{'fraction_non_alphanumeric': 0.04087561651239781, 'fraction_numerical': 0.017093439632457266, 'mean_word_length': 5.193305439330544, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Expression of Mutant Ubiquitin and Proteostasis Impairment in Kii Amyotrophic Lateral Sclerosis/Parkinsonism-Dementia Complex Brains

Kii amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) is a progressive neurodegenerative disorder that is endemic to the Kii peninsula of Japan. The disorder is clinically characterized by a variable combination of parkinsonism, dementia, and motor neuron symptoms. Despite extensive investigations, the etiology and pathogenesis of ALS/PDC remain unclear. At the neuropathological level, Kii ALS/PDC is characterized by neuronal loss and tau-dominant polyproteinopathy. Here, we report the accumulation of several proteins involved in protein homeostasis pathways, that is, the ubiquitin-proteasome system and the autophagylysosome pathway, in postmortem brain tissue from a number of Kii ALS/PDC cases (n1⁄4 4). Of particular interest is the presence of a mutant ubiquitin protein (UBBþ1), which is indicative of disrupted ubiquitin homeostasis. The findings suggest that abnormal protein aggregation is linked to impaired protein homeostasis pathways in


INTRODUCTION
The Kii peninsula of Japan is one of 3 high-incidence foci of amyotrophic lateral sclerosis (ALS) and parkinsonismdementia complex (PDC) in the Western Pacific (1). Similar foci have been found in the Pacific island of Guam (Marianas Islands) (2,3) and in West New Guinea (4). Kii ALS/PDC is a heterogeneous disorder in which patients show motor neuron signs and/or parkinsonian features with dementia. The Western Pacific variant of ALS is clinically indistinguishable from ALS in other parts of the world, but PDC appears to be a unique disease in the endemic areas. At the neuropathological level, Kii ALS/PDC is characterized by tau-dominant polyproteinopathy without abundant senile plaques (5)(6)(7)(8).
The cause of Western Pacific ALS/PDC is unclear, although both genetic and environmental factors have been considered. A hexanucleotide repeat expansion with the sequence (guanine) 4 (cytosine) 2 in the C9ORF72 gene partly accounts for ALS in the Kii peninsula (9). There is tremendous interest in understanding the etiopathogenesis of Western Pacific ALS/PDC because insight might be gained regarding neurodegenerative diseases found elsewhere (10,11).
We previously demonstrated the presence of ubiquitin-B þ1 (UBB þ1 ) protein in Guam PDC brains (12,13). UBB þ1 is a frameshift mutant of ubiquitin that is generated by molecular  . Immunohistochemical detection of mutant ubiquitin (UBB þ1 ) and protein homeostasis machinery in Kii amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) brains. (A) UBB þ1 is a frameshift mutant of ubiquitin that is generated by molecular misreading, a type of transcriptional mutagenesis. Occasional dinucleotide deletions in Ubiquitin-B (UBB) mRNA lead to the formation of a dysfunctional ubiquitin protein that inhibits the ubiquitin-proteasome system (UPS). (B) UBB þ1 contains an abnormal C-terminal extension that can be recognized by specific antibodies. UBB þ1 can be truncated from its C-terminus by deubiquitinating enzymes (DUBs), that is, ubiquitin carboxyl-terminal hydrolase L3 (UCHL3), resulting in another dysfunctional ubiquitin (Ub G76Y ) (23) (M.H. Glickman, personal communication). Preventing truncation results in increased UBB þ1 toxicity, hinting at a protective effect of DUB cleavage (34). Inhibition of DUBs, for example, by oxidative stress, prevents UBB þ1 truncation. (C-F) Immunostaining for MC1 (Tau 5-15/312-322 conformational antibody) and CP13 (tau phosphoepitope Ser202) reveals abundant neurofibrillary tangles (NFTs) in Kii ALS/PDC brain. (G, H) UBB þ1 can also be detected in cytoplasmic inclusions in ALS/PDC brain sections. Furthermore, specific components of the UPS, that is, the proteasomal AAA-misreading, a type of transcriptional mutagenesis (14) (Fig. 1A). UBB þ1 is a dose-dependent inhibitor of the ubiquitin-proteasome system (UPS), the major pathway for intracellular proteolysis, and accumulation of UBB þ1 in cells is a marker for impairment of the UPS (15) (Fig. 1B). It has been reported that UBB þ1 accumulates in the neuropathological hallmarks of several tauopathies (e.g., Alzheimer disease [AD] and Down syndrome) and in polyglutamine repeat disorders (14)(15)(16). Abnormalities in protein homeostasis (proteostasis) are implicated in the pathogenesis of a number of neurodegenerative conditions and it was hypothesized that accumulation of UBB þ1 may also be observed in Kii ALS/PDC brains. To evaluate expression of UBB þ1 and specific components of the proteostasis network, immunohistochemical analyses were performed on Kii ALS/PDC postmortem brain tissue.

