An inhibitor of the proteasomal deubiquitinating enzyme USP14 induces tau elimination in cultured neurons

Article abstract of DOI:10.1074/jbc.M117.815126

The ubiquitin-proteasome system (UPS) is responsible for most selective protein degradation in eukaryotes and regulates numerous cellular processes, including cell cycle control and protein quality control. A component of this system, the deubiquitinating enzyme USP14, associates with the proteasome where it can rescue substrates from degradation by removal of the ubiquitin tag. We previously found that a small-molecule inhibitor of USP14, known as IU1, can increase the rate of degradation of a subset of proteasome substrates. We report here the synthesis and characterization of 87 variants of IU1, which resulted in the identification of a 10-fold more potent USP14 inhibitor that retains specificity for USP14. The capacity of this compound, IU1-47, to enhance protein degradation in cells was tested using as a reporter the microtubule-associated protein tau, which has been implicated in many neurodegenerative diseases. Using primary neuronal cultures, IU1-47 was found to accelerate the rate of degradation of wild-type tau, the pathological tau mutants P301L and P301S, and the A152T tau variant. We also report that a specific residue in tau, lysine 174, is critical for the IU1-47–mediated tau degradation by the proteasome. Finally, we show that IU1-47 stimulates autophagic flux in primary neurons. In summary, these findings provide a powerful research tool for investigating the complex biology of USP14.

Full text of DOI:10.1074/jbc.M117.815126

JBC Papers in Press. Published on September 26, 2017 as Manuscript M117.815126
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M117.815126
Enhancement of proteasome function in primary neurons
An inhibitor of the proteasomal deubiquitinating enzyme USP14 induces tau elimination
in cultured neurons
Monica Boselli1§, Byung-Hoon Lee1,2§, Jessica Robert1, Miguel A. Prado1, Sang-won Min3,
Chialin Cheng4, M. Catarina Silva4, Changhyun Seong1,5, Suzanne Elsasser1, Ketki M.
Hatle1, Timothy C. Gahman6, Steven P. Gygi1, Stephen J. Haggarty4, Li Gan3, Randall W.
King1* and Daniel Finley1*
Running title: Enhancement of proteasome function in primary neurons
§Equal contribution
*To whom correspondence should be addressed: Dept. of Cell Biology, Harvard Medical School,
240 Longwood Ave., Boston, MA 02115
Tel.: 617-432-3492 (DF), 617-432-3692 (RWK) Fax: 617-432-1144
E-mail: daniel_finley@hms.harvard.edu, randy_king@hms.harvard.edu
1Department of Cell Biology, Harvard Medical School, Boston, MA 02115.
2Department of New Biology, DGIST, Daegu, Korea
3Gladstone Institute of Neurological Diseases, Department of Neurology, University of
California, San Francisco, CA 94158
4Chemical Neurobiology Laboratory, Center for Genomic Medicine, Department of Neurology,
Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114.
5Regeneron Pharmaceuticals, Tarrytown, NY 10591
6Small Molecule Discovery Program, Ludwig Institute for Cancer Research, La Jolla, CA 92093
Keywords: proteasome, ubiquitin, IU1-47, tauopathy, USP14, small molecule, neurodegenerative
disease.
ABSTRACT
The ubiquitin-proteasome system
(UPS) is responsible for most selective
protein degradation in eukaryotes and
regulates numerous cellular processes,
including cell cycle control and protein
quality control. A component of this
system, the deubiquitinating enzyme
USP14, associates with the proteasome,
where it can rescue substrates from
degradation by removal of the ubiquitin
tag. We previously found that a small-
molecule inhibitor of USP14, known as
IU1, can increase the rate of degradation
of a subset of proteasome substrates. We
tested using as
microtubule-associated protein tau, which
a
reporter the
has
been
implicated
in
many
Using
neurodegenerative
diseases.
