This process is also a major degradation pathway for many aggregate-prone, disease-cau-sing proteins associated with neurodegenerative disorders, such as mutant huntingtin in Huntington’
Trang 1Huntington’s disease: degradation of mutant huntingtin
by autophagy
Sovan Sarkar and David C Rubinsztein
Department of Medical Genetics, University of Cambridge, Cambridge Institute for Medical Research, Addenbrooke’s Hospital, UK
Autophagy
Degradation of cellular proteins occurs by two
path-ways The proteasomes predominantly degrade
short-lived nuclear and cytosolic proteins These substrates
are generally selected for degradation after they are
tagged with polyubiquitin chains The narrow pore of
the proteasome precludes entry of protein complexes
and organelles The bulk degradation of cytoplasmic
proteins or organelles is mediated largely by
macro-autophagy, generally referred to as autophagy [1]
Autophagy substrates generally have long half-lives and can include protein complexes or damaged cellular organelles This process involves the formation of small double-membrane structures of unknown ori-gin(s) called phagophores, which elongate to form autophagosomes Autophagosomes ultimately fuse with mammalian lysosomes (or yeast vacuoles) to form autolysosomes, where their contents are degraded by acidic lysosomal hydrolases [1] (Fig 1)
During autophagosome formation, the elongation of the phagophore involves a ubiquitin-like conjugation
Keywords
autophagy; Huntington’s disease; lithium;
mTOR; polyglutamine; rapamycin
Correspondence
S Sarkar, Department of Medical Genetics,
University of Cambridge, Cambridge
Institute for Medical Research,
Addenbrooke’s Hospital, Hills Road,
Cambridge CB2 0XY, UK
Fax: +44 1223 331206
Tel: +44 1223 331139
E-mail: ss457@cam.ac.uk
D C Rubinsztein, Department of Medical
Genetics, University of Cambridge,
Cambridge Institute for Medical Research,
Addenbrooke’s Hospital, Hills Road,
Cambridge CB2 0XY, UK
Fax: +44 1223 331206
Tel: +44 1223 762608
E-mail: dcr1000@hermes.cam.ac.uk
(Received 29 February 2008, accepted 9
May 2008)
doi:10.1111/j.1742-4658.2008.06562.x
Autophagy is a nonspecific bulk degradation pathway for long-lived cyto-plasmic proteins, protein complexes, or damaged organelles This process is also a major degradation pathway for many aggregate-prone, disease-cau-sing proteins associated with neurodegenerative disorders, such as mutant huntingtin in Huntington’s disease In this review, we discuss factors regu-lating the degradation of mutant huntingtin by autophagy We also report the growing list of new drugs⁄ pathways that upregulate autophagy to enhance the clearance of this mutant protein, as autophagy upregulation may be a tractable strategy for the treatment of Huntington’s disease
Abbreviations
3-MA, 3-methyladenine; AD, Alzheimer’s disease; ALS, amyotrophic lateral sclerosis; Ab, amyloid-b; GSK-3b, glycogen synthase kinase-3b;
HD, Huntington’s disease; IMPase, inositol monophosphatase; IP 3 , inositol 1,4,5-trisphosphate; LC3, microtubule-associated protein 1 light chain 3; mTOR, mammalian target of rapamycin; SMER, small-molecule enhancer of rapamycin.
