Citrullination a potential post-translational modification linked to TDP-43 pathology Patricia Rocha-Rangel1, Zainuddin Quadri1, Dale Chaput3, Daniel C. Lee PhD2, Maj-Linda B. Selenica PhD1 1Sanders Brown Center on Aging, Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY, United States; 2Sanders Brown Center on Aging, Department of Neuroscience, University of Kentucky, Lexington, KY, United States; 3Proteomics and Mass Spectrometry Core Facility, Florida Center of Excellence for Drug Discovery and Innovation (CDDI), University of South Florida, 3720 Spectrum Blvd, Suite 303, Tampa, FL 33612, USA.

Induced Citrullinated TDP-43 levels in Tar transgenic INTRODUCTION OBJECTIVES animal model TDP-43 is a nuclear RNA/DNA binding protein that in its pathological form, . Investigate PAD4 expression and activity in neurons of TAR transgenic A. Non-Tg TAR4 TAR4/4 mislocalizes and aggregates into the cytoplasm as insoluble cellular mice citR83 inclusions. TDP-43 inclusions are the histological hallmarks of . Evaluate the affinity of different CitTDP-43R antibodies during pathology 50µm frontotemporal lobar degeneration with TDP-43 (FTLD-TDP) and progression in TAR mouse models amyotrophic lateral sclerosis (ALS). Currently, mechanisms responsible for . Determine the CitTDP-43R protein structure in the neurons of TAR 50µm TDP-43 mislocalization and aggregation remain unclear, but it is transgenic mice hypothesized that post translational modifications (PTMs) play an active citR268/272 role. Citrullination is an irreversible PTM in which peptidyl arginine Identification of Citrullinated TDP-43 by mass 50µm deiminases (PADs) catalyze the conversion of arginine to citrulline. Little is spectrometry known about PADs in neurodegeneration, but there is few evidence of 50µm increased PAD4 expression in Alzheimer’s Disease (AD) and ALS motor A. LC-MS/MS TDP-43 citR83 neurons. From analyses of recombinant TDP-43, we identified 11 out of 20 B. NonTg TAR4 TAR4/4 C. TDP-43 citR268/272 arginine (R) residues citrullinated by PAD4. We developed custom 19 Unmodified 170 kda 1.0 ✱ Non-Tg 130 kda **** 0.8 TAR4 min TDP-43 SEM antibodies for each site, undergoing validation in recombinant, cellular and *** ± ✱ TAR4/4 98 kda 0.6 animal models. Here, we demonstrate the validation of CitR antibodies 72 kda ** 0.4 using western blot and immunohistochemistry in a TDP-43 transgenic 55 kda 0.2 43 kda TDP-43 animal model; TAR4 and TAR4/4 mouse model. Our results demonstrate 0.0 34 kda citR83 that PAD4 expression increases in TDP-43 transgenic animal models 20 Modified Ratio/GAPDH -0.2 min TDP-43 36 kda GAPDH following disease progression. We further demonstrate, via CitR 83 and TDP-43 citR83 NonTg citR 268/272 antibodies profile, the occurrence of citrullination as a “bona +PAD4 TAR4 TAR4/4 4.0 ✱ 170 kda fide” PTM. Overall, we posit that citrullination is a potential therapeutic SEM 3.0

130 kda **** ± target for treating TDP-43 proteinopathy and provides a novel approach for ✱ 98 kda *** 2.0 studying AD and LATE. 72 kda ** B. C. D. E. 55 kda 1.0 43 kda TDP-43 0.0 34 kda citR268/272 METHODS Ratio/GAPDH -1.0 36 kda GAPDH

Mass Spectrometry. Mass Spectrometry was used to identify citrullination Figure 4. A. Cortical neurons labeled with citR83 and citR268/272 antibodies. sites of recombinant TDP-43 protein. TAR4 and TAR4/4 anterior cortex compared to anterior cortex of non-tg Western blot. Western blot was used to measure the protein levels. controls; positive staining for both citRTDP-43 antibodies increased in a gene Immunohistochemistry. Fixed brains from non-tg, TAR4, and TAR 4/4 dose-dependent manner. B, C. Western blots probed with TDP-43 CitR83 and littermates were sectioned into horizontal sections, 25 µm thick. Brain Figure 2. A. Identification of 11 citR sites using MS/MS spectra scanning. Sites were CitR268/272 confirm that protein expression for both CitRTDP-43 oligomers sections incubated in primary antibody (CitTDP-43R83) (1:10,000) confirmed in both unmodified and modified CitR83TDP-43. B. Two novel antibodies increases with pathology, when compared to non-tg samples. One-way overnight at RT with gentle agitation and incubated in secondary antibody against citR TDP-43 at sites R83 and R268/272 are validated using (C, D) ANOVA was performed with Tukey’s as post-hoc (*p<0.05). (1:3000) for 2 hours at RT the next day. This protocol was modified for recombinant TDP-43. High-order oligomers at 130 and 170 kDa were recognized in TDP-43+PAD4 conditions. E. Total TDP-43 antibody confirmed the oligomeric folding antibodies CitTDP-43R268/272 (1:1000) and PAD4 (1:1000) to include of citR TDP-43, with bands present at 98, 130 and 170 kDa (green stars). CONCLUSION antigen retrieval in 70% formic acid prior to incubation in peroxidase blocker. Induced PAD4 levels in TAR transgenic animal model . PAD4 expression increases with pathological TDP-43 progression HYPOTHESIS A. Non-Tg TAR4 TAR4/4 . PAD4 expression significantly increases in the neuronal cytoplasm of TAR4/4 mice, compared to TAR4 and non-tg mice. . Citrullination of TDP-43 significantly increases in pathological TDP-43 phenotypes and similarly accumulates in neuronal cytoplasm. 50µm 50µm . CitR83TDP-43 provided more suitable results in IHC of TAR B. PAD4 tissue 6.0 ✱✱ . PAD4 and CitRTDP-43 antibodies express higher abundancy ✱ Non-Tg TAR4 of these proteins within the anterior cortex of diseased brain SEM 4.0 ✱ . Future directions: Investigate whether citRTDP-43 is a toxic ± TAR4/4 subspecies of TDP-43 and how it contributes to cognitive 2.0 decline and the development of AD, LATE, and FTLD-TDP/ALS % Area Figure 1. We hypothesize that PAD4-induced citR unfolds TDP-43, leading to an 0.0 accumulation of unfolded TDP-43 oligomers and soluble aggregates. These Figure 3. A. PAD4 expression and activity increased in the neuronal cytoplasm of conformers contribute to hallmarks of TDP-43 pathology including nucleocytoplasmic TAR4 and TAR 4/4 mice as pathology progresses compared to non-tg littermates. Funded from SBCoA/COM start-up trafficking and accumulation in stress granules. Our findings suggest that B. Quantification of % PAD4 positive area in the anterior cortex of non-tg and TAR citrullination may be a factor responsible for TDP-43 aggregation in neuronal mice. One-way ANOVA was performed with Tukey’s as post-hoc (*p<0.05, funds cytoplasm. **p<0.01).