primary neuronal cultures, IU1-47 was
found to accelerate the rate of
degradation of wild-type tau, the
pathological tau mutants P301L and
P301S, and the A152T tau variant. We
also report that a specific residue in tau,
lysine 174, is critical for the IU1-47
mediated tau degradation by the
proteasome. Finally, we show that IU1-47
stimulates autophagic flux in primary
neurons. In summary, these findings
provide a powerful research tool for
investigating the complex biology of
USP14.
report
here
the
synthesis
and
characterization of 87 variants of IU1,
which resulted in the identification of a
ten-fold more potent USP14 inhibitor that
retains specificity for USP14. The
capacity of this compound, IU1-47, to
enhance protein degradation in cells was
1
Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Enhancement of proteasome function in primary neurons
The ubiquitin-proteasome system
ubiquitin-protein conjugate (8); lacking the
ubiquitin tether, the substrate may be
released prematurely. While most USP14
substrates are likely to be docked on the
proteasome, there is evidence that USP14
suppresses autophagy as well, through an
unknown mechanism (10).
(UPS) is the major cellular pathway
responsible for selective protein degradation
within the eukaryotic cell (1-4). Proteins
destined for elimination typically carry
covalent ubiquitin modifications, which can
serve as recognition signals for the
proteasome as well as for selective
autophagy. The formation of a substrate-
bound ubiquitin chain requires the activity
of three classes of enzymes: ubiquitin
activating enzyme (E1), which activates the
carboxyl terminus of ubiquitin; a ubiquitin-
conjugating enzyme (E2), which receives
ubiquitin from E1, forming a thioester
adduct; and a ubiquitin-protein ligase (E3),
which binds substrates and guides the
donation of ubiquitin from the E2. The serial
addition of ubiquitin to a substrate through
this pathway, often forming ubiquitin
chains, generates substrates of progressively
higher affinity for the proteasome, and thus
more rapid rates of degradation. The
proteasome recognizes substrates via
specific ubiquitin receptors within its 19-
subunit regulatory particle. Subsequently, in
an ATP-dependent process, the proteasome
translocates the substrate into the 28-subunit
core particle to be degraded (2, 5, 6).
The phenotype of mouse Usp14
mutants indicates that it is particularly
important in neurons (11-13) though
phenotypic severity is highly strain-
dependent (14). Consistent with
a
noncatalytic function of the enzyme, as
described originally for the yeast ortholog
(9, 15), the Usp14 loss-of-function
phenotype in the mouse may not entirely
reflect loss of deubiquitinating activity, as
indicated by studies involving transgenic
overexpression of a catalytically inactive
form of the enzyme (13, 16).
We previously identified specific
small-molecule inhibitors of human USP14
by high-throughput screening. One such
compound, known as IU1, abrogates the
catalytic activity of USP14 while apparently
not affecting its noncatalytic regulatory
function (8). IU1 is cytoprotective under
various conditions, including ischemia-
reperfusion and ER stress (17, 18). Using
MEF and HEK293 cells, IU1 was shown to
accelerate the degradation of some but not
all substrates of the proteasome (8).
Consistent with the selectivity of USP14’s
effect on protein degradation in cells,
preferred in vitro substrates of USP14 are
modified by multiple ubiquitin chains (8,
19). USP14 removes chains en bloc until a
single chain remains, but will not remove
the last chain.
