©2008 Landes Bioscience. Do not distribute.
230Autophagy2008; Vol. 4 Issue 2
lysosomal sorting.2,3 The protein complexes required for MVB
formation, called endosomal sorting complex required for transport‑I
(ESCRT‑I), ESCRT‑II and ESCRT‑III, were recently characterized in
detail in yeast.2,3 These heteromeric protein complexes are thought
to act sequentially. ESCRT‑I binds to ubiquitinated cargoes, activates
ESCRT‑II, and thereby recruits ESCRT‑III. ESCRT‑III contains the
coiled‑coil proteins Vps20, Snf7, Vps2 and Vps24 and is involved
in cargo concentration on late endosomes. ESCRT‑III also binds to
other factors, such as the AAA‑type ATPase Vps4, which is necessary
for ESCRT dissociation before the next round of sorting. Failure of
ESCRT‑III assembly or dissociation disrupts MVB formation.
The ESCRT machinery is involved in multiple disease processes,
including HIV budding4,5 and tumor suppression.6‑8 However, the
role of ESCRT‑III in neuronal development and neurodegeneration
is poorly understood. Reduced activity of Shrub, the Drosophila
ortholog of the yeast ESCRT‑III component Snf7, is required for
the proper dendritic development in Drosophila.9 The hereditary
spastic paraplegia protein spastin interacts with CHMP1B, an
ESCRT‑III‑associated endosomal protein.10 Moreover, a single splice
site mutation in CHMP2B, the gene encoding the human ortholog
of the yeast ESCRT‑III component Vps2, is mutated in a rare form
of autosomal dominant frontotemporal dementia (FTD3) and
amyotrophic lateral sclerosis (ALS).11‑13 However, detailed molecular
mechanisms of neurodegeneration associated with FTD remain
ESCRT‑III Dysfunction Causes Neurodegeneration
mSnf7‑2, one of the two mouse orthologs of the yeast protein Snf7,
is highly expressed in most brain neurons and essential for embryonic
development.14 Using both loss‑ and gain‑of‑function approaches
in cultured mature cortical neurons, we examined the consequences
of ESCRT‑III dysfunction as a result of either mSnf7‑2 depletion
or ectopic expression of the mutant protein CHMP2BIntron5. We
find that dendritic retraction and neurodegeneration occurr in both
cases.14 In contrast, other reported mutant proteins CHMP2BD10
and CHMP2BD148Y do not cause this effect. CHMP2BIntron5 was
associated more avidly with mSnf7‑2 than CHMP2BWT, thereby
preventing proper dissociation of ESCRT‑III and leading to neuro‑
degeneration. This notion was further supported by the finding that
expression of a dominant‑negative form of SKD1, the AAA‑family
ATPase required for ESCRT‑III dissociation,15 shows a phenotype
similar to CHMP2BIntron5 expression.14 These results, together
with our finding that not all mutant CHMP2B proteins cause
Autophagy is a regulated pathway for bulk degradation of cyto-
plasmic contents and organelles, an important process involved in
many physiological and pathological conditions in multiple organs,
including the nervous system. It has been proposed that devel-
oping autophagosomes fuse with late endosomal compartments
before their fusion with lysosomes; however, little is known about
the functional relationship between the autophagy and endocytic
pathways. In the endosomal-lysosomal pathway, a key step in
sorting transmembrane cargo proteins is regulated by multimeric
complexes called ESCRT (endosomal sorting complex required for
transport). We recently reported that dysfunction of ESCRT-III,
either by depletion of its essential subunit mSnf7-2 or by expres-
sion of a mutant CHMP2B protein associated with frontotemporal
dementia linked to chromosome 3 (FTD3), caused autophagosome
accumulation and dendritic retraction before neurodegeneration in
cultured mature cortical neurons. This defect is likely a result of an
abnormal fusion process between autophagosomes and endosomal
compartments or lysosomes. This study suggests that defects in the
late steps of the autophagy pathway may contribute to the patho-
genesis of FTD and potentially other neurodegenerative diseases.
Endocytosis modulates many transmembrane receptor‑mediated
signaling pathways, which are especially important in mature neurons.
Unlike other cell types, many neurons have highly branched dendritic
and axonal processes, whose structural integrity must be maintained
throughout adulthood. Defects in the endosomal‑lysosomal pathway
have been implicated in a number of neurodegenerative disorders,
but the underlying mechanisms are largely unknown.1
Transmembrane cargoes destined for lysosomal degradation are
sorted into vesicles within the lumen of multivesicular bodies (MVBs);
often, monoubiquitination of cargo proteins is a positive signal for
Roles of ESCRT in autophagy-associated neurodegeneration
Jin-A Lee and Fen-Biao Gao*
Gladstone Institute of Neurological Disease and Department of Neurology; Neuroscience Graduate Program; University of California, San Francisco; San Francisco, California
Key words: autophagy, endosomes, multivesicular body, ESCRT, frontotemporal dementia
*Correspondence to: Fen-Biao Gao; Gladstone Institute of Neurological Disease and
Department of Neurology; University of California, San Francisco; San Francisco,
California 94158 USA; Tel: 415.734.2514; Fax: 415.355.0824; Email: fgao@
Submitted: 12/04/07; Revised: 12/06/07; Accepted: 12/06/07
Previously published online as an Autophagy E-publication:
Addendum to: Lee J-A, Beigneux A, Ahmad ST, Young SG, Gao F-B. ESCRT-III dysfunc-
tion causes autophagosome accumulation and neurodegeneration. Curr Biol 2007;
[Autophagy 4:2, 230‑232; 16 February 2008]; ©2008 Landes Bioscience
©2008 Landes Bioscience. Do not distribute.
