cells
Review

Ubiquitin, Autophagy and
Neurodegenerative Diseases
Yoshihisa Watanabe 1, *, Katsutoshi Taguchi 2 and Masaki Tanaka 2, *1
2*

Department of Basic Geriatrics, Graduate School of Medical Science, Kyoto Prefectural University of
Medicine, Kyoto 602-8566, Japan
Department of Anatomy and Neurobiology, Graduate School of Medical Science, Kyoto Prefectural
University of Medicine, Kyoto 602-8566, Japan; ktaguchi@koto.kpu-m.ac.jp
Correspondence: y-watana@koto.kpu-m.ac.jp (Y.W.); mtanaka@koto.kpu-m.ac.jp (M.T.)

Received: 5 August 2020; Accepted: 2 September 2020; Published: 2 September 2020


Abstract: Ubiquitin signals play various roles in proteolytic and non-proteolytic functions. Ubiquitin
signals are recognized as targets of the ubiquitin–proteasome system and the autophagy–lysosome
pathway. In autophagy, ubiquitin signals are required for selective incorporation of cargoes, such asproteins, organelles, and microbial invaders, into autophagosomes. Autophagy receptors possessing
an LC3-binding domain and a ubiquitin binding domain are involved in this process. Autophagy
activity can decline as a result of genetic variation, aging, or lifestyle, resulting in the onset of various
neurodegenerative diseases. This review summarizes the selective autophagy of neurodegenerative
disease-associated protein aggregates via autophagy receptors and discusses its therapeutic application
for neurodegenerative diseases.
Keywords: ubiquitin; autophagy; neurodegenerative diseases; ubiquitin–proteasome system;
autophagy–lysosome pathway

1. Introduction
Many neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD),amyotrophic lateral sclerosis (ALS), and Huntington’s disease (HD), involve accumulation of harmful
and aggregation-prone proteins. These aggregated proteins are known to be ubiquitinated in manyneurodegenerative diseases. Although harmful proteins are immediately degraded by proteolytic
systems in healthy individuals, any perturbation of these systems caused by genetic variation, aging,or lifestyle results in accumulation of harmful protein aggregates and the onset of various diseases
including neurodegenerative diseases. Ubiquitination is the most important targeting signal for
proteolytic systems [1]. Indeed, pathological analyses show that most of the protein inclusions andaggregates in the brains of neurodegenerative disease cases are positive for ubiquitin [2]. Recent
advances in mass spectrometry technology have contributed to the characterization of ubiquitin chains
and the decoding of ubiquitin signals. Ubiquitin signals are categorized as mono-ubiquitin, homotypic
poly-ubiquitin, and heterotypic poly-ubiquitin [3]. Homotypic poly-ubiquitin chains are generated
by conjugation of two or more ubiquitin molecules via their seven lysine residues (Lys-6, Lys-11,
Lys-27, Lys-29, Lys-33, Lys-48, and Lys-63) or the initiation methionine residue (Met-1), whereas
heterotypic poly-ubiquitin chains are formed by linkages of two or more different Lys residues [3].
These ubiquitin signals have roles in proteolytic functions and non-proteolytic functions, such astranscription regulation, membrane trafficking, DNA repair, and cell signaling [4]. Mutations in several
autophagy related proteins, such as Parkin, PINK1, p62, and OPTN, are linked to neurodegenerative
diseases. Autophagy receptors function in the selective autophagic clearance of disease-related proteins
via ubiquitin signals. Thus, augmentation of autophagy is potentially a good therapeutic approach forCells 2020, 9, 2022; doi:10.3390/cells9092022

