Znf179 E3 ligase-mediated TDP-43 polyubiquitination is involved in TDP-43- ubiquitinated inclusions (UBI) (+)-related neurodegenerative pathology

Hui-Ching Lin,Hsin-Chuan Lin,Jiann-Her Lin,Tzu-Jen Kao,Wan-Chen Huang,Kuen-Haur Lee,Yi-Chao Lee,Chi-Chen Huang,Che-Kun James Shen,Wen-Chang Chang

doi:10.1186/s12929-018-0479-4

Abstract

BackgroundThe brain predominantly expressed RING finger protein, Znf179, is known to be important for embryonic neuronal differentiation during brain development. Downregulation of Znf179 has been observed in motor neurons of adult mouse models for amyotrophic lateral sclerosis (ALS), yet the molecular function of Znf179 in neurodegeneration has never been previously described. Znf179 contains the classical C3HC4 RING finger domain, and numerous proteins containing C3HC4 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.MethodsWe used in vivo and in vitro ubiquitination assay to examine the E3 ligase autoubiquitination activity of Znf179 and its effect on 26S proteasome activity. To search for the candidate substrates of Znf179, we immunoprecipitated Znf179 and subjected to mass spectrometry (MS) analysis to identify its interacting proteins. We found that ALS/ FTLD-U (frontotemporal lobar degeneration (FTLD) with ubiquitin inclusions)-related neurodegenerative TDP-43 protein is the E3 ligase substrate of Znf179. To further clarify the role of E3 ubiquitin ligase Znf179 in neurodegenerative TDP-43-UBI (ubiquitinated inclusions) (+) proteinopathy, the effect of Znf179-mediated TDP-43 polyubiquitination on TDP-43 protein stability, aggregate formation and nucleus/cytoplasm mislocalization were evaluated in vitro cell culture system and in vivo animal model.ResultsHere we report that Znf179 is a RING E3 ubiquitin ligase which possesses autoubiquitination feature and regulates 26S proteasome activity through modulating the protein expression levels of 19S/20S proteasome subunits. Our immunoprecipitation assay and MS analysis results revealed that the neuropathological TDP-43 protein is one of its E3 ligase substrate. Znf179 interactes with TDP-43 protein and mediates polyubiquitination of TDP-43 in vitro and in vivo. In neurodegenerative TDP-43 proteinopathy, we found that Znf179-mediated polyubiquitination of TDP-43 accelerates its protein turnover rate and attenuates insoluble pathologic TDP-43 aggregates, while knockout of Znf179 in mouse brain results in accumulation of insoluble TDP-43 and cytosolic TDP-43 inclusions in cortex, hippocampus and midbrain regions.ConclusionsHere we unveil the important role for the novel E3 ligase Znf179 in TDP-43-mediated neuropathy, and provide a potential therapeutic strategy for combating ALS/ FTLD-U neurodegenerative pathologies.

Highlights

The brain predominantly expressed Really Interesting New Gene (RING) finger protein, Znf179, is known to be important for embryonic neuronal differentiation during brain development
Znf179 possesses E3 ubiquitin ligase activity Numerous proteins containing RING finger domains act as E3 ubiquitin ligases [21, 22], and autoubiquitination is a notable feature for RING E3 ubiquitin ligases, resulting in proteasome degradation or functional alteration of the E3 ligase protein itself [31]
We found that the expression levels of insoluble polyubiquitinated full-length 43 kDa nuclear protein TAR DNAbinding protein (TDP-43), and the truncated 35 kDa/ 25 kDa fragments were increased in the urea-soluble fraction from Znf179 knockout mice cortex at 4 months of age when comparing with the wild-type control (Fig. 6e)

Summary

Introduction

The brain predominantly expressed RING finger protein, Znf179, is known to be important for embryonic neuronal differentiation during brain development. The non-amyloidogenic ubiquitinated inclusions (UBI) are found to be the common neuropathology in both FTLD-U (frontotemporal lobar degeneration (FTLD) and ALS (amyotrophic lateral sclerosis) neurodegenerative diseases [1]. A wide spectrum of pathological processing-related events that contribute to TDP-43 aggregate formation have been reported, including mutations within the C-terminal glycine-rich region [13], hyperphosphorylation [14, 15], ubiquitination [6], nucleus/cytoplasm mislocalization [16], abnormal cleavage into 25/35 kDa C-terminal fragments [10, 17], changes in solubility [18], prolonged protein half-life [13], formation of detergent-insoluble cytosolic aggregates in the central nervous system [6, 7], and blockage of the ubiquitin proteasome system (UPS) or autophagy degradation pathways [19, 20].

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