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.
