Targeting the glycine-rich domain of TDP-43 with antibodies prevents its aggregation in vitro and reduces neurofilament levels in vivo

Cytoplasmic aggregation and concomitant nuclear clearance of the RNA-binding protein TDP-43 are found in similar to 90% of cases of amyotrophic lateral sclerosis and similar to 45% of patients living with frontotemporal lobar degeneration, but no disease-modifying therapy is available. Antibody therapy targeting other aggregating proteins associated with neurodegenerative disorders has shown beneficial effects in animal models and clinical trials. The most effective epitopes for safe antibody therapy targeting TDP-43 are unknown. Here, we identified safe and effective epitopes in TDP-43 for active and potential future passive immunotherapy. We prescreened 15 peptide antigens covering all regions of TDP-43 to identify the most immunogenic epitopes and to raise novel monoclonal antibodies in wild-type mice. Most peptides induced a considerable antibody response and no antigen triggered obvious side effects. Thus, we immunized mice with rapidly progressing TDP-43 proteinopathy (rNLS8 model) with the nine most immunogenic peptides in five pools prior to TDP-43 Delta NLS transgene induction. Strikingly, combined administration of two N-terminal peptides induced genetic background-specific sudden lethality in several mice and was therefore discontinued. Despite a strong antibody response, no TDP-43 peptide prevented the rapid body weight loss or reduced phospho-TDP-43 levels as well as the profound astrogliosis and microgliosis in rNLS8 mice. However, immunization with a C-terminal peptide containing the disease-associated phosphoserines 409/410 significantly lowered serum neurofilament light chain levels, indicative of reduced neuroaxonal damage. Transcriptomic profiling showed a pronounced neuroinflammatory signature (IL-1 beta, TNF-alpha, Nf-kappa B) in rNLS8 mice and suggested modest benefits of immunization targeting the glycine-rich region. Several novel monoclonal antibodies targeting the glycine-rich domain potently reduced phase separation and aggregation of TDP-43 in vitro and prevented cellular uptake of preformed aggregates. Our unbiased screen suggests that targeting the RRM2 domain and the C-terminal region of TDP-43 by active or passive immunization may be beneficial in TDP-43 proteinopathies by inhibiting cardinal processes of disease progression

Fluid biomarkers for amyotrophic lateral sclerosis: a review

Abstract Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of upper and lower motor neurons. Presently, three FDA-approved drugs are available to help slow functional decline for patients with ALS, but no cure yet exists. With an average life expectancy of only two to five years after diagnosis, there is a clear need for biomarkers to improve the care of patients with ALS and to expedite ALS treatment development. Here, we provide a review of the efforts made towards identifying diagnostic, prognostic, susceptibility/risk, and response fluid biomarkers with the intent to facilitate a more rapid and accurate ALS diagnosis, to better predict prognosis, to improve clinical trial design, and to inform interpretation of clinical trial results. Over the course of 20 + years, several promising fluid biomarker candidates for ALS have emerged. These will be discussed, as will the exciting new strategies being explored for ALS biomarker discovery and development

Generation and characterization of monoclonal antibodies against pathologically phosphorylated TDP-43.

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

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

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

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

Three rabbit mABs exhibit high specificity and sensitivity to pS409/410-TDP-43.

(A) ELISA analysis of phospho- and nonphospho-TDP-43 peptides using the indicated rabbit mAbs. (B) Dot blot analysis of rTDP43 treated with or without CK1 using the indicated rabbit mAbs. (C) Immunoblot analysis of HEK293T cell lysates expressing GFP, GFP-TDP-25 or GFP-TDP-25mut (S409/410A) using the indicated rabbit mAbs. GAPDH was used as a loading control.</p

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

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