Ethics Approval
This study was approved by the ethics committee of Mie University (Approval No. 2592).

Immunohistochemistry on Human Postmortem Brain Tissue
Formalin-fixed paraffin-embedded brain tissue sections (6 lm thick) from several Kii ALS/PDC cases and a nonneurological control (Table 1) were immunostained using standard procedures. Briefly, formalin-fixed paraffin-embedded sections were deparaffinized in xylene and rehydrated in a descending alcohol series. Sections were then treated with formic acid for 1 hour, followed by washes in tap water for . All antibodies were diluted in SuMi buffer (50 mM Tris, 150 mM NaCl, 0.25% gelatin, and 0.5% Triton X-100, pH 7.6). Next, sections were rinsed in TBS and incubated with biotinylated horse anti-mouse or horse anti-rabbit antibodies (1:400) (Vector Laboratories, Burlingame, CA) for 1 hour at RT. After incubation with secondary antibodies, sections were rinsed in TBS and incubated with avidin-biotin-peroxidase complex (1:800) (Elite ABC kit; Vector Laboratories) for 1 hour at RT. After final washes in TBS, sections were reacted with 0.5 mg/ mL 3,3 0 -diaminobenzidine (Merck, Darmstadt, Germany) containing 0.2% nickel ammonium sulfate and 0.01% hydrogen peroxide (H 2 O 2 ; Merck) in TBS (pH 7.6). Reactions were stopped in distilled water. Stained sections were dehydrated in an ascending alcohol series, cleared in xylene, and coverslipped with Entellan (Merck). AD patient brain sections were used as positive controls in all experiments.

RESULTS
To explore whether UBB þ1 and other proteins involved in proteostasis mechanisms accumulate in Kii ALS/PDC patient brains, immunohistochemical analyses were carried out on postmortem brain tissue from a number of Kii ALS/PDC cases (n ¼ 4) ( Table 1). Kii ALS/PDC sections showed numerous neurofibrillary tangles (NFTs) (Fig. 1C-F), in agreement with previous observations (5)(6)(7)(8). Importantly, immunohistochemistry revealed marked cytoplasmic deposition of UBB þ1 in neurons of the hippocampus and frontal/temporal lobe of Kii ALS/PDC cases ( Table 2). UBB þ1 was specifically present in NFT-like inclusions in all evaluated sections (Fig. 1G,  H). Interestingly, reactivity for UBB þ1 was also observed in glial cells, that is, astrocytes (Supplementary Data Fig. S1A). In addition, particular components of the UPS, that is, the proteasomal AAA-ATPase subunit RPT3(/S6b) and the deubiquitinating enzyme (DUB) ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), were detected in protein aggregates in Kii ALS/ PDC sections (Fig. 1I-L). In contrast, no reactivity for NFTs, UBB þ1 , and the specified UPS components was observed in brain sections from a nonneurological control (Table 2).
Another protein that was found to be present in aggregates in Kii ALS/PDC brain sections is phosphorylated protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (pPERK) ( Table 2). pPERK is a marker for activation of the unfolded protein response (UPR), a stress response pathway that reacts to disturbances in ER homeostasis (17). Immunoreactivity for pPERK was evident in tangle-like inclusions and in granular structures (Fig. 1M, N). The granular structures that were seen may represent granulovacuolar degeneration (GVD) bodies (18) (Supplementary Data Fig. S1B). pPERK was not detected in control brain (Table 2).
Lastly, immunostaining experiments were performed for the ubiquitin-binding protein p62, ATG8, and ATG12. All of these proteins are involved in the autophagy-lysosomal pathway (ALP), another key homeostatic mechanism in cells (19). All tested Kii ALS/PDC sections contained aggregates that stained positive for p62, ATG8, and ATG12 proteins, indicating abnormalities in the autophagy pathway (Fig. 1O-T). Control brain did not show reactivity for any of these proteins ( Table 2).