Integral subunits of the proteasome
are complemented by a variety of proteins
that associate with it in a salt-sensitive
manner and that are not stoichiometric
within the complex (7). These proteasome-
associated
proteins
regulate
protein
degradation in diverse ways; they include
both inhibitors and activators of the
proteasome as well as factors that alter its
specificity. Among these factors,
a
deubiquitinating enzyme (DUB) known in
mammals as USP14, and in yeast as Ubp6,
shows particularly strong regulation of the
proteasome. USP14 and Ubp6 are
profoundly activated upon association with
proteasome (7-9). Substrate deubiquitination
by USP14 and Ubp6 can be very fast, and
accordingly the suppression of proteasome
activity by deubiquitination appears to result
from the removal of ubiquitin from the
substrate before the proteasome commits to
degrading the substrate component of the
The availability of IU1 has led to
the identification of a growing number of
proteins identified as apparent targets of
USP14’s deubiquitinating activity. Proteins
such as the androgen receptor, cGAS,
vimentin, GFPu, CD3δ, and most notably the
prion protein, PrpC, show accelerated
degradation or reduced levels upon IU1
treatment, most simply accounted for by
reduced deubiquitination at the proteasome
(17, 20-24). Interestingly, IU1 specifically
reduces the level of a phosphorylated form
2

Enhancement of proteasome function in primary neurons
of tyrosine hydroxylase (25). Thus, USP14
inhibition enhances protein degradation in
vivo and in vitro (8, 19) though, likely
because of the sharply restricted substrate
specificity of USP14 (19), its inhibition does
not enhance the degradation of proteins
generally. Consistent with this view, USP14
knockdown resulted in reduced levels of 87
proteins in H4 neuroglioma cells (10). In
addition, murine embryonic fibroblasts
(MEFs) that are null for USP14 showed
accelerated bulk degradation of proteins
(26). Assuming that these effects are direct,
they might be due to abrogation of
deubiquitination or of the noncatalytic effect
of USP14.
phosphorylation,
acetylation,
and
ubiquitination. Tau toxicity appears closely
linked to its acetylation and phosphorylation
(35, 36).
Studies of tau-P301L transgenic
mice harboring an inducible tau expression
system showed that simple reduction in tau
level is sufficient to restore performance in
behavioral tests of memory and to prevent
neuron loss (37). It is therefore of interest to
investigate the use of small molecules which
may be capable of selectively decreasing tau
levels, several of which have been described
(8, 35, 38). In the case of IU1, the molecular
scaffold contains functional moieties that
can be subjected to combinatorial chemical
modifications to improve potency (Fig. 1A).
We describe here 87 variants of IU1, among
them IU1-47, a more potent inhibitor of
USP14. Using tau as a reporter, we
demonstrate that IU1-47 enhances protein
degradation in neurons.
Recent work has begun to explore
the integration of USP14 into cellular
signaling
pathways.
USP14
is
phosphorylated by AKT at S432, within the
BL2 loop of USP14 (10), which occludes
the USP14 active site in the inactive state of
the enzyme (27). This phosphorylation event
appears to increase the activity of
proteasome-bound USP14 (10), though it
may be insufficient to activate USP14 to
disassemble ubiquitin-protein conjugates in
the absence of the proteasome (19). In
addition to AKT, the JNK and WNT
signaling pathways have been linked to
USP14 (13, 28).
RESULTS
IU1-47 is a Potent and Selective Inhibitor
of USP14
In order to develop a more potent
USP14 inhibitor, with improved selectivity,
we prepared 87 analogs of IU1 (Fig. 1;
Suppl. Tables 1 and 2). To evaluate
selectivity, each derivative was tested in
parallel for inhibition of USP14 and of
IsoT/USP5, a closely related DUB, in both
cases using the Ub-AMC hydrolysis assay
(8). Screening of IU1 variants was carried
Several key proteins involved in
neurodegenerative diseases appear to be
proteasome substrates (18, 29, 30). An
example is the microtubule-associated
protein tau (MAPT), which regulates
microtubule assembly and stability (31, 32).
Point mutations at several sites in the MAPT
gene lead to familial FTDP-17 (Fronto-
temporal dementia and parkinsonism linked
to chromosome 17). Other diseases
characterized by the accumulation of tau-
containing protein aggregates include
Alzheimer’s disease, chronic traumatic
encephalopathy, progressive supranuclear
palsy, agyrophilic grain disease, corticobasal
degeneration, and Pick’s disease (33). Tau
aggregates spread progressively through
different brain regions, depending on the
tauopathy (34). Tau is subject to extensive
post-translational modification, including
out in duplicate using
a
real-time
fluorescence assay at a single concentration
of compound. For those analogs that proved
more potent than the parental compound,
IC50 values were also determined (Suppl.