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2 of 15neurodegenerative diseases. This review focuses on the role of ubiquitin signals in autophagy andtheir relevance to the onset of neurodegenerative diseases.
2. Neurodegenerative Diseases and Protein Aggregates
AD is a progressive neurodegenerative disorder that leads to cognitive decline [5]. The main
hallmarks of AD are deposition of β-amyloid protein (Aβ) outside neurons, termed senile plaques,
and in the vascular walls of the brain, and the accumulation of hyperphosphorylated Tau-protein asneurofibrillary tangles inside neurons [6]. Aβ is generated from amyloid precursor protein (APP)
through sequential cleavage by β-secretase and γ-secretase complexes [7]. Studies of familial AD
show that AD-causing variants in genes encoding APP and presenilins, catalytic components of theγ-secretase complex, elevate relative levels of the Aβ1–42 or Aβ1–43 isoforms of Aβ1–40 [8]. Aβ1–42
and Aβ1–43 are more aggregation-prone and cytotoxic compared with the Aβ1–40 peptide [9]. Initially,Aβ deposits are found exclusively in the neocortex and subsequently expand into the hippocampus,
striatum, and brainstem [10]. Aβ is produced from APP and is mainly secreted into the extracellularspace [11]. However, Aβ oligomers also accumulate intracellularly through endocytosis of secreted
Aβ [12]. A triple-transgenic model of familial AD harboring transgenes expressing PS1(M146V),
APP(Swe), and Tau(P301L) was defective for synaptic plasticity, including long-term potentiation,
because of the accumulation of intraneuronal Aβ [13]. These findings provide evidence that extra- andintra-cellular Aβ accumulation causes cognitive impairment.
Aβ accumulation is the initial event in AD progression. Subsequently, Tau pathology develops
in a delayed fashion [14]. Tau promotes the assembly of tubulin into microtubules, a component of
the cytoskeleton, and lends support to neuronal morphology [15]. In normal brain, Tau contains two
phosphates per molecule, while in AD, fibrillary Tau is abnormally phosphorylated (approximately eightphosphates per molecule) [16]. This hyper-phosphorylation is mediated by GSK3β, and it affects theinteraction of Tau with microtubules, leading to neurodegeneration and cognitive impairment [17,18].
Accumulation of abnormally phosphorylated Tau is also causative for other neurodegenerative
disorders, including frontotemporal lobar degeneration (FTD), corticobasal degeneration, andprogressive supranuclear palsy [19]. Intracellular Tau aggregates have been suggested to spreadthough synaptic circuits in a prion-like manner [20]. Neurofibrillary tangles, a major pathologicalhallmark of AD, are found at early stages in the transentorhinal cortex and the entorhinal cortex, aregion providing input to the hippocampal circuitry [19]. Subsequently, Tau pathology propagates tothe hippocampus, the temporal cortex, and then progresses to primary motor/sensory areas [19].
Parkinson’s disease and dementia with Lewy bodies are neurodegenerative diseases that arecharacterized by the presence of intracellular abnormal deposits called Lewy bodies and Lewy neurites.
These deposits mainly consist of α-synuclein, which is a natively unfolded protein localized to the
nucleus and presynaptic nerve terminals [21–23]. Several α-synuclein variants, such as missense andmultiplication variants, are responsible for familial PD, suggesting that increased expression andabnormal structure of α-synuclein cause its aggregation and neurodegeneration [24,25]. Althoughaccumulated α-synuclein is usually modified by phosphorylation, nitrosylation, glycation, andglycosylation, it remains unclear whether the aggregation is linked to its modification [26–28]. Fibrillar
α-synuclein can propagate Lewy body and Lewy neurite pathology through cell-to-cell transmission
leading to synaptic dysfunction and death of dopaminergic neurons in in vitro primary neurons andin the mouse brain [29–31]. Indeed, Lewy body-like inclusions were propagated in grafted embryonicnigral neurons that were transplanted into PD patients [32,33]. α-Synuclein is detected in cerebrospinal
fluid of subjects with or without PD [34]. The mechanism by which α-synuclein is secreted remains
unclear although the involvement of exosomal release and exocytosis via vesicles or multivesicularbodies has been reported [35–37]. Uptake of extracellular α-synuclein has been proposed to be
mediated by pinocytosis and receptor-mediated endocytosis. Extracellular α-synuclein fibrils bindcell surface heparan sulfate proteoglycans and are intracellularly taken up through pinocytosis [38].
Tau fibrils are also incorporated into cells in the same manner [38]. In addition, lymphocyte-activation

