Additional file 1 of Quantitative patterns of motor cortex proteinopathy across ALS genotypes
Creators: name: Nolan, Matthew
name: Scott, Connor
name: Menuka Pallebage Gamarallage
name: Lunn, Daniel
name: Kilda Carpenter
name: McDonough, Elizabeth
name: Meyer, Dan
name: Sireesha Kaanumalle
name: Santamaria-Pang, Alberto
name: Turner, Martin R.
name: Talbot, Kevin
name: Ansorge, Olaf
Publisher: name: figshare
Year: 2020
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: Pharmacology
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Hematology
subject: Plant Biology
subject: 60506 Virology

description: Additional file 1 Supp Fig. 1: Additional staining of pathology in the motor cortex and spinal cord. Staining for OPTN was highly positive in all neurons in controls (A), but absent in an OPTN mutation case (D), reflecting the truncation of the protein upstream of the antibody epitope. Asterisks in (B) mark the presence of Betz cells. Arrows in (E) mark mislocalized and aggregated FUS inclusions. Arrows in (F) mark the presence of aggregated SOD1 protein in a case with the SOD1 D101G mutation (case 31). As discussed in the text, antibody SOD1 SPC-206 did highlight solid compact and skein aggregates but had strong background staining (C), making it unsuitable for quantitative automated image analysis. P62 was used instead as a marker for compact SOD-1 associated protein aggregates; granular aggregation of misfolded wild-type SOD1, which has been suggested to be present in all genotypes of ALS, was not revealed by p62 immunohistochemistry [22]. Scale bar applicable to all panels = 50 μm. Supp Fig. 2: Significant variation in the extent of pTDP-43 pathology between TARDBP and OPTN ALS mutation cases. pTDP-43 pathology in the TARDBP case was sparse, and consisted almost exclusively of compact NCI (a,b, red arrows highlight pathology). In contrast, pathology was severe and widespread in the homozygous ALS-OPTN case (c,d), with NCI, oligo inclusions and dystrophic neurites in all layers, including the subcortical white matter (e). Supp Fig. 3: CD68 staining between the primary motor cortex (a) and lumbar spinal cord (b) white and grey matter is positively correlated in all the genotypes tested; Pearson r, results as on figure. Best fit lines are manually added for illustrative purposes. Supp Fig. 4: Assessment of anterior horn neuron size. Anterior horn degeneration and shrinkage was most prominent in FUS (b) and SOD1 cases, and noticeably less severe in the single ALS-OPTN case (c), however there was significant intraindividual differences within genotypes (d). pTDP-43 aggregation in the anterior horn did not correlate with anterior horn neuron shrinkage/loss in either sporadic (blue dots) or C9ORF72 disease (red squares) (e). Supp Fig. 5: Calculated predominance ratios for single-IHC cohort using CD68 as a surrogate marker of neurodegeneration. Ratios were calculated by dividing the log expression of motor cortex CD68/mm2 by the log expression of anterior horn CD68/mm2. Lower ratios therefore represent a higher LMN burden of activated microglia. Supp Fig. 6: Betz cells occasionally display pTDP-43 aggregation but also occasionally nodular microgliosis and neuronophagia. MxIF analysis did not reveal significant evidence of pTDP-43 within Betz cells (a,b, green lined arrows indicate unaffected Betz cells), but it can occasionally be seen in some cases (c,d). However we did also find evidence of nodular microgliosis surrounding large layer V neurons in some cases (e). UL = upper layers, DL = deeper layers. Scale bars where not indicated (μm): a = 40, b,e = 50. Supp Table 3: Numerical results for Olig and TPPP/p25 quantification.