Table 1).
Of the initial set of analogs tested,
most showed a severe reduction in USP14
inhibition, indicating that USP14 contacts
across the landscape of IU1 must be
preserved in order to retain activity (Suppl.
Table 1). A strong preference was observed
for substitution at the 4-position of the
3

Enhancement of proteasome function in primary neurons
fluorophenyl group (A-ring, Fig. 1A), with
proteasome) (Fig. 1D), consistent with our
previous findings on the parental compound
(8). The Ub-AMC hydrolytic activity of
USP14 is enhanced 800-fold or more in the
presence of proteasomes, indicating that
proteasome-associated USP14 is in an
activated state (8). Nonetheless, the Ub-
AMC assay is sufficiently sensitive to
monitor the activity of free USP14. In
summary, failure to inhibit free USP14 is a
shared feature of IU1 series compounds,
evident over a range of potencies. Most
likely these compounds probe a specific
conformation of USP14 that is assumed
upon proteasome binding.
electron-withdrawing groups being favored,
and an improvement in potency was
correlated with increased lipophilicity (F to
Cl). On the pyrrole core (B-ring, Fig. 1A),
replacement of the methyl groups by
hydrogen or ethyl moieties greatly reduced
activity. Reduction of the ketone to either an
alcohol or methylene completely eliminated
USP14 inhibitory activity. Modification of
the C-ring was tolerated, with the tertiary
amine favored over a secondary amine,
although a secondary amine was not devoid
of activity (Fig. 1A; Suppl. Table 1, see IU1-
4, IU1-6, IU1-8, IU1-14, IU1-15, and IU1-
17). We found that piperidine (6-membered
amine) was slightly more potent than
IU1-47 Enhances in Vitro Degradation of
Known Proteasome Substrates
pyrrolidine
Replacement of the basic amine with an
aniline abolished activity at the
(5-membered
amine).
We next tested the effect of IU1-47
on
a proteasome substrate, the CDK
concentrations tested (Suppl. Table 1, IU1-5
and IU1-16).
Among first 37 analogs synthesized,
we selected the most potent variations of the
A and C rings and combined them to create
inhibitor Sic1 (Fig. 2A). The Sic1 assay
allows one to monitor in vitro the coupled
USP14 activities of deubiquitination and
modulation of substrate degradation rate. It
has been shown that USP14 can inhibit the
degradation of ubiquitinated Sic1PY (8), an
engineered form of Sic1 in which the PY
element signals ubiquitination (39). We
therefore tested whether IU1-47 could
alleviate or reverse this effect. Initially, we
assayed USP14-dependent deubiquitination
under conditions that prevent substrate
degradation, namely the proteasome was
purified and assayed in the presence of
ADP. Under these conditions, the
deubiquitination of Sic1PY is very rapid,
almost reaching an apparent endpoint within
two minutes (Fig. 2A). IU1-47 blocked this
reaction, indicating that it inhibits USP14
activity against not only Ub-AMC but also
against true ubiquitin-protein conjugates. In
a similar experiment, we assayed the effect
of IU1-47 on Sic1PY in the presence of ATP,
where deubiquitination and substrate
degradation proceed in parallel, thus
competing to determine the fate of the
substrate. As expected, USP14 strongly
inhibited Sic1PY degradation, and IU1-47
antagonized this effect (Fig. 2B).
Comparable results were obtained using
IU1-47,
1-(1-(4-Chlorophenyl)-2,5-
dimethyl-1H-pyrrol-3-yl)-2-(piperidin-1-yl)
ethanone (Fig. 1B; Suppl. Table 1). IU1-47
has an IC50 value of 0.6 µM for proteasome-
associated USP14 (Fig. 1C). This represents
an approximately 10-fold increase in affinity
for USP14 over IU1, accompanied by a
modest increase in selectivity over
IsoT/USP5 to approximately 33-fold (Fig.