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gene 3 (LAG3), a transmembrane protein, has high affinity for α-synuclein fibrils [39]. Interestingly,
pathological Tau and Aβ species do not bind to LAG3, indicating that LAG3 is a specific receptor for
α-synuclein fibrils [39]. LAG3 deficiency effectively reduces the endocytosis of α-synuclein fibrils andthe propagation of PD pathology [39].
Amyotrophic lateral sclerosis (ALS) and FTD are neurodegenerative diseases characterized by
motor and cognitive impairment, respectively. Both diseases have genetic and pathological overlaps.
For example, variations in various genes, such as TDP-43, FUS, p62 and C9 or f72, are attributed to the
etiology of both diseases, and TDP-43 pathology is often observed in both diseases (in ~97% of ALS
and ~50% of FTD cases) [40,41]. In most cases of familial and sporadic ALS, immunohistochemical
analysis shows that TDP-43 is included in ubiquitin-positive round and skein-like inclusions [42].
However, TDP-43 pathology is negative in other cases of familial ALS, such as in cases with SOD1
and FUS variations where inclusions are composed of SOD1 and FUS, respectively [43]. Numerous
studies demonstrate that these mutant forms are aggregate-prone proteins that are cytotoxic bycausing dysfunction to various cellular processes [44,45]. TDP-43, SOD1, and FUS aggregates also
have prion-like seeding activity, which propagates ALS pathology [27,46–48]. TDP-43 and FUS are
RNA-binding proteins that are involved in RNA and protein quality control [49]. Exposure to various
stresses, such as heat shock, oxidative stress, and endoplasmic reticulum stress, induces formationof stress granules, which are dynamic assemblies of proteins and RNAs [50]. Stress granules aremembrane-less organelles that contain translationally stalled mRNAs associated with translation
initiation factors and multiple RNA-binding proteins, suggesting that stress granules regulate mRNAtranslation and stability and protect from environmental stresses [51,52]. Recent reports have indicatedthat variations linked to ALS-FTD in TDP-43, FUS, TIA-1, and C9 or f72 cause abnormal stress granuleassembly and disassembly. For example, the TDP-43A382T ALS-FTD variation causes a significant
reduction in stress granule assembly in human fibroblasts [53]. In contrast, the TIA-1P362L ALS-FTD
variation delays stress granule disassembly and promotes the accumulation of non-dynamic stressgranules [54]. These results indicate that dysfunctional stress granule dynamics might contribute to
ALS pathogenesis. Moreover, mutations of p62 and OPTN were also identified in familial and sporadic
ALS-FTD [55]. Both proteins are known as a ubiquitin binding protein shuttling ubiquitinated proteinsfor their degradation [55]. The FUS-containing inclusions are also immunoreactive with antibodies to
p62 and OPTN in spinal anterior horn neurons in all sporadic ALS and in non-SOD1-familial ALS
cases [55]. Recently, it was reported that ALS-FTLD-linked mutations of p62 disrupt autophagy andanti-oxidative stress pathway underlying the neurotoxicity in ALS-FTLD [56].
Other neurodegenerative diseases are also characterized by neuronal protein aggregates. Expanded
polyglutamine (polyQ) tracts are aggregation-prone and expanded polyQ-containing proteins, such as
huntingtin and ataxins, cause HD and spinocerebellar ataxia, respectively [57]. Huntingtin is a 348 kDa
protein, and its N-terminal region contains the expandable polyQ tract [58,59]. Huntingtin undergoes
post-translational modifications at multiple sites, such as phosphorylation, acetylation, sumoylation
and ubiquitination, and is then cleaved by various proteases [58,60]. Cleaved N-terminal fragmentswith an expanded polyQ tract are released and form fibrillary aggregates or inclusion bodies [61].
Pathogenic polyQ-expanded huntingtin also has prion-like properties. Mutant huntingtin aggregateswere detected in the extracellular matrix of grafted neurons in HD patient brains, indicating that
pathological huntingtin can spread within the brain [62,63].
3. Ubiquitination in Protein Degradation
Accumulation of harmful proteins is a hallmark of various neurodegenerative diseases, as
described above. Cells are protected from harmful proteins by protein quality control mechanisms,
including molecular chaperone and protein degradation systems [64]. Eukaryotes have two major
protein degradation systems, the ubiquitin–proteasome system (UPS) and the autophagy–lysosome
pathway (ALP). In the UPS, ubiquitin-tagged proteins are targeted by a multi-subunit proteasecomplex, the proteasome. The proteasome consists of a multi-catalytic proteinase complex (20S)Cells 2020, 9, 2022

