**7. Conclusions and Perspectives**

In this review, we have summarized the recent advances in identifying the regulators of LUBAC-mediated linear ubiquitination and its pathophysiological functions. We have highlighted the "linear ubiquitin code", and its "writer" (LUBAC), "erasers" (DUBs such as OTULIN and CYLD), and "decoders" (linear ubiquitin-binding proteins such as UBAN domain-containing proteins and A20). LUBAC principally activates the canonical NF-κB pathway and suppresses apoptosis. Therefore, the impaired LUBAC activity and the aberrant functions in linear ubiquitin decoders are associated with autoinflammatory and neurodegenerative diseases, and cancers. In particular, it is worthwhile to focus on the fact that linear ubiquitin is present in the protein aggregates of various neurodegenerative diseases, including ALS.

From these studies, we hypothesized that the aggregable proteins in wisp inclusions may be initially conjugated with K48-linked ubiquitin chains; however, they seem to be resistant to the proteasomal degradation due to misfolding (Figure 6). Concomitant with aging, maturation, and liquid–liquid phase separation (LLPS), LUBAC-mediated linear ubiquitin and/or K63-linked ubiquitin chains will be conjugated to NCI. We speculated that it may further generate complex-types of ubiquitin chains, such as K48/linear-branched chains, in the thick bundles. The linear polyubiquitin may serve as a sca ffold to recruit the IKK complex and OPTN via the UBAN domain, thus inducing neuroinflammation followed by cell death and selective autophagy, such as mitophagy and aggrephagy (Figure 6). Interestingly, agitation experiments showed that linear polyubiquitin forms fibrillar aggregates more easily than K48-linked polyubiquitin [157]. Therefore, the linear ubiquitination may facilitate the formation of thick inclusions by LLPS. Thus, linear ubiquitination may have pleiotropic e ffects on neuroinflammation, protein folding, and proteostasis. Although we identified HOIPIN-8 as a potent inhibitor of LUBAC, further studies are necessary to generate potent and specific inhibitors of LUBAC for therapeutic treatments of neurodegenerative diseases.

**Figure 6.** Proposed schema for the involvement of multiple ubiquitin chains, including linear ubiquitin, in neurodegenerative diseases, and their potential functions.

**Author Contributions:** D.O., Y.S., H.I., and F.T. performed literature research and wrote the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by MEXT/JSPS KAKENHI grants (Nos. JP16H06575, JP18H02619, and JP19K22541 to F.T., and JP18K06967 and JP19H05296 to D.O.), Osaka City University Strategic Research Grant 2017 for top priority research (F.T.), Takeda Science Foundation (F.T.), a Grant for Research Program on Hepatitis from the Japan Agency for Medical Research and Development (AMED – 19fk0210050h0001 to F.T.), GSK Japan Research Grant 2017 (D.O.), and a gran<sup>t</sup> from the Nakatomi Foundation (D.O.).

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
