*5.3. Linear Ubiquitination in Neurodegenerative Diseases*

Neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, ALS, FTD, and related tauopathies, are characterized by the progressive degeneration of the central or peripheral nervous system [141], and at present, no disease-modifying therapies or medicines have been developed. The accumulation of misfolded, aggregated, and ubiquitinated proteins is a common mechanism underlying neurodegenerative diseases. Disease-specific misfolded and aggregated proteins have been identified, such as amyloid-β and tau in Alzheimer's disease, α-synuclein in Parkinson's disease, huntingtin in Huntington's disease, and superoxide dismutase 1 and TAR DNA-binding protein 43 (TDP-43) in ALS [142]. Although the aggregates or inclusions of these proteins are ubiquitin-positive, the types of ubiquitin linkages within these protein aggregates have not been identified.

ALS is a fatal progressive neurodegenerative disease caused by the loss of motor neurons. Although most ALS cases are sporadic, around 10% are familial, and mutations in approximately 20 genes encoding proteins involved in protein/RNA aggregation (*SOD1*, *TDP-43*, *hnRNPA1*/*2,* and *FUS*), neuroinflammation (*TBK1*), the ubiquitin-proteasome pathway (*UBQLN2*), and autophagy (*C9orf72, OPTN*, *SQSTM1*/*p62,* and *VCP*) have been identified [143]. Although OPTN reportedly functions as an autophagy receptor, we determined that the ALS-associated *OPTN* mutations, *E478G* and *Q398X*, abrogated the inhibitory effects of OPTN on LUBAC-mediated NF-κB activation, and accelerated TNF-induced cell death in HEK293T and HeLa cells [121]. Importantly, the intracytoplasmic inclusions in neurons from patients with the heterozygous *E478G* and homozygous *Q398X* mutations reacted with an anti-linear ubiquitin antibody, and they were co-localized with TDP-43 and phosphorylated p65, which is an activated form of NF-κB [121]. Moreover, these spinal anterior cells are positive to anti-cleaved caspase 8 and 3 antibodies, suggesting that OPTN regulates neuroinflammation and cell death. We recently showed that the linear ubiquitination of not only the *OPTN*-associated ALS, but also TDP-43-containing inclusions was detected in the cases of sporadic ALS [144]. Interestingly, all of the neuronal cytoplasmic inclusions (NCIs) in spinal cords, including fine "wisps", were immunolabeled by the anti-K48-linked ubiquitin antibody (Figure 4A,C), whereas the linear ubiquitin was mainly detected in the intermediate and thick bundles of TDP-43-positive inclusions (Figure 4B,E). Similarly, we showed that K63-linked ubiquitin was predominantly detected in intermediate and thick bundles, and linearand K63-linked ubiquitin immunoreactants were not always co-localized [144]. Moreover, OPTN and phosphorylated p65 were co-localized with these inclusions derived from sporadic ALS patients. Furthermore, we showed that K48-linked ubiquitination is present in all of the tau neurofibrillary tangles, including small ones, whereas a subset of thick neurofibrillary tangles, dystrophic neurites of senile plaques, and neuropil threads were immunopositive for linear ubiquitin in Alzheimer's disease [145]. These results suggested that various ubiquitinations, including linear ubiquitin, is involved in protein aggregates of neurodegenerative diseases.

Importantly, Winklhofer's group reported that linear ubiquitin is indispensable for protein quality control [146]. They showed that LUBAC is recruited to the aggregates derived from overexpressed huntingtin-derived polyglutamine (Htt-polyQ) in human neuroblastoma SH-SY5Y cells, and linear ubiquitin also co-localized with the Htt-polyQ aggregates. During the recruitment of LUBAC, AAA-ATPase p97/VCP, which binds to the PUB domain of HOIP, plays an important role to suppress the proteotoxicity, since the linear ubiquitination of Htt-polyQ modulates the aberrant interaction of Htt-polyQ with the transcription factor Sp1. They further indicated that linear ubiquitination is involved in various disease-associated aggregable proteins, such as ataxin-3 (Machado-Joseph disease), and SOD1, TDP-43, and OPTN (ALS). Thus LUBAC, together with its linear ubiquitination activity, seems to be a crucial regulator of various neurodegenerative diseases.

**Figure 4.** Thick bundles of inclusions are immunopositive for anti-K48 and anti-linear ubiquitin antibodies, whereas fine wisps exclusively reacted with anti-K48 ubiquitin, but not anti-linear ubiquitin. The double immunofluorescence staining using anti-K48-linked ubiquitin antibody (**A**,**D**), anti-linear ubiquitin antibody (**B**,**E**), and their merged images (**C**,**F**) are shown. Arrows; neuronal cytoplasmic inclusions co-localized with K48- and linear-ubiquitins. Arrowheads; K48-ubiquitin-positive inclusions, which lack immunoreactivity for anti-linear ubiquitin antibody (Bars = 20 μm, modified from Ref. [144] with permission).