1C). IU1-47 is more lipophilic than IU1, as
reflected in its higher cLogP value (5.9 as
opposed to 4.7 in the case of IU1). The
backbone of IU1-47 was further varied,
though an analysis of 49 additional
compounds yielded none that were
appreciably improved in their properties
(Suppl. Table 2). Analytical studies,
including 1H-NMR spectroscopic data of
IU1-47 (Suppl. Fig.1), as well as LC-MS
analysis of IU1-47 and of its stability in
presence of USP14 (Suppl. Figs. 2 and 3,
respectively), were also performed to
confirm the purity and stability of the
compound.
IU1-47 was essentially inactive on
free USP14 (USP14 that is not bound to the
4

Enhancement of proteasome function in primary neurons
ubiquitinated cyclin B (19) as a substrate
(data not shown).
Hippocampal primary neurons were
treated with IU1-47 for 48 hours and total
tau, phospho-tau, and nonphospho-tau levels
were assessed by quantitative immuno-
blotting (Fig. 3B). Phospho-tau species were
monitored because mutant forms of tau are
known to be preferentially phosphorylated,
and the pattern of tau phosphorylation
correlates with the loss of neuronal integrity
(43). We therefore assessed whether IU1-47
could promote the elimination of phospho-
tau species such as those detectable using
the PHF1 and AT8 antibodies, which
correlate with the later stages of disease
progression in AD (43). As a result of IU1-
47 treatment, all tau forms in primary
IU1-47 Decreases Endogenous Wild-Type
and Human Wild-Type Tau Levels in
Cultured Cells
IU1 is cell-permeable and stable in
cultured cells (8). To determine whether the
degradation of a proteolytic substrate could
be stimulated in cells by inhibiting USP14
with IU1-47, we focused on tau
(Microtubule-Associated-Protein-Tau),
a
protein implicated in neurodegenerative
diseases (40-42). First, we transfected a
plasmid that expresses wild-type, untagged
human tau into both wild-type MEFs and
MEFs lacking USP14. IU1-47 treatment
increased tau degradation in wild-type
MEFs, indicating that IU1-47 retains the
capacity of IU1 to stimulate the degradation
of specific proteasome substrates, in that we
have previously found IU1 to affect tau
degradation in MEFs (8). IU1-47 had no
effect in this assay in Usp14 null MEFs,
confirming that USP14 inhibition underlies
stimulated tau degradation in this cell type
(Suppl. Fig. 4). Additionally, cell viability
measured by MTT assay showed that IU1-
47 is well tolerated in MEFs (Suppl. Fig. 5).
The promotion of tau degradation by IU1-47
was independently observed using an
adenoviral vector expressing wild-type
human tau in MEFs (data not shown).
neurons
were
significantly
reduced,
including the phosphorylated forms (Fig.
3B), presumably due to accelerated
degradation of the phosphorylated species.
Although all phosphorylated forms tested
were reduced upon treatment, the IU1-47
effect was stronger for specific variants,
such as the Ser202-phosphorylated species,
in comparison to those modified at Ser396
and Ser404. Similar results were obtained
using cortical neurons, and IU1-47 proved
significantly more potent than IU1 in these
tau-clearance assays (data not shown).
To confirm by an independent
method that tau level was indeed decreased
upon IU1-47 treatment, we employed
AQUA (Absolute Quantification) for
quantitative determination of total tau in cell
lysates by mass spectrometry (LC–MS). For
this approach, proteins present in extract
were digested with Lys-C, and the
abundance of a specific peptide from tau
was compared to that of a synthetic heavy-
atom-labeled internal standard peptide of
identical sequence. The analysis confirmed
that the total tau level was indeed decreased
upon IU1-47 treatment (Fig. 3C).
The toxic effect of mutant or
misfolded tau is exerted in neurons (40, 42).