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and two regulatory complexes (19S and 11S) [65]. Misfolded proteins and short-lived proteins
undergo ubiquitination by a multi-step process that requires a ubiquitin-activating (E1) enzyme, aubiquitin-conjugating (E2) enzyme, and a ubiquitin ligase (E3) [66]. Initially, ubiquitin is activatedby the E1 enzyme in an ATP-dependent manner, and activated ubiquitin is then transferred to an E2
enzyme. E3 then ligates the ubiquitin to the target protein [3]. Ubiquitinated proteins are recruited to
the regulatory complex of the proteasome and are then deubiquitinated and unfolded [67]. Linearized
proteins then translocate into the proteolytic chamber of the 20S proteasome and are cleaved by its six
proteolytic sites [68].
The ALP is an intracellular metabolic process in which cytoplasmic proteins and organelles
sequestrated by autophagosomes are degraded in lysosomes [69]. Autophagy is regulated bymore than 30 autophagy regulated proteins and its core machinery is classified in four subgroups:(1) The ATG1/ULK1 complex; (2) ATG9 and its cycling system; (3) the phosphatidylinositol 3-kinase
complex; (4) two ubiquitin-like conjugation systems (ATG8/LC3 and ATG12) [69]. In mammals, theULK1 complex has an essential role in the initiation of autophagy and is directed to the endoplasmic
reticulum together with ATG9 vesicles and the phosphatidylinositol 3-kinase complex [69]. DFCP1 andWIPIs are recruited to the endoplasmic reticulum membrane and promote the formation of isolation
membrane [69]. Autophagosome formation is mediated by two ubiquitin-like conjugation systems,
conjugation of ATG12 to ATG5 and conversion of LC3 to a phosphatidylethanolamine-conjugatedmembrane-bound form [69]. Finally, mature autophagosomes fuse with lysosomes, resulting indegradation of cellular components [69].
Mono-ubiquitination functions in the regulation of protein interaction, trafficking, andtranscriptional activity [70] and mono-ubiquitinated proteins are degraded by the proteasome inboth mammalian and yeast cells [71]. Multi-ubiquitin is generated by the sequential conjugation ofubiquitin to ubiquitin via Lys residues (Lys-6, Lys-11, Lys-27, Lys-29, Lys-33, Lys-48, and Lys-63) or
Met residues (Met-1, Met-14, Met-20) [3]. Lys-11-, Lys-48-, and Lys-63-linked poly-ubiquitination act asproteolytic signals for the proteasome and autophagy (Figure 1). Lys-48-linked poly-ubiquitin is the
major signal of numerous short-lived proteins and unfolded proteins for proteasomal degradation [72].
Lys-11-linked poly-ubiquitin is also involved in degradation of short-lived cell cycle proteins and in
the ERAD (endoplasmic reticulum-associated degradation) pathway upon ER stress [73]. Furthermore,
small aggregated proteins are selectively degraded via the autophagy–lysosome system. Lys-48and Lys-63-linked poly-ubiquitin is required for selective sequestration of aggregated proteins into
autophagosomes through autophagy receptor proteins [3]. Furthermore, accumulation of Lys-11-,
Lys-48-, Lys-63-linked poly-ubiquitinated insoluble proteins was observed in the brains of Atg5- andAtg7-null mice, indicating that multiple ubiquitin signals might be involved in autophagic degradationof various cargoes (Figure 1) [74]. Autophagy receptor proteins possess both a ubiquitin-binding
domain and an LC3-interacting region (LIR), and bind to various cargoes, such as protein aggregates,
intracellular organelles and microbial invaders [75,76]. Ubiquitinated protein aggregates are selectively
recognized by autophagy receptor p62, NBR1, OPTN, and TOLLIP [77,78]. These complexes arethen associated with autophagosome protein LC3 though an LIR domain, resulting in sequestration
into an isolation membrane and degradation in lysosomes [78]. Ubiquitination is also often required
for selective clearance of organelles. For example, damaged mitochondria in PD are eliminated
via ubiquitin-dependent PINK1-Parkin-mediated mitophagy [79]. Upon mitochondrial damage,
Parkin, an E3 ubiquitin ligase, conjugates Lys-6-, Lys-11-, Lys-48-, and Lys-63-linked poly-ubiquitin to
mitochondrial outer membrane proteins, and then mitochondria bind to autophagy receptors [79–81].
Furthermore, PINK1 coordinately acts upstream of Parkin in this process. Phosphorylated ubiquitin
by PINK1 is required for Parkin activation [82]. These findings indicate that ubiquitin signals also
have an important role in the autophagic clearance of organelles.