Trang 2system, in which mammalian Atg12 is conjugated to
Atg5 The Atg12–Atg5 conjugate then forms a
com-plex with Atg16L This comcom-plex associates with the
isolation membrane for the duration of
autophago-some formation, but dissociates upon its completion
[2] (Fig 1) The function of the Atg12 system is closely
linked to another ubiquitin-like system involving
microtubule-associated protein 1 light chain 3 (LC3),
which is the mammalian ortholog of yeast Atg8 and
the only known mammalian protein that specifically
associates with the autophagosome membrane [3] LC3
is cleaved to form cytosolic LC3-I After autophagy induction, LC3-I is conjugated with phosphatidyletha-nolamine, resulting in the LC3-II species that associ-ates with autophagosomes [3] The membrane targeting of LC3 depends on Atg5 [4]
The formation of autophagosome precursors is prevented by 3-methyladenine (3-MA) or wortmanin, which are inhibitors of phosphatidylinositol-3-kinases, and class III phosphatidylinositol-3-kinase is required for autophagy [5–7] (Fig 1) Autophagy is negatively regulated by the mammalian target of rapamycin (mTOR) Inhibition of mTOR by rapamycin induces autophagy, but its mechanism of action in mammalian cells is still unknown [8] At a physiological level, auto-phagy is induced by amino acid deprivation [9] Autophagy regulates the clearance of aggregate-prone disease-causing proteins associated with various neurodegenerative disorders, such as mutant huntingtin [causing Huntington’s disease (HD)], ataxin-3 (causing spinocerebellar ataxia 3), forms of tau (causing fronto-temporal dementias), the A53T and A30P a-synuclein mutants (causing familial Parkinson’s disease), and mutant forms of superoxide dismutase 1 [causing famil-ial amyotrophic lateral sclerosis (ALS)] [10–14] Two recent landmark studies highlighted the strong link between autophagy and neurodegeneration, where loss
of basal autophagy in mouse neuronal cells mediated
by knockdown of the essential autophagy genes, Atg5
or Atg7, resulted in progressive motor deficits, cytoplas-mic aggregates, and neurodegeneration [15,16]
Autophagy in HD
HD is a progressive, autosomal dominant, neurode-generative disorder caused by the expansion of a CAG trinucleotide repeat (> 35 repeats) in the huntingtin gene, which is translated into an expanded polygluta-mine tract in the N-terminus of the huntingtin protein Mutant huntingtin toxicity is believed to be expressed after it is cleaved to form N-terminal fragments com-prising the first 100–150 residues with the expanded polyglutamine tract, which are also the toxic species found in aggregates (also called as inclusions) [17] Although the polyglutamine disorders are associated with intraneuronal aggregates, it is debatable whether the aggregates are toxic or protective [18,19] Recent studies and reviews have implicated the preaggregate oligomers as the most toxic species in neurodegenera-tive diseases [20–25] However, induction of autophagy results in decreases of both aggregated and soluble
‘monomeric’ huntingtin species, and results in decreased toxicity in cell, fly and mouse models of HD [26] Phosphorylation of various mutant proteins, such
Autophagosome
Lysosome
Signal
Induction
Formation
Fusion
Breakdown and
recycling
Baf
Degradation of aggregate-prone proteins
Phagophore
LC3 Atg12-Atg5.Atg16L
3-MA
Aggregate-prone proteins, e.g., mutant huntingtin
Autolysosome
Fig 1 The mammalian autophagy–lysosomal pathway A signal
(such as starvation under physiological conditions) induces the
for-mation of double-membrane structures (phagophores) that
seques-ter portions of cytoplasm along with proteins or damaged cell
organelles to be degraded Aggregate-prone proteins such as
mutant huntingtin can also be sequestered in this way The Atg12–
Atg5–Atg16L complex and LC3 localize to the phagophore
through-out its elongation process Upon completion of autophagosome
formation, the Atg12–Atg5–Atg16L complex dissociates from the
membrane, whereas LC3-II remains on it The autophagosome
ulti-mately fuses with the lysosome to form an autolysosome, where
its contents are degraded by acidic proteases Breakdown within
the autolysosome allows recycling of the degraded cargo (amino
acids, fatty acids, sugars, and nucleotides) during starvation
condi-tions Autophagy can be inhibited by drugs such as 3-MA at the
formation of autophagic vacuole stage, and by bafilomycin A1 (baf)
at the fusion stage between autophagic vacuole and lysosome.
Trang 3as huntingtin, ataxin-1, and ataxin-3, may regulate
neurodegeneration in these disease conditions [27–32],
but does not primarily influence the process of
auto-phagy, as far as we are aware However,
hyperphosph-orylation of tau, causing neurofibrillary tangles in
Alzheimer’s disease (AD) [33], may influence its
loca-tion, dependence on autophagy, and accessibility to
autophagy
Increased autophagy has been reported in HD
Mouse clonal striatal cells transiently transfected with
truncated and full-length human wild-type and mutant
huntingtin show the presence of both normal and
mutant proteins in dispersed and perinuclear vacuoles
