in blocking buffer. Three 5 min washes with PBS preceded a 2-h incubation at room temperature with 4 μg/mL Alexa Fluor 647-conjugated secondary antibody (ThermoFisher A21244) prepared in blocking buffer. Three 5 min wash steps with PBS were then preformed followed by incubation with 1.2 μg/mL C-terminal TDP-43 Antibody with TDP-43APT (CGGUGUUGCU with a 3' Biotin-TEG modification, ATDBio, Southampton, UK) [0.67 μM] prepared in blocking buffer for 4h at RT in the dark. A wash step was performed with blocking buffer with aptamer E2108 [0.67 μM i.e. 1:50]. Slides were then incubated with 4% PFA in PBS (30min), washed with PBS and then incubated with DAPI 1:10,000 (10min), washed with PBS and mounted with Vectashield anti-fade. Three 5 min wash steps with PBS were then preformed followed by 4h covered incubation at room temperature with 1.2 μg/mL CoraLite Plus 488-conjugated C-terminal TDP-43 antibody (Proteintech, CL488-12892) and 0.67 μM TDP-43APT (CGGUGUUGCU with a 3' Atto 590 modification, ATDBio, Southampton, UK) prepared in blocking buffer. A wash step was performed with blocking buffer supplemented with 0.67 μM Atto 590-conjugated TDPAPT. Slides were then incubated with 4% PFA in PBS (30min), washed with PBS and then incubated with DAPI 1:10,000 (10min), washed with PBS and mounted with Vectashield anti-fade. Sections were imaged using a Zeiss AxioScan Z1 slide scanner with identical acquisition settings for all images.

Quantitative digital and manual pathology analysis

Ten (400 × 400 pixel) regions of interest (ROIs) were taken from each whole slide scanned image. Neuropil, neuronal nuclei, and neuronal cytoplasm were manually segmented using QuPath software [4]. Mean DAB intensity for neuropil, neuronal nuclei and neuronal cytoplasm were calculated and exported for subsequent analysis. DoG superpixel segmentation analysis was then carried out on the neuropil, neuronal nuclei, and neuronal cytoplasm separately to quantify aptamer-positive foci. Here, compartments were split into superpixels generated from pixels with similar intensities and textures for further classification, and each superpixel was classified as positive or negative for aptamer dependent on pre-set DAB intensity thresholds. The area of positive superpixels and total area were then exported. Weighted mean DAB intensity and weighted mean area of positive superpixels (where total area was used to weight) were then calculated per case (10 ROIs) to prevent pseudo replication. ROIs were then blinded and manually rated for (i) number of neurons with rod features (Nrods), (ii) more than one rod feature (N > 1rod + 1), (iii) nuclear morphologies consistent with aggregation (Nnuclearaggregation + 1), (iv) membrane pathology (Nmembranepath + 1), and (v) punctate cytoplasmic stain (Ncytoplasmicpuncta + 1). Blinded ROIs were also manually rated for the numbers of glia with (i) nuclear pathology and (ii) with cytoplasmic pathology. The product score for all neuronal features was calculated as

$$(\left( }\, + \,} \right)*\left( }\, > \,}\, + \,} \right)*\left( }\, + \,} \right)*\left( }\, + \,} \right)*\left( }\, + \,} \right))$$

Weighted mean neuronal features (where total number of neurons was used to weight) and weighted mean glial features (where total number of glia were used to weight) were calculated per case (10 ROIs) to prevent pseudoreplication. Data were visualized using RStudio with the “ggplot2” package [23]. ANOVA was used to determine differences between controls, discordant and concordant groups. Pearsons or Spearman’s correlation tests were used to determine correlations between STMN-2 counts and aptamer scores.