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FigureFigure1.1.Degradation
Degradationof
ofubiquitinatedubiquitinatedcargoes.
cargoes.Harmful
Harmfulproteinsproteinsand
andmitochondria
mitochondriacan
canbebemodified
modifiedbyubiquitin
additions,suchsuchas mono-ubiquitin
and Lys-11-,
Lys-48-, Lys-48-,
and Lys-63-poly-ubiquitin
byvarious
variousubiquitin
additions,as mono-ubiquitin
and Lys-11-,and Lys-63-polychains.
proteasome
preferentiallydegradesmono-ubiquitinatedproteins
and Lys11and
ubiquitinThe
chains.
The proteasome
preferentiallydegradesmono-ubiquitinatedproteins
and Lys11Lys-48-linked
proteins,
whereasautophagy
preferentiallyeliminates
Lys-48-,andLys-63decoratedand Lys-48-linked proteins, whereas autophagy preferentially eliminates Lys-48-, and Lys-63protein
aggregatesmitochondria.
decorated
protein andaggregatesand mitochondria.

4. Ubiquitination of Neurodegenerative Disease-Associated Proteins
4. Ubiquitination of Neurodegenerative Disease-Associated ProteinsHarmful proteins causing neurodegenerative diseases undergo ubiquitination and pathologicalHarmful proteins causing neurodegenerative diseases undergo ubiquitination and pathologicalanalyses using anti-ubiquitin antibodies identify various protein aggregates and inclusions. Paired
analyses using anti-ubiquitin antibodies identify various protein aggregates and inclusions. Paired
helical filament-Tau (PHF-Tau) is modified by Lys-6-, Lys-11-, Lys-48-, Lys-63-poly-ubiquitin chainshelical filament-Tau (PHF-Tau) is modified by Lys-6-, Lys-11-, Lys-48-, Lys-63-poly-ubiquitin chainsand mono-ubiquitin in AD brains or cultured cells (Figure 2) [83–85]. CHIP, a HSP70 co-chaperone,and mono-ubiquitin in AD brains or cultured cells (Figure 2) [83–85]. CHIP, a HSP70 co-chaperone,is an E3 ubiquitin ligase of PHF-Tau [84,86]. Lys-254, 257, 311, and 317 of PHF-Tau are acceptors foris an E3 ubiquitin ligase of PHF-Tau [84,86]. Lys-254, 257, 311, and 317 of PHF-Tau are acceptors for
ubiquitin [85]. Lys-724, 725, 726, 751, and 763 of APP is intracellularly conjugated with ubiquitin in
ubiquitin [85]. Lys-724, 725, 726, 751, and 763 of APP is intracellularly conjugated with ubiquitin inmouse brain [87] and impairment of this ubiquitination leads to accumulation of both secreted andmouse brain [87] and impairment of this ubiquitination leads to accumulation of both secreted andintracellular Aβ40 [87].
intracellular Aβ40 [87].
α-Synuclein undergoes ubiquitination by various E3 ubiquitin ligases (Figure 2). Seven in absentia
homolog (SIAH), an E3 ubiquitin ligase, mono-ubiquitinates α-synuclein at Lys-12, 21, and 23, resulting
in an increase in the aggregation of α-synuclein and apoptotic cell death [88,89]. NEDD4 ubiquitin
ligase also targets α-synuclein and mediates Lys-63-poly-ubiquitin [90]. Ubiquitinated α-synuclein isdegraded by the endosomal-lysosomal pathway, suggesting that this process might have a protective
effect against the pathogenesis of PD and other α-synucleinopathies [90]. Furthermore, CHIP is also
involved in α-synuclein mono-ubiquitination or poly-ubiquitination, similarly of PHF-Tau [91,92].
CHIP-mono-ubiquitinated α-synuclein is deubiquitinated by USP9X. USP9X knockdown promotes
accumulation of mono-ubiquitinated α-synuclein and enhances the formation of α-synuclein inclusions
upon proteolytic inhibition [92]. Ubiquitin ligase E6-AP is localized to Lewy bodies in the PD brain,
and is involved in α-synuclein ubiquitination and proteasome-dependent degradation [93].

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FigureFigure2.2. Ubiquitination
Ubiquitinationofofneurodegenerative
neurodegenerativedisease-associateddisease-associatedproteins.
proteins.Neurodegenerative
Neurodegenerative
disease-associatedproteins,
such
as
Tau,
APP
(β-amyloid
precursor
protein),disease-associated proteins, such as Tau, APP (β-amyloid precursor protein),α-synuclein,
α-synuclein,TDP43,
TDP43,
SOD1,
areubiquitinatedat atindividual
targetsites.
Specificubiquitinligases
involved
in
SOD1,and
andHuntingtin
Huntingtin
areubiquitinatedindividual
targetsites.
Specificubiquitinligases
involved
thisubiquitinationand theof ubiquitin
chains chains
can be identified
by various
studies.
in thisubiquitination
andpattern
the patternof ubiquitin
can be identified
by biochemicalvarious biochemical

studies.