The question of whether IU1-47 can be used
to control tau levels in a physiologically
relevant setting was assessed using primary
neurons derived from either mice or rat.
Cortical primary neuronal cultures were
infected with a lentiviral vector expressing
wild-type human tau. At DIV (day in vitro)
9-12, cells were treated with either IU1-47
and or MG-132 for 48 hours. Total tau
levels were assessed by immunoblotting.
IU1-47-treated cells showed a significantly
lower level of tau and the effect was
reversed in presence of the proteasome
inhibitor MG-132, suggesting that IU1-47
stimulates tau degradation principally via
the ubiquitin-proteasome system (Fig. 3A).
As expected, tau mRNA level was
unaffected by IU1-47 treatment (Fig. 3D)
and neuronal toxicity measured at the end of
the 48-hr treatment showed that murine
primary neurons can tolerate IU1-47
treatment at the assayed concentrations (Fig.
3E).
5

Enhancement of proteasome function in primary neurons
Treatment with IU1-C (8),
a
mutations in human genes associated with
familial AD, including the human amyloid
precursor protein (APP) with the following
mutations: K670N, M671L, I716V, V717I,
and a human presenilin (PSEN1) transgene
carrying the M146L and L286V mutations
(49). By six months of age these mice
exhibit amyloid deposits, and they
subsequently develop severe decreases in
cognitive function. We isolated primary
cortical neurons from 5XFAD mice and
treated the resulting cultures with IU1-47 as
described above. Under these conditions,
both total tau and phospho-tau were
significantly decreased upon 48 hr treatment
with IU1-47 (Fig. 4D).
structural analog of IU1-47 that fails to
inhibit Ub-AMC hydrolysis, did not induce
tau degradation (Fig. 3F). IU1, the
progenitor of IU1-47, was much less
effective than IU1-47 in this and other cell-
based assays (data not shown).
IU1-47 Stimulates Degradation of Human
Wild-Type and Pathological Tau Forms
in Murine Primary Neurons and in
Human iPSC-Derived Neurons
Having studied the effects of IU1-47
on the turnover of endogenous wild-type
tau, we examined the compound’s effects on
mutant forms of tau that cause neuronal
toxicity. We tested IU1-47 treatment in
primary neuronal cultures expressing human
tau via an AAV system that codes for a
pathogenic mutation: P301S, which has been
shown to be causative for autosomal
dominant FTDP-17 (44, 45). IU1-47
treatment decreased tau levels in these
cultures as well (Fig. 4A). We then tested
IU1-47 treatment using an AAV vector that
expresses the A152T human tau variant
(Fig. 4B), which has been proposed as a risk
factor for both FTD and AD (46). A
comparable reduction in tau level to what is
shown in panel A was also observed for this
tau variant (Fig. 4B).
Finally, we tested whether tau
reduction upon IU1-47 treatment was also
observed in neurons derived from human
induced pluripotent stem cells (iPSC). Tau
elimination upon IU1-47 treatment was also
observed in human iPSC-derived neurons
(Fig. 4E). Higher concentrations (~2-3x) and
longer treatment times (96 hr vs 48 hr) with
IU1-47 were required for reduction of
phospho-tau levels human iPSC-derived
neurons, as compared to rodent primary
neurons. These higher exposure levels were
still well-tolerated (Suppl. Fig. 6). While
these quantitative differences between
rodent primary neurons and human iPSC-
derived neurons in their responsiveness to
IU1-47 may be attributable to species
differences, they may alternatively reflect
differences in the overall level of maturity of
these cell populations. The former were
We proceeded to use transgenic
mice carrying the human tau mutation
P301L (tau-P301L), and two additional
mutations in the APP and in the PSEN1
genes, both well studied in the context of
AD (47, 48). These mice progressively
develop both amyloid β and tau pathology,
phenocopying both aspects of AD. We
isolated primary cortical neuronal cultures
from 3XFAD embryos and treated the
cultures with IU1-47 as described above.
