Characterize the E3 Ligase Activity of Znf179 and Its Effect on the Proteinopathies of TDP-43 Protein

Rationale: A predominantly brain-expressed novel RING finger protein Znf179 plays an important role for neuronal differentiation. During brain development, Znf179 is gradually increased and maintains in high levels in adult stage. Microarray database revealed that Znf179 is significantly downregulated in motor neurons of amyotrophic lateral sclerosis (ALS) mice, implying the role of Znf179 in neurodegenerative diseases. However, the molecular mechanism of Znf179 remains unclear. Znf179 contains the classic C3HC4 RING finger domain, and numerous proteins containing C3HC4 or C3H2C3 RING finger domain act as E3 ubiquitin ligases. Hence, we are interested to identify whether Znf179 possesses E3 ligase activity and its role in ALS neuropathy. Preliminary results: We first identified the RING finger protein Znf179 possessed the E3 ubiquitin ligase activity that exhibiting autoubiquitylation characteristics. Znf179 also regulated the proteasome activity through modulating the protein expression levels of 19S/20S proteasome subunits. To search for the candidate substrates of Znf179, we immunoprecipitated Znf179 and subjected to Mass analysis to identify its interacting proteins. We found that TDP-43 is one of the substrates of Znf179 E3 ligase. Znf179 interactes with TDP-43 protein and mediates polyubiquitylation of TDP-43 in vitro and in vivo. In ALS and FTLD-U patients, TDP-43 has been identified as the major components in the ubiquitin (+) inclusion. And we found that knockout of Znf179 in mice brain would lead to the puctate-like TDP-43 inclusion. These results implicated that depletion of Znf179 may result in cytosolic aggregates formation of TDP-43 protein. Hypothesis: According to our preliminary results, we anticipate that the E3 ligase Znf179-mediated TDP-43 polyubiquitylation may trigger the proteasomal or autophagic-lysosomal degradation pathways and regulates the turn over rates of TDP-43 protein. Lack of Znf179-mediated TDP-43 polyubiquitylation may decrease the metabolism rates of TDP-43 and enhance the aggregation and loss-of function of TDP-43 proteins. Specific Aims: To examine the E3 ligase activity of Znf179 and its role in neurodegenerative TDP-43-UBI (+) proteinopathy, we will first dissect the effect of autoubiquitylation on Znf179 E3 ligase activity and Znf179-mediated proteasome activity. Second, we will clarify the impact of E3 ubiquitn ligase Znf179-mediated TDP-43 polyubiquitylation on TDP-43 metabolism and proteinopathy, including TDP-43 protein stability, aggregates formation, nucleus/cytoplasm mislocalization, and RNA stability/ exon skipping activity in cell culture model. To further confirm the results from cell culture system, we will examine whether overexpression of Znf179 will reverse the pathological progression and consequences in FTLD/ALS-TDP-43 transgenic mice. Novelty and application: Exploring the role of Znf179 E3 ligase on TDP-43 proteinopathy would expand our understating about TDP-43-UBI (+) related neurodegenerative pathology and provides a useful therapeutic strategy in ALS/FTLD diseases.