ALS-causing TDP-43 and SOD1 aggregates are also detected by anti-ubiquitin antibodies. TDP-43
is targetedby Znf179
ubiquitin ubiquitination
ligase and is modified
by poly-ubiquitin
Znf179α-Synuclein
undergoesby various
E3 ubiquitinchainsligases[94].
(Figure2).knockout
Seven insuppresses
TDP-43proteosomal
turnover,resultinginaccumulationofinsoluble
TDP-43andcytosolicabsentia homolog (SIAH), an E3 ubiquitin ligase, mono-ubiquitinates α-synuclein at Lys-12,
21, and
TDP-43inclusions
inthecortex,hippocampus
andmidbrainregions[94].
In
additionto
Znf179,CUL2
23, resulting in an increase in the aggregation of α-synuclein and apoptotic cell death [88,89]. NEDD4
ubiquitin
TDP-43
with poly-ubiquitin,coordinatelyvon Hippelαubiquitinligase
ligasecan
alsomodify
targetsmisfolded
α-synuclein
and mediates
Lys-63-poly-ubiquitin[90].with
Ubiquitinated
Lindauprotein
(VHL)
[95].
Mass
spectrometry
analysisidentified
TDP-43ubiquitinationsites tohavebesynuclein is degraded by the endosomal-lysosomal pathway, suggesting that this process might
Lys-84,
Lys-95effectLys-160,
Lys-181,and Lys-263 residues
andotherthat its
poly-ubiquitin chainslink
via Lys-48
a protective
againstthe pathogenesisof PD andα-synucleinopathies[90].
Furthermore,andLys-63
[96].
SOD1
is
targeted
by
NEDL1
and
gp78
ubiquitinligases.
NEDL1
colocalizes
CHIP is also involved in α-synuclein mono-ubiquitination or poly-ubiquitination, similarly of with
PHFSOD1
inclusions
inthespinalcord
ventralhorn
motor
neuronsofboth
ALS
patients
andmutantTau [91,92]. CHIP-mono-ubiquitinated α-synuclein is deubiquitinated by USP9X. USP9X knockdown
SOD1
transgenic
mice [97].ofgp78
ubiquitin ligase is α-synuclein
also involvedandin theubiquitinationof SOD1 of[83].
promotes
accumulation
mono-ubiquitinatedenhancesthe formation
αgp78
is
a
protein
with
at
least
five
membrane-spanningdomains,
includingaRINGfingerconsensus
synuclein inclusions upon proteolytic inhibition [92]. Ubiquitin ligase E6-AP is localized to Lewy
sequence,
an importantrole in ERAD[98]. Interestingly,
this ubiquitin
ligase also mediates
bodies inandthe plays
PD brain,and is involvedin α-synuclein
ubiquitinationand proteasome-dependentubiquitinationofspinocerebellar
ataxia-associatedataxin-3[99].
gp78
overexpression
promotes thedegradation [93].
ubiquitination
anddegradation
of
SOD1
and
ataxin-3
incultured
cells,
whereasknockdownof gp78
ALS-causing TDP-43 and SOD1 aggregates are also detected by anti-ubiquitin antibodies.
TDPstabilizes
them
[99].
43 is targeted by Znf179 ubiquitin ligase and is modified by poly-ubiquitin chains [94]. Znf179
Turnoverof huntingtin
is proteosomal
regulated byturnover,ubiquitination,
viainLys-48and Lys-63-poly-ubiquitin.
knockout
suppresses
TDP-43
resultingaccumulationof insoluble TDP-43
Althoughaggregatedmutanthuntingtin
mainlyincludesLys-63-poly-ubiquitin
chains,overexpression
and cytosolic TDP-43 inclusions in the cortex, hippocampus and midbrain regions [94].
In addition
oftoLys-48-specificreduces
Lys-63-ubiquitination
huntingtin aggregation,
Znf179, CUL2ubiquitin
ubiquitinligase,ligaseUbe3a,
can modify
misfolded
TDP-43 with andpoly-ubiquitin,coordinately
enhancingdegradation
via theLys-48(VHL)
ubiquitin–proteasome
system [100].
Similarly,ubiquitinTDP-43
ligase,with vonitsHippel
Lindauprotein[95]. Mass spectrometry
analysisidentified
UBR5,
is also involved
Lys-48-proteasomal
degradationof bothandmutantandhuntingtin
ubiquitinationsites toinbe
Lys-84, Lys-95 Lys-160,
Lys-181,
andnormal
Lys-263
residuesthat its[101].
polyHowever,tumor
necrosisfactorreceptor-associatedfactor6(TRAF6)canpromotetheLys-63-ubiquitin
ubiquitin chains link via Lys-48 and Lys-63 [96]. SOD1 is targeted by NEDL1 and gp78 ubiquitin
chain
on mutantmight
contributeto autophagic
clearanceof huntingtin
aggregates[102].
ligases.
NEDL1huntingtin
colocalizesand
with
SOD1
inclusions
in the spinal
cord ventral
horn motor
neuronsofboth ALS patients and mutant SOD1 transgenic mice [97]. gp78 ubiquitin ligase is also involved inCells 2020, 9, 2022

