Rabbit mAbs against pS409/410-TDP-43 detect TDP-43 pathology in brain tissues from FTLD and ALS patients and rNLS8 mice.

(A‒C) Representative images of immunohistochemical analysis using the indicated rabbit mAbs against pS409/410-TDP-43 in the frontal cortex of normal controls (A), FTLD-TDP type A patients (B), and FTLD-TDP type B patients (C), and in the motor cortex of ALS patients (C). Black arrows indicate neuronal cytoplasmic inclusions (NCI), and red arrows mark dystrophic neurites (DN). Inserts in B are higher magnifications of neuronal intranuclear inclusions (NCII). (D) Representative images of immunohistochemical analysis using the indicated rabbit mAbs against pS409/410-TDP-43 in the cortex of non-transgenic (nTg) and rNLS8 mice. Inserts are higher magnifications of NCI. (E) Representative images of immunohistochemical analysis using the indicated rabbit mAbs against pS409/410-TDP-43 in the cortex of AAV-2R and AAV-149R mice. Inserts are higher magnifications of inclusions. For all panels, scale bars are 20 μm.</p

Schematic representation of the methods and procedures to generate, screen and characterize monoclonal antibodies against pS409/410-TDP-43.

Schematic representation of the methods and procedures to generate, screen and characterize monoclonal antibodies against pS409/410-TDP-43.</p

26H10 rabbit mAb exhibits high specificity to pS409/410-TDP-43.

26H10 rabbit mAb exhibits high specificity to pS409/410-TDP-43.</p

26H10 rabbit mAb detects TDP-43 pathology in FTLD-TDP brain tissues.

26H10 rabbit mAb detects TDP-43 pathology in FTLD-TDP brain tissues.</p

Characteristics of patients with FTD/ALS.

Inclusions containing TAR DNA binding protein 43 (TDP-43) are a pathological hallmark of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). One of the disease-specific features of TDP-43 inclusions is the aberrant phosphorylation of TDP-43 at serines 409/410 (pS409/410). Here, we developed rabbit monoclonal antibodies (mAbs) that specifically detect pS409/410-TDP-43 in multiple model systems and FTD/ALS patient samples. Specifically, we identified three mAbs (26H10, 2E9 and 23A1) from spleen B cell clones that exhibit high specificity and sensitivity to pS409/410-TDP-43 peptides in an ELISA assay. Biochemical analyses revealed that pS409/410 of recombinant TDP-43 and of exogenous 25 kDa TDP-43 C-terminal fragments in cultured HEK293T cells are detected by all three mAbs. Moreover, the mAbs detect pS409/410-positive TDP-43 inclusions in the brains of FTD/ALS patients and mouse models of TDP-43 proteinopathy by immunohistochemistry. Our findings indicate that these mAbs are a valuable resource for investigating TDP-43 pathology both in vitro and in vivo.</div

Validation of representative B cell clones confirms their specificity for pS409/410-TDP-43.

(A) Dot blot analysis of rTDP43 treated with or without CK1 using the indicated supernatants from B cell clones. (B) Immunoblot analysis of HEK293T cell lysates expressing GFP, GFP-TDP-25, or GFP-TDP-25mut (S409A/S410A) using the indicated supernatants from B cell clones. GAPDH was used as a loading control. (C) Representative images of immunohistochemical analysis using the supernatants from indicated B cell clones in the hippocampus of human FTLD patients. Arrows mark TDP-43 inclusions. Scale bars are 20 μm.</p

Immunoreactivity of B cell clones against TDP-43 species as measured by ELISA immunoassay.

Immunoreactivity of B cell clones against TDP-43 species as measured by ELISA immunoassay.</p

Rabbit mAbs against pS409/410-TDP-43 detect the pathological accumulation of phosphorylated TDP-43 in FTLD-TDP brain tissues.

(A) Immunoblot analysis of urea-soluble fractions from the frontal cortex of FTLD-TDP patients and normal controls using the indicated rabbit mAbs. (B) MSD analysis of phosphorylated TDP-43 protein levels in urea-soluble fractions from the frontal cortex of FTLD-TDP patients and normal controls using the indicated rabbit mAbs (n = 4−5 per group). Data shown as the mean ± SEM. ** (left to right) P  =  0.0050, 0.0091 and 0.0075, unpaired two-tailed t-test.</p

S1 Raw images -

Inclusions containing TAR DNA binding protein 43 (TDP-43) are a pathological hallmark of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). One of the disease-specific features of TDP-43 inclusions is the aberrant phosphorylation of TDP-43 at serines 409/410 (pS409/410). Here, we developed rabbit monoclonal antibodies (mAbs) that specifically detect pS409/410-TDP-43 in multiple model systems and FTD/ALS patient samples. Specifically, we identified three mAbs (26H10, 2E9 and 23A1) from spleen B cell clones that exhibit high specificity and sensitivity to pS409/410-TDP-43 peptides in an ELISA assay. Biochemical analyses revealed that pS409/410 of recombinant TDP-43 and of exogenous 25 kDa TDP-43 C-terminal fragments in cultured HEK293T cells are detected by all three mAbs. Moreover, the mAbs detect pS409/410-positive TDP-43 inclusions in the brains of FTD/ALS patients and mouse models of TDP-43 proteinopathy by immunohistochemistry. Our findings indicate that these mAbs are a valuable resource for investigating TDP-43 pathology both in vitro and in vivo.</div

The rabbit bleed specifically detects pS409/410-TDP-43.

(A) Immunoblot analysis of rTDP43 with or without CK1 treatment using the indicated sera and antibody. (B) Immunoblot analysis of HEK293T lysates expressing GFP, GFP-TDP-25, or GFP-TDP-25mut (S409A/S410A) using the indicated sera and antibodies. GAPDH was used as a loading control. (C) Representative images of immunohistochemical analysis using the indicated sera in the frontal cortex of human FTLD-TDP patients. Arrows mark TDP-43 inclusions. Scale bars are 20 μm.</p