Additional file 1 of Targeting the glycine-rich domain of TDP-43 with antibodies prevents its aggregation in vitro and reduces neurofilament levels in vivo
Creators: name: Riemenschneider, Henrick
name: Simonetti, Francesca
name: Sheth, Udit
name: Katona, Eszter
name: Roth, Stefan
name: Hutten, Saskia
name: Farny, Daniel
name: Michaelsen, Meike
name: Nuscher, Brigitte
name: Schmidt, Michael K.
name: Flatley, Andrew
name: Schepers, Aloys
name: Gruijs da Silva, Lara A.
name: Zhou, Qihui
name: Klopstock, Thomas
name: Liesz, Arthur
name: Arzberger, Thomas
name: Herms, Jochen
name: Feederle, Regina
name: Gendron, Tania F.
name: Dormann, Dorothee
name: Edbauer, Dieter
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Neuroscience
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer

description: Additional file 1: Figure S1. TDP-43 epitopes and preimmune control serum. A: Overview of peptide antigens conjugated with ovalbumin through the N-terminal cysteine residue. Note that OVA conjugates of peptides 7, 8, 10, and 11 showed some aggregation in vitro. Peptides in bold were highly immunogenic in C57BL/6J wild-type mice and were thus used for the treatment study in rNLS8 mice. Novel monoclonal antibodies were raised in C57BL/6J mice, except of 26A2 and 26F12, which were generated from immunized Balb/c mice. B: Preimmune serum contains no detectable antibodies against human recombinant TDP-43 or pTDP-43 peptide as determined by ELISA. Background-corrected mean optical density at 450 nm was measured in duplicates per animal. All sera were diluted 1:100. Figure S2. Combined TDP-43 peptides are equally immunogenic and safe in three inbred mouse strains. Antibody response plateaus already after two immunizations. A: Timeline of immunization with pooled TDP-43 peptides, blood sampling, and body weight measurement of C57BL/6J, C3H/HeOuJ, and CD-1 mice. For each strain, six animals per treatment group were used. B: A dilution series of pooled sera was analyzed by ELISA against recombinant full-length TDP-43 or pTDP-43 peptide. Background-corrected mean OD450 was measured in duplicates. Triangular shapes indicate CD-1, circles represent C57BL/6J, and squares depict C3H/HeOuJ animals. Open shapes correspond to first blood sampling, while b2 is illustrated by filled shapes. C: Kaplan–Meier plot indicating survival probability of mice from different strains. No antigen combination caused statistically significant lethality in any of the strains investigated. D: Development of body weight over 14-week study period. Mean values are plotted group-wise. Only age, but not treatment or the interaction between treatment and age, had a statistically significant effect on the body weight as revealed by a two-way repeated measures ANOVA. C57BL/6J: p = 1.74*10–24; C3H/HeOuJ: p = 2.56*10–33; CD-1: p = 1.37*10–29. E: Dilution series of pooled rNLS8 sera from different time points were analyzed by ELISA against recombinant full-length TDP-43 or pTDP-43 peptide. Background-corrected mean OD450 was measured in duplicates. Note that the antibody response plateaued after two immunizations. Age of animals is annotated for each timepoint. Figure S3. Additional immunofluorescence stainings of pooled antisera confirm specificity towards nuclear and mislocalized TDP-43. Double immunofluorescence stainings of pooled antisera and with either phosphorylated TDP-43 or pan-TDP-43 were performed on frontal cortex sections of an FTLD case and a healthy control. A: Antisera from TDP1 + 3# and TDP10 + 12 animals reliably stained nuclear TDP-43 but no cytoplasmic inclusions in a control case negative for pTDP-43. B: Double immunofluorescence with a commercial TDP-43 antibody validates specificity of pooled antisera in patients. Antisera from TDP1 + 3# and TDP10 + 12 mice detected both nuclear and cytoplasmic TDP-43, while TDP15 sera showed strong preference for cytoplasmic TDP-43 with little affinity to nuclear TDP-43. Representative images are shown. All scale bars = 10 µm. Figure S4. Additional comparisons of gene expression profiles and cytokine assays in rNLS8 mice. A: Expression patterns of genes characterizing early, late, and recovery stage in isolated microglia [19] in rNLS8 mice/ALS patients. Asterisks denote significant changes. LFC: log2-fold change. B: Concordant gene expression changes between rNLS8 mice and ALS patients. Only statistically significant genes with a LFC value ≥ 1 are shown. The 27 genes upregulated in both conditions cluster into different GO categories as depicted in Fig. 6C. C: Cytokine levels determined by multiplex assay from cortical tissue of vaccinated rNLS8 mice and controls as in Fig. 6E. Non-affected analytes are shown. From the panel INF-γ, IL-2, IL-4, IL-10, IL-12p70 could not be detected at all. IL-5 and IL-6: Control PBS n = 6, ctrl VAX n = 6, TDP-PBS n = 10, TDP5 + 7 n = 6, TDP8 + 9 n = 9, TDP10 + 12 n = 9, and TDP15 n = 7; IL-9, IL-15, IL-17A/F, IL-27p28/IL-30, IL-33, and MIP-2: Control PBS n = 10, ctrl VAX n = 10, TDP-PBS n = 10, TDP5 + 7 n = 13, TDP8 + 9 n = 12, TDP10 + 12 n = 12, and TDP15 n = 12. MIP-2 levels were only statistically significantly elevated in TDP-PBS compared to ctrl PBS mice (p = 0.004), but not ctrl VAX mice as indicated by pairwise t-test with Benjamini–Hochberg correction. D: RNAseq-based gene expression of all detected cytokines from the cytokine/chemokine panel in rNLS8 mice and ALS patients compared to controls as in Fig. 6A. Asterisks denote significant changes. Figure S5. Characterization of TDP-43 mAbs on droplet formation, affinity, and patient inclusions. A: Representative time-lapse images of Alexa 488-labeled TDP-43 condensates obtained by spinning disc confocal microscopy. TDP-43 treated with isotype control antibody does not fuse, while mAbs targeting the C-terminal glycine-rich region increase droplet mobility, leading to slow fusion events and enlarged TDP-43 droplets. Scale bar = 2.5 μm. B: Novel TDP-43 mAbs were analyzed by ELISA against recombinant TDP-43 in dilution series. Background-corrected OD450 are shown. C: mAbs which most potently inhibited TDP-43 aggregation and LLPS detected pTDP-43-positive inclusions in a FTLD patient and additionally stained nuclear TDP-43 by immunofluorescence.