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Collectively, numerous neurodegenerative disease-associated proteins undergo ubiquitination by avariety of ubiquitin ligases. These ubiquitin signals mainly serve to eliminate pathogenic proteins,
although the ubiquitin signal on neurodegenerative disease-associated proteins can be pathogenic.
5. Autophagic Degradation of Neurodegenerative Disease-Associated Proteins
Small protein aggregates are thought to be degraded by ALP. Autophagy receptors recognize
ubiquitin chains bound to cargoes and transport them to autophagosomes (Figure 3A). Recent
advances in mass spectrometry technology have contributed to the decoding of ubiquitin signals
and have revealed the diversity of ubiquitin chains. In addition, the ubiquitin binding-domains ofautophagy receptors have been categorized (Figure 3B). p62, Nbr1, and c-Cbl have a UBA domain,a small domain of about 40 residues [103]. The UBA domain of p62 and Nbr1 binds strongly toboth Lys-48- and Lys-63-poly-ubiquitin [104]. Moreover, the UBA domain of ubiquilin-1 and yeast
Ede1, other ubiquitin binding proteins, have a high affinity for mono-ubiquitin, indicating that
autophagy receptors with the UBA domain might bind to mono-ubiquitin [105,106]. OPTN has two
ubiquitin binding domains, a UBAN and a zinc finger domain. A UBAN domain can interact not
only with Lys-63-linked poly-ubiquitin but also with linear ubiquitin chains, which are generated
between the N-terminal methionine of one ubiquitin and the C-terminal glycine of the next in the
chain. (Figure 3B) [107,108]. However, the zinc finger domain of OPTN recognizes various protein
aggregates in a ubiquitin-independent manner [109], although the same domain of NDP52 canrecognize mono-ubiquitin, Lys-48-, and Lys-63-poly-ubiquitin [110]. NDP52 is a selective autophagy
receptor for cytosolic bacteria (xenophagy) and damaged mitochondria (mitophagy), which aredecorated with ubiquitin [111,112]. Although preferences of ubiquitin binding domains for ubiquitin
codes remain unclear, individual autophagy receptors might selectively recognize disease-associatedaggregates by the ubiquitin code. Indeed, the UBA domain of p62 can bind both Lys-48-linked andLys-63-linked ubiquitin chains but has a higher affinity for Lys-63 chains [113]. In addition, the UBA
domain of NBR1 is structurally distinct from the p62 UBA domain, resulting in a different interaction
with ubiquitin. NBR1 has significantly higher affinity for mono-ubiquitin compared with p62 [114].
Accordingly, substrate preference of autophagy receptors might be dependent on the ubiquitin codeson cargoes (Figure 3A).
Activation of autophagy receptors is mediated by various kinases. For example, p62 isphosphorylated by various kinases, such as mTORC1, casein kinase 1, and TBK1 [64,115,116]. OPTN and
NBR1 activities are also regulated by TBK1 and GSK3β, respectively [117,118]. Several phosphorylationsites are located in the ubiquitin binding domain, whose status alters the affinity for ubiquitinatedproteins. Indeed, inhibition of several phosphorylation sites reduces cargo-binding potential [119].
This evidence indicates that structural alterations to autophagy receptors by phosphorylation controls
autophagic clearance of various cargoes.
Autophagy activation accelerates elimination of neurodegenerative disease-associated protein
aggregates and inclusions. The mTORC1 inhibitor, rapamycin, is well known to induce autophagy
activity [120]. The effect of rapamycin has been investigated using various neurodegenerative diseasemodels. For example, accumulation of Tau, huntingtin, and α-synuclein aggregates was significantly
decreased in cultured cell and Drosophila models of AD, HD, and PD [121,122]. Moreover, themTORC1-independent autophagy inducer, trehalose, also reduced protein aggregation and neuronaldegeneration in ALS and tauopathy model mice [123,124]. Moreover, progression of PD-like pathology
was investigated in autophagy suppressor Rubicon-KO mice, in which basal autophagy is constitutively
activated. Spread of Lewy body-like α-synuclein aggregates was significantly reduced in the brain of
this mouse [125]. These results indicate that autophagy induction may be an effective treatment forvarious neurodegenerative diseases.