[34] Furthermore, huntingtin-labeled vacuoles display
the ultrastructural features of early and late
autophago-somes, and huntingtin-enriched cytoplasmic vacuoles
appear to be more abundant in cells expressing mutant
huntingtin [35] Similar features have been seen in
brains from HD patients and transgenic mice, where
there are excessive endosomal–lysosomal-like
organ-elles, tubulovesicular structures, and multiple vesicular
bodies [36,37] Increased autophagosome–lysosomal
bodies have also been found in primary striatal neurons
from HD mice expressing truncated mutant huntingtin
following dopamine-stimulated oxidative stress [38]
Moreover, increased numbers of autophagosomes have
been found in lymphoblasts of HD patients as
com-pared to the control lymphoblasts [39]
Degradation of mutant huntingtin by autophagy
Previous work from our laboratory demonstrated that mutant huntingtin is an autophagy substrate [11] Inhi-bition of autophagy at the level of autophagosome formation by 3-MA [6], or at the level of autopha-gosome–lysosome fusion using bafilomycin A1 [40], slo-wed mutant huntingtin clearance and increased the levels of soluble and aggregated mutant huntingtin in
HD cell models [11] Furthermore, rapamycin treatment increased mutant huntingtin clearance and decreased the levels of soluble proteins and aggregates [11] (Fig 2) Yuan and colleagues have demonstrated that autophagy clears full-length mutant huntingtin [41]
No discernible perturbation of wild-type huntingtin clearance was seen with these autophagy modulators [11,42] These data suggest that the aggregate-prone mutant form of huntingtin, unlike the wild-type huntingtin, is strongly dependent on autophagy for its clearance
Interestingly, we found that mTOR was sequestered
in mutant huntingtin aggregates in HD cell models, transgenic mice, and patients’ brain This sequestration impaired mTOR kinase activity, thereby inducing autophagy Therefore, this study identified a new protective role for mutant huntingtin aggregates in inducing autophagy for their self-destruction by
β-catenin-Tcf transcription
Cytoprotection Autophagy
Rap
Clearance
of mutant huntingtin
Additive protective effects
LiCl
Autophagy
Clearance of mutant huntingtin
GSK-3 β
Ins
IP1
IP2
IP3
Phospho-inositol signaling
CBZ, VPA
IMPase mTOR
mTOR pathway
?
?
SMERs, Trehalose
Autophagy
?
Clearance
of mutant huntingtin
Additive protective effects
Fig 2 Schematic representation of autophagy-inducing compounds ⁄ pathways that facilitate the clearance of mutant huntingtin in mamma-lian cells Autophagy is classically induced with rapamycin (rap), which inhibits mTOR Upregulation of autophagy enhances the clearance of mutant huntingtin and reduces toxicity in various HD models Autophagy can also be induced with drugs that decrease IP3levels in the phosphoinositol signaling pathway in an mTOR-independent fashion, such as lithium (LiCl), which inhibits inositol monophosphatase (IMPase), and carbamazepine (CBZ) and valproic acid (VPA), which inhibit inositol (Ins) synthesis Although lithium also inhibits glycogen syn-thase kinase-3b (GSK-3b) in the wingless (Wnt) signaling pathway that activates mTOR and inhibits autophagy, the autophagy-inducing effect
of lithium is attributed to IMPase inhibition Combination treatment with lithium and rapamycin alleviates the block in autophagy by GSK-3b inhibition, and hence additively enhances autophagy and facilitates greater clearance of mutant huntingtin Furthermore, GSK-3b inhibition by lithium increases b-catenin–Tcf-mediated transcription, which is cytoprotective and can contribute to additional protective effects in this com-bination treatment for HD SMERs and trehalose have also been shown to induce mTOR-independent autophagy, and thus can additively upregulate autophagy when used together with rapamycin by enhancing autophagy through two independent pathways The precise mecha-nisms by which all the autophagy-inducing drugs trigger the autophagic machinery are still unclear.
Trang 4enhancing the clearance of the mutant protein [12] A
recent study has shown that expanded polyglutamine
with 72 repeats induced autophagy dependent on
eukaryotic translation initiation factor 2a, and this
protected against polyglutamine-induced endoplasmic
reticulum stress-mediated cell death [43]
Inducing autophagy for enhancement
of mutant huntingtin clearance
Autophagy upregulation may be a therapeutic strategy
for HD and related conditions, where the mutant
aggre-gate-prone proteins are autophagy substrates [8]
(Fig 2) The autophagic clearance of mutant huntingtin
aggregates is likely to be a consequence of degrading the
aggregate precursors (soluble and oligomeric species),
rather than large aggregates that are much larger than
typical autophagosomes [8,12] In this review, we will
restrict our discussion to studies investigating
modula-tion of autophagy for mutant huntingtin degradamodula-tion
Inducing autophagy by mTOR inhibition
In addition to showing that rapamycin or its analog
CCI-779 was protective in cells, Drosophila and mouse
models of HD, it was also shown that raised
intracel-lular glucose or glucose 6-phosphate induced
