MINIREVIEW
Huntington’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,

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.
FEBS Journal 275 (2008) 4263–4270 ª 2008 The Authors Journal compilation ª 2008 FEBS 4263
system, in which mammalian Atg12 is conjugated to
Atg5. The Atg12–Atg5 conjugate then forms a com-
plex with Atg16L. This complex 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].

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

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

2
IP
3
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 IP
3
levels 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-

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 (IP
3
) 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

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].
Degradation of mutant huntingtin by autophagy S. Sarkar and D. C. Rubinsztein
4266 FEBS Journal 275 (2008) 4263–4270 ª 2008 The Authors Journal compilation ª 2008 FEBS
In order to counteract the autophagy inhibitory

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

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.
S. Sarkar and D. C. Rubinsztein Degradation of mutant huntingtin by autophagy
FEBS Journal 275 (2008) 4263–4270 ª 2008 The Authors Journal compilation ª 2008 FEBS 4267
Further 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-

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