DISCUSSION
In the present study, we demonstrated that UBB þ1 , a frameshift mutant of ubiquitin and dose-dependent inhibitor of the UPS, is expressed in Kii ALS/PDC brains. Specifically, UBB þ1 was shown to be present in NFT-like structures in ALS/PDC brain sections. This staining pattern is intriguing, because disturbances of the UPS have been strongly associated with tau pathology in previous work (15,20). Accumulation of UBB þ1 and tauopathy might be mechanistically linked and it would be interesting, for example, to test whether UBB þ1 can modify the aggregation and cytotoxicity of tau in experimental models. Of note, UBB þ1 was also identified in astrocytes in Kii ALS/PDC sections. Such UBB þ1 -positive astrocytes were previously observed in Guam PDC brains (12). The significance of these glial inclusions is unclear, but similarities between granular or fuzzy astrocytes in ALS/PDC and aging-related tau astrogliopathy have been noticed (7,21). It was surmised that ALS/PDC might actually represent a kind of accelerated aging (7), and perhaps involves a glial senescence-like mechanism. This hypothesis could be tested in future studies by measuring the expression of senescence markers in patient brains. Preliminary analysis did not indicate clear changes in microglia density and morphology; although this needs to be examined further (Supplementary Data Fig. S2).
In addition to UBB þ1 , specific components of the UPS, that is, the proteasomal ATPase subunit RPT3 and the DUB UCHL1, were found in aggregates in Kii ALS/PDC brains. Aggregate structures containing these proteins were also observed in Guam PDC cases (12,13). Accumulation of the neuron-specific DUB UCHL1 is of particular interest, because UBB þ1 has been reported to be a DUB inhibitor in vitro (22). UCHL1 normally hydrolyzes small C-terminal adducts of ubiquitin to generate ubiquitin monomers, but appears to be unable to truncate UBB þ1 (23) (M.H. Glickman, personal communication) (Fig. 1B). We speculate that an interaction effect between UBB þ1 and select DUBs, like UCHL1, might contribute to abnormalities in the UPS and neurodegeneration.
Furthermore, we provided evidence for activation of the UPR (PERK arm) in Kii ALS/PDC brains. The UPR is a cellular stress response pathway that, analogous to the heat shock response (an important regulator of nuclear and cytoplasmic proteostasis), is involved in homeostatic regulation in the ER (17). The activated UPR represses global protein translation and induces expression of effector genes that allow cells to cope with stress (e.g., via stimulation of autophagy) or, alternatively, induces cell death when the level of stress cannot be handled. pPERK, an activated ER stress sensor, was frequently located in tangle-like structures. Intriguingly, pPERKpositive granules were noted in the evaluated sections and may reflect GVD bodies (18). Evidence for increased pPERK and GVD has also been found in Guam PDC brains (13,24). It has recently been established that GVD bodies are neuronselective lysosomal structures that can be induced by intracellular tau pathology (25,26). Strikingly, activated necrosome components could be detected in GVD bodies in AD neurons and it was hypothesized that the observed lesions might represent a delayed type of necroptosis (27). It remains to be determined whether necrosome components can also be identified in granular bodies in ALS/PDC brain. Longitudinal single-cell analysis of NFT formation and its relationship to GVD would assist in clarifying a mechanistic link.
Finally, we revealed that multiple proteins of the ALP, that is, p62, ATG8, and ATG12, accumulate in Kii ALS/PDC brains. Again, immunoreactivity for these proteins was previously observed in Guam PDC patient brains (13). The ALP plays crucial roles in several physiological processes (e.g., protein and organelle turnover), and deficiencies in the ALP can lead to disease (19). ATG8 and ATG12 are involved in vesicle expansion and completion in the autophagic process, and the ubiquitin-binding protein p62 is an adaptor that acts at the intersection between the UPS and autophagy (19). Inclusions containing another adaptor protein involved in autophagy and vesicle trafficking, that is, optineurin, were detected in Kii ALS/PDC tissue as well (28). Excitingly, tau pathology could be reduced by pharmacological activation of lysosomes in mice, suggesting that the ALP may represent a therapeutic target in tauopathies (29).
It is important to stress that altered protein degradation pathways, and in particular expression of UBB þ1 , are not limited to the diseases mentioned hereinbefore. Other tauopathies, including argyrophilic grain disease, show similar alterations (15,30). This feature is in contrast with the inclusions found in synucleinopathies (15), except for cases with mixed tau and a-synuclein pathology (31). Moreover, these alterations are not limited to the nervous system; similar pathology is also encountered in striated muscle in myofibrillar myopathies and related conditions (32,33). These findings illustrate the role of UBB þ1 in a heterogeneous group of disorders encompassing diverse pathologies and clinical manifestations.
In summary, we showed that UBB þ1 and several components of proteostasis pathways are accumulated in diseaseassociated aggregates in both Kii ALS and Kii PDC brains. This indicates a potential role for impairment of the proteostasis network in the pathogenesis of ALS/PDC. The results also support a similar pathogenesis of Kii and Guam ALS/PDC variants. Proteostasis mechanisms critically contribute to the cellular response to toxic proteins and pathogenic protein seeding. Therefore, improved understanding of proteostasis dysfunction in neurodegenerative disease may offer clues for therapeutic interventions that prevent or reverse aggregation. Kii ALS and Kii PDC also show TDP-43 and a-synuclein pathology in brain and spinal cord (7). It will be interesting to investigate the relation of impaired proteostasis to Kii ALS/PDC multiple proteinopathy in more detail. Double-labeling experiments to show colocalization of different abnormal proteins in aggregates and the addition of more cases would strengthen the significance of these findings.