auto-phagy by mTOR inhibition, thereby reducing mutant
huntingtin aggregates⁄ toxicity in HD cell models
[11,12,44] The mechanism by which mTOR regulates
autophagy remains unclear, and this kinase controls
several cellular processes besides autophagy, probably
contributing to the complications seen with its
long-term use over many months mTOR is an important
signaling molecule that regulates diverse cellular
func-tions, such as initiation of mRNA translation,
ribo-some biogenesis, transcription, cell growth, and
cytoskeletal reorganization [45] Inhibition of mTOR
by rapamycin causes cell cycle arrest and leads to poor
wound healing and mouth ulcers [46] Thus,
com-pounds that induce autophagy by mTOR-independent
mechanisms may be more suitable for the treatment of
such neurodegenerative disorders, which may require
drugs to be taken for decades
Inositol-lowering agents trigger
autophagy independently of mTOR
We previously showed that lithium induced autophagy
by inhibiting inositol monophosphatase (IMPase; an
intracellular target of lithium), leading to free inositol
depletion, which, in turn, decreased inositol
1,4,5-tris-phosphate (IP3) levels [47,48] (Fig 2) This effect on
autophagy was mimicked by a specific IMPase inhibi-tor, L-690,330 Induction of autophagy by these agents reduced the proportion of cells with mutant huntingtin aggregates and enhanced the clearance of soluble aggregate-prone proteins Mood-stabilizing drugs such
as carbamazepine and valproic acid, which deplete inositol levels, also enhanced the clearance of mutant proteins (Fig 2) The autophagy-enhancing effect of lithium was most likely to be mediated at the level of,
or downstream of, lowered IP3, as it was abrogated by pharmacological treatments that increased the level of
IP3 Induction of autophagy by IMPase inhibition was mTOR-independent Moreover, IP3 levels had no effect on the autophagy-inducing property of mTOR inhibition by rapamycin, suggesting that these two pathways are independent of each other [47] There-fore, agents that reduce inositol or IP3 levels may be possible therapeutic candidates where autophagy is a protective pathway
The autophagy-inducing property of lithium has recently been suggested to contribute to its protective effects in ALS patients and mouse models, where the drug treatment increased survival and delayed disease progression [14] Remarkably, all the ALS patients on lithium treatment for 15 months survived, whereas approximately 30% of control patients matched for age, disease duration and sex receiving riluzole died [14] However, lithium may also be mediating its effects via autophagy-independent pathways
Combination treatment with lithium and rapamycin has additive effects
on autophagy
Although we demonstrated that lithium induced mTOR-independent autophagy by inhibiting IMPase [47], we have recently shown that glycogen synthase kinase-3b (GSK-3b), another intracellular target of lithium, has opposing effects on autophagy in an mTOR-dependent fashion [49] (Fig 2) Inhibition of GSK-3b by SB216763 inhibited autophagy and resulted in increased mutant huntingtin aggregation;
an effect that was also observed in GSK-3b knockout mouse embryonic fibroblasts This effect was indepen-dent of the GSK-3b target, b-catenin Indeed, inhibi-tion of GSK-3b activated mTOR by phosphorylating the tuberous sclerosis complex protein TSC2 [50], which impaired autophagy However, lithium or IMPase inhibitor (L-690,330) reduced the proportion
of cells with mutant huntingtin aggregates even in GSK-3b null cells, suggesting that induction of auto-phagy by lithium due to IMPase inhibition occurred even in the absence of GSK-3b [49]
Trang 5In order to counteract the autophagy inhibitory
effects of mTOR activation resulting from lithium
treatment due to GSK-3b inhibition, we used the
mTOR inhibitor rapamycin in combination with
lith-ium This combination enhances autophagy by
mTOR-independent (IMPase inhibition by lithium)
and mTOR-dependent (mTOR inhibition by
rapamy-cin) pathways [47,49] (Fig 2) Combination treatment
with lithium and rapamycin had additive protective
effects on the autophagic clearance of mutant
hunting-tin, as compared to either drug alone We have further
demonstrated proof-of-principle for this rational
com-bination treatment approach in vivo by showing
greater protection against neurodegeneration in an HD
Drosophilamodel with TOR inhibition and lithium, as
compared to inhibition of either pathway alone [47,49]
Furthermore, this approach may also benefit from the
cytoprotective effects of GSK-3b inhibition, due to
activation of the b-catenin–Tcf pathway (Fig 2)
Although treatment with lithium on its own is also
likely to mediate antiapoptotic effects in HD models
[51,52], the autophagy-inhibitory effect of GSK-3b
may explain the previous equivocal effects of lithium
in an HD mouse model [53]
The rational combination treatment of HD or
related disorders may be beneficial where the mutant
aggregate-prone proteins are autophagy substrates
Combination therapy with more moderate IMPase and
mTOR inhibition may also be safer for long-term
