Emerging role of PAD4 mediated TDP-43 citrullination in the neuropathology of LATE and Alzheimer’s Disease Related Dementias - ABSTRACT TAR DNA-binding protein 43 (TDP-43) pathology is associated with a spectrum of clinical dementias. Despite the enormous risk that TDP-43 proteinopathies present to public health, there is a lack of strategies against TDP-43 pathology. Our laboratory has discovered that peptidyl arginine deiminase 4 (PAD4) induces the conversion of arginine (positive) to citrulline (neutral), a novel and irreversible posttranslational modification of TDP-43. We identified elevated PAD4 expression and TDP-43 citrullination (citR) at the outset of Limbic-Predominant Age-related TDP- 43 Encephalopathy (LATE) and in AD Related Dementias (ADRDs). We cross-validated our findings in a novel TDP-43 mouse model and will utilize it to investigate the mechanistic impact of PAD4 on TDP-43 phenotype. To date, we know very little about the role of PAD4 in the progression of LATE and ADRD. Notably, as citR TDP-43 remains elusive, our studies suggest domain-specific effects of citR moieties on TDP-43 nuclear import, cytoplasmic accumulation, and phase-separation dynamics. We will address the significant gap and define mechanisms of Ca2+-coordinated PAD4 activity on TDP-43 toxic gain-of-function in the human brain, mouse models, and neuronal cells. We state that PAD4-mediated TDP-43 citrullination impairs TDP- 43 nucleocytoplasmic shuttling and accelerates cytoplasmic accumulation, ultimately leading to neuropathology and cognitive decline in LATE. In Aim 1, we will establish how genetic PADi4 (gene) gain- and loss-of-function regulates TDP-43 nuclear import and cytoplasmic accumulation and determine PAD4-restricted TDP-43 epitopes in primary neurons. We will also assess how neuronal PADi4 expression or deletion promotes TDP-43 phenotype, synaptic dysfunction, and neurobehavioral deficits in a mouse model. Aim 2 will investigate the direct effects of NLS and LCD-directed pseudo-citR mimetics on TDP-43 nuclear import, accumulation, and solubility in primary neurons. We will also determine how these citR moieties regulate TDP- 43 liquid-solid separation kinetics. Lastly, we will determine the functional role of pseudo-citR mimetics on the TDP-43 phenotype in vivo. Aim 3 will determine if PAD4 activity and citR TDP- 43 epitope restriction signatures follow regional vulnerability of the human brain in LATE and ADRD. The proposed studies will elucidate how PAD4 activity and this novel PTM define functional consequences that lead to the neuropathogenesis of TDP-43 proteinopathies.