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Figureubiquitinatedcargoes.
Figure3. 3.Autophagy
Autophagyreceptors
receptorsand
andselective
selectiveautophagy
autophagyofof
ubiquitinatedcargoes.(A)(A)Autophagy
Autophagy
receptorsplay
animportantroleinselective
autophagy.
Appropriate
autophagy
receptors
receptors play an important role in selective autophagy. Appropriate autophagy receptorsbindbindtotovariousvariousubiquitinatedubiquitinatedcargoes
cargoes(Lys-48-,(Lys-48-,Lys-63-,
Lys-63-,linear-poly-ubiquitin
linear-poly-ubiquitinchain,chain,and
andmono-ubiquitin)mono-ubiquitin)
throughtheirubiquitinbinding
domain.
Many
autophagy
receptors
areregulatedby various
through their ubiquitin binding domain. Many autophagy receptors are regulated by various
kinases,kinases,andthenautophagy
receptor-cargocomplexes
interactwiththeautophagosome
and then autophagy receptor-cargo complexes interact with the autophagosome protein,protein,
LC3. (B)
LC3.
(B) Structureof major
autophagy
receptors.
Autophagy
receptors
consistofofan
an LIR
LIR domain
Structureof major
autophagy
receptors.
Autophagy
receptors
consistdomainandanda aubiquitinbinding
domain.
Ubiquitinbinding
domains
(UBA,
UBAN,
and
ZnF)
are
classified
basedonubiquitin binding domain. Ubiquitin binding domains (UBA, UBAN, and ZnF) are classified based
motifsequences.
Biochemical
studiesshowshowpreferencesof autophagy
receptors
for ubiquitin
types.
on motif
sequences.
Biochemical
studies
preferencesof autophagy
receptors
for ubiquitin
types.

Activation of autophagy receptors is mediated by various kinases. For example, p62 isphosphorylated by various kinases, such as mTORC1, casein kinase 1, and TBK1 [64,115,116]. OPTN

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9 of 15

6. Concluding Remarks
Protein quality control systems, such as UPS and ALP, decline with age, which is a leading causeof neurodegenerative diseases. Clinical trials of several autophagy activators have been conducted
for AD and ALS patients. For example, resveratrol is a natural polyphenol that induces autophagy
activity by directly inhibiting mTOR [126]. In individuals with mild to moderate AD, decline of
cerebrospinal fluid and plasma Aβ1–40 levels were observed in a resveratrol-treated group comparedwith a placebo-treated group [127]. However, improvement of cognitive function was not reported
in this trial. The existing drugs, metformin and lithium, are also autophagy inducers, and clinical
trials of these drugs for AD patients have also been conducted [128–130]. Currently, clinical trials of
rapamycin are planned for ALS patients but not for AD patients, although it is hoped that further
evidence warranting trials in AD patients will be forthcoming. While, it has been revealed thatdeubiquitinating enzymes such as UCL-L1 and ubiquitin-specific proteases are also involved in PD
and AD through proteostasis [131]. Various inhibitors of deubiquitinating enzymes might be a new
therapeutic target [131]. Collectively, detailed knowledge of ubiquitin chains in neurodegenerative
disease-associated proteins and structural analyses of their interactions with ubiquitin binding domains
will be beneficial for the development of novel therapies for neurodegenerative diseases.
Author Contributions: Y.W. wrote the paper. M.T. and K.T. edited and revised the manuscript. All authors readand approved the final manuscript.
Funding: This work was funded by Grants-in-Aid for Scientific Research from the Japan Society for the Promotionof Science (Y.W.: 18K07505, K.T.: 18K07371, and M.T.: 17H03553) and by the Shimizu Foundation for Immunology
and Neuroscience Grant for 2018.
Acknowledgments: We thank Jeremy Allen, from Edanz Group for editing a draft of this manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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