treatment than using doses of either inhibitor that
result in more severe perturbations of a single
path-way This alternative strategy may help to lessen the
drug-specific side-effects
GSK-3b is also known to hyperphosphorylate tau,
and inhibitors of GSK-3b such as lithium may be used
for preventing accumulation of hyperphosphorylated
tau in AD [33,54] Furthermore, GSK-3a has been
shown to facilitate amyloid precursor protein
process-ing at the c-secretase step and thereby regulate
amy-loid-b (Ab) production [55] Lithium reduced Ab
production by inhibiting GSK-3a [55] Thus, GSK-3
inhibition by lithium may be a tractable therapeutic
strategy in AD, as it reduces the formation of both
neurofibrillary tangles and amyloid plaques
Further-more, lithium may also potentially enhance autophagic
clearance of mutant tau, as autophagy induction with
rapamycin has this effect [10]
Trehalose induces mTOR-independent
autophagy
Trehalose, a disaccharide present in many
nonmamma-lian species, functions as a chemical chaperone and
protects cells against various environmental stresses by preventing protein denaturation [56] Trehalose has been shown to alleviate polyglutamine-induced pathol-ogy in an HD mouse model, and this protective effect was suggested to be mediated by trehalose binding to the expanded polyglutamines, thus stabilizing the partially unfolded mutant protein [57] We have recently reported a novel function of trehalose in inducing autophagy independently of mTOR [42] (Fig 2) Trehalose increased autophagic flux in various cell lines, thereby enhancing the clearance of mutant huntingtin and a-synuclein mutants and reducing the toxicity of these mutant proteins Furthermore, treha-lose facilitated the clearance of endogenous autophagy substrates as assessed by reduced mitochondrial load, and this protected cells against proapoptotic insults by decreasing active caspase-3 levels [42] The dual protec-tive properties of trehalose (‘autophagy induction’ for enhancing clearance and ‘chemical chaperone’ for inhibiting aggregation), coupled with its lack of toxic-ity, suggest that it may be a valuable drug for further development
Screens for autophagy modulators
In order to identify further autophagy modulators, we recently carried out a primary small-molecule screen in yeast in collaboration with Schreiber and colleagues [58] First, novel small-molecule enhancers (SMERs) and small-molecule inhibitors of the cytostatic effects
of rapamycin were identified in a yeast screen with
50 729 compounds Three SMERs induced mTOR-independent autophagy in the absence of rapamycin, thereby enhancing the clearance of mutant huntingtin and A53T a-synuclein in mammalian cells, and attenu-ated mutant huntingtin fragment toxicity in HD cells and Drosophila models [58] These three SMERs also had additive effects with rapamycin, and the combined treatment facilitated greater clearance of mutant proteins than either of the treatments alone (Fig 2) A further screen of structural analogs of these three SMERs identified 18 additional candidate drugs that reduced the proportion of cells with mutant huntingtin aggregates [58]
Yuan and colleagues recently performed an image-based screen for autophagy inducers by analyzing 480 bioactive compounds in a stable human glioblastoma cell line expressing green fluorescent protein (GFP)– LC3 [59] Analysis of autophagy was performed by using GFP–LC3 punctate structures with high-throughput fluorescence microscopy, and the screen hits were classified into three groups depending on the number, size and intensity of the GFP–LC3 vesicles
Trang 6Further analysis of the hits was carried out, from
which eight compounds were identified that induced
autophagic degradation without notable cellular
dam-age These compounds are fluspirilene, trifluoperazine,
pimozide, niguldipine, nicardipine, amiodarone,
lopera-mide, and penitrem A, which did not affect mTOR
activity and reduced the numbers of expanded
polyglu-tamine aggregates in a cell-based assay with the
excep-tion of nicardipine Some of these new targets may be
beneficial for the treatment of HD, as seven out of the
eight final hits were FDA-approved drugs [59]
Conclusion
Autophagy is a major degradation route for mutant
huntingtin and other aggregate-prone proteins
associ-ated with neurodegenerative disorders Furthermore,
autophagy induction may also be a valuable strategy
in the treatment of infectious diseases, including
tuber-culosis [60] Since the first discovery of autophagic
clearance of mutant huntingtin by rapamycin was
reported [11], studies have identified novel
autophagy-inducing pathways⁄ drugs Although various small
mol-ecules have been identified since then, the key question
now is to understand their targets regulating
mamma-lian autophagy This remains a daunting task, as it is
still unclear how mTOR regulates autophagy
Acknowledgements
We are grateful to the Wellcome Trust, Medical
Research Council (MRC), EUROSCA and the National
Institute for Health Research, Biomedical Research
Centre at Addenbrooke’s Hospital for funding
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