ESCRT‑III function. However, in our in vitro neuronal culture
system, we detected no signs of apoptotic cell death when mSnf7‑2
was depleted or CHMP2BIntron5 was expressed.
Role of ESCRT in autophagy
neurodegeneration, suggest that some reported CHMP2B mutations
may not be pathogenic due to their inability to disrupt ESCRT‑III
function. This explanation may help to resolve the conundrum
regarding the genetic basis of FTD3.12
In cortical neurons containing dysfunctional ESCRT‑III, we
observe an accumulation of ubiquitinated protein aggregates, which
was confirmed in a recent report that also shows an accumula‑
tion of ubiquitin‑positive aggregates in ESCRT‑I, ESCRT‑II, and
ESCRT‑III‑depleted HeLa cells or in cells expressing mutant
CHMP2B proteins.16 Moreover, cytosolic ubiquitin‑positive inclu‑
sions were found in the hippocampal and cortical regions in FTD3
patient brains of the Danish family with the CHMP2B muta‑
tion,17 suggesting that dysfunctional ESCRT‑III is indeed likely to
contribute to the pathogenesis of FTD3. Compromised ESCRT
function may also be involved in other neurodegenerative processes.
For instance, ESCRT‑I subunit TSG101 is a substrate of the E3
ubiquitin ligase Mahogunin, whose null mutation causes spongiform
Role of ESCRT in the Autophagy Pathway
How does ESCRT dysfunction contribute to neurodegeneration?
To identify the underlying mechanism, we investigated whether the
apoptosis pathway was activated in neurons with a compromised
Another important cellular pathway thought to be associated with
cell death is the autophagy pathway, a bulk degradation system with
multiple functions in development and disease.19,20 Using multiple
approaches, including GFP‑LC3 localization, LC3‑I/LC3‑II western
blot, and electron microscopy analysis, we find that ESCRT‑III
dysfunction causes autophagosome accumulation in mature cortical
neurons.14 Depletion of ESCRT‑I, ESCRT‑II and ESCRT‑III
similarly cause autophagosome accumulation in HeLa cells and in
flies.16,21 Interestingly, a recent report shows that lysosomal storage
disorders are also associated with autophagosome accumulation in
affected brains, suggesting that the autophagy pathway is involved in
neurodegenerative diseases more broadly than previously thought.22
How does ESCRT dysfunction cause autophagosome accumu‑
lation, and what is the mechanism underlying the link between
MVBs and autophagy? The endocytic pathway may converge with
the autophagic pathway and the maturation of autophagosomes
requires their fusion with MVBs.23‑25 Our recent data indicate that
ESCRT‑III dysfunction blocks the maturation of autophagosomes
into LAMP‑1‑positive autolysosomes in mature cortical neurons
(Fig. 1). Our findings are consistent with the observations that
depletion of ESCRT subunits results in the inhibition of autophagic
clearance of cytosolic proteins and organelles and accumulation of
protein aggregates.16,21 These findings together suggest that ESCRT
is required for the proper turnover of autophagosomes. However,
further investigation is needed to dissect where ESCRT acts exactly
in the autophagy pathway.
Figure 1. ESCRT-III dysfunction blocks the maturation of autophagosomes in primary cortical neurons. 15 days in vitro (DIV) mature cortical neurons were
incubated for 1 h with DMSO, or vinblastine in DMSO (final concentration 50 mM), a microtubule depolymerizing agent that induces the accumulation of
autophagosomes by preventing their fusion with lysosomes and ultimate degradation, and cultured without B27 medium for another 24 h to induce starva-
tion. In parallel experiments, mSnf7‑2 siRNA and CHMP2BIntron5 were transfected into mature cortical nurons during the 24 h starvation. (A) Western blot
analysis of LC3-I/LC3-II expression in mSnf7‑2 depleted-, or vinblastine-treated mature cortical neurons after starvation. (B) GFP-LC3 was transfected into
15 DIV cortical neurons together with pSuper-mSnf7-2 or Flag-CHMP2BIntron5. Anti-LAMP-1 antibody was used for lysosome staining. (C) Quantification of
the percentages of LC3-, LAMP-1-positive compartments among LC3-positive autophagosomes in mature cortical neurons. All neurons were starved and more
than 10 neurons were analyzed in each experimental condition. Values are mean ± SEM on the basis of two independent experiments (p < 0.001).
©2008 Landes Bioscience. Do not distribute.
E3 ligase Mahogunin ubiquitylates TSG101 and regulates endosomal trafficking. Mol Biol
Cell 2007; 18:1129‑42.
19. Klionsky DJ. Autophagy: From phenomenology to molecular understanding in less than a
decade. Nat Rev Mol Cell Biol 2007; 8:931‑7.
Role of ESCRT in autophagy
232 Autophagy2008; Vol. 4 Issue 2
Increasing evidence indicates that autophagy plays a critical role
in neurodegenerative diseases. Although inhibition of basal neuronal
autophagy in the developing mouse brain through depletion of atg5
or atg7 causes neurodegeneration,26,27 the exact role of autophago‑
somes in age‑dependent neurodegenerative diseases and how this
process is regulated in mature neurons remain to be further charac‑
terized.28,29 Functional maturation of autophagosomes by ESCRT
may be a critical step for proper degradation of cytosolic proteins
and organelles in both basal autophagy and induced autophagy in
diseased neurons. Abnormal accumulation of autophagosomes may
be detrimental to neuronal survival as well.
We thank G. Howard for editorial assistance and Lei Liu for
technical help. This work was supported by the Korea Research
Foundation and the California Institute for Regenerative Medicine
(J.L.) and by Pfizer/American Federation for Aging Research and the
National Institutes of Health (F.‑B.G.).
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