Misregulation of RNA processing is a major component of the related neurodegenerative diseases Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration. TDP-43 is an RNA binding protein and the primary pathological finding in both diseases. Post-translational modification of TDP-43 by direct S-nitrosylation of cysteines 173 and 175 leads to disulfide bond formation and loss of solubility in ALS / FTD models. Further, TDP-43 aggregation may be able to induce nitrosative stress within the cell, leading to further loss of solubility. We present models of TDP-43 aggregation that demonstrate TDP-43 is capable of propagating between cells quantifiably, spreading disease-associated aggregates. C9orf72 is the most common genetic cause of ALS / FTD, and here we establish a CRISPR / Cas9 technology capable of tracking and degrading RNA foci found in in myotonic dystrophy and C9 ALS. Our findings provide a novel framework for therapeutics targeted at TDP-43 aggregation and RNA toxicity.

Amyotrophic lateral sclerosis (ALS) is a severely debilitating and fatal adult-onset neurodegenerative disease marked by the progressive loss of nerve cells in the brain and spinal cord leading to loss of voluntary muscle movement. A unifying feature of the majority of familial and sporadic ALS cases is TDP-43 proteinopathy, or the abnormal expression and function of trans-activation response DNA-binding protein (TDP-43 protein encoded by the TARDBP gene). The loss-of-function and gain-of-function associated with TDP-43 nuclear depletion, mislocalization, and cytoplasmic accumulation is being heavily investigated. Further, aberrant post-translational modifications (PTMs) to TDP-43 may contribute to disease mechanisms and toxicity. Most research has focused on the cytoplasmic TDP-43 aggregate formation, which is believed to be a toxic driver of disease progression. Phosphorylation has been implicated alongside cytoplasmic TDP-43 accumulation and aggregation, with contrasting results as to whether serves a protective or toxic role in this context. Further, multiple candidate kinases have been proposed to be the primary facilitator for TDP-43 phosphorylation in ALS disease. This dissertation seeks to provide insight into the role of CK1-dependent TDP-43 phosphorylation in protein solubility, aggregation, and toxicity, and ultimately determine whether targeting this mechanism holds promise for ALS and TDP-43 proteinopathy therapy. Chapter 1 provides an overview of our current understanding on ALS pathophysiology, neuropathology, disease mechanisms, ALS research models, and strategies that are being investigated for therapeutic development. In Chapter 2, we use in vitro assays and cellular models to demonstrate that CK1δ and CK1ε are the primary kinases that regulate ALS disease-relevant TDP-43 phosphorylation sites, and that RNAi and pharmacologic strategies that target CK1δ and CK1ε effectively reduce phosphorylated TDP-43 (pTDP-43) levels. Chapter 3 extends the previous findings of Chapter 2 into an in vivo inducible ALS mouse model and reveal that Csnk1e genetic deletion delays pTDP-43 formation but was insufficient in reducing cellular TDP-43 proteinopathy and motor behavior deficits. Finally, in Chapter 4, we study the efficacy of CK1δ/ε inhibitors in vivo and determine that manipulation of the S409/410 site on TDP-43 by selective CK1δ/ε inhibition is not a candidate therapeutic target on its own.