Tau levels in primary neurons derived from
these mice were significantly decreased
upon treatment with IU1-47, including the
phospho-tau species S202/T205 (Fig. 4C).
The level of USP14 itself did not change
upon IU1-47 treatment of 3xFAD primary
neurons (Fig. 4C), as is generally the case.
Similar experiments were also performed
using 5xFAD transgenic mice carrying six
treated
with
compound
following
approximately one week of in vitro culture
(DIV4-6) whereas the latter were cultured
for six weeks and under different conditions
than primary neurons. In particular, human
iPSC-derived neurons differ from their
counterparts in the nature of the
predominant tau species (whether 4R or 3R).
The Effect of IU1-47 on Proteasome-
Mediated Degradation of Tau is
Dependent on Lysine 174
Recent work has shown the
importance of lysine 174 in tau toxicity and
turnover (50). We therefore tested whether
6

Enhancement of proteasome function in primary neurons
IU1-47 can induce tau degradation in
was found to be an off-target effect of IU1,
and thus not relevant to USP14 inhibition.
Using IU1-47, we did not observe the 17-
kDa calpain-dependent cleavage fragment of
tau detected with tau5 antibody (Suppl. Fig.
7A), nor with the tau1 (data not shown). In
addition, we assessed whether calpain
inhibition in primary neurons could abrogate
the reduction in tau protein by IU1-47.
Samples treated with both IU1-47 and
calpeptin, a calpain inhibitor, were harvested
and analyzed by immunoblotting. We found
that samples treated with both IU1-47 and
calpeptin showed a similar reduction in tau
level to those treated with IU1-47 alone
(Fig. 6B). Thus, IU1-47 does not appear to
reduce tau levels through calpain activation
at the concentrations and conditions used in
this study. Our conditions differed from
those of Kiprowska et al. not only in the use
of a different compound, but also in the use
of a different culture medium. For our
experiments, all treatments were performed
with Neurobasal since this neuronal-specific
medium has previously been shown to
improve neuronal cell viability compared to
DMEM (56, 57). In accord with previous
work, when we measured cell viability after
subjecting cultures of primary neurons to
cytotoxic stress using staurosporine, we
found that viability was substantially higher
in neurons subjected to stress in presence of
Neurobasal medium as compared to DMEM
(Suppl. Fig. 7B). The results suggest that
media formulation can influence neuronal
response to chemical inhibitors.
primary neuronal cultures expressing human
tau mutant K174Q. Interestingly, IU1-47
induced the degradation of human wild-type
tau but not its K174Q counterpart (Fig. 5A).
We additionally performed an in vivo
ubiquitination assay in HEK293 cells
transiently expressing FLAG-tagged tau
constructs
(wild-type,
tauK174Q
or
tauK274Q) and HA-tagged ubiquitin. Cells
were treated with MG-132 to inhibit
proteasomal degradation and ubiquitinated
tau was immunoprecipitated and detected
with an antibody against the HA-tag (Fig.
5B). The amount of polyubiquinated
tauK174Q was significantly lower than that
of its wild-type counterpart, confirming the
importance of K174 in proteasome-mediated
degradation (Fig. 5B, right panel).
A
substitution of lysine 274 had no significant
effect on the amount of polyubiquinated tau
in HEK293 cells, confirming the specificity
of lysine 174 for tau-mediated degradation
by the proteasome (Fig. 5B).
USP14 Inhibition Causes an Increase in
Autophagic Flux in Primary Neurons
Inhibition of USP14 has been shown
to induce increased autophagic flux in
neuroglioma cells (10). We therefore
assessed whether IU1-47 also induced
autophagy in primary neuronal cells. We
observed a perturbation in autophagic flux
as shown by elevation of the level of
lipidated LC3 in primary neuronal cultures
treated with IU1-47 (Fig. 6A). We did not
observe a marked difference of activated
caspase-3 between vehicle-treated and IU1-
47 treated samples, suggesting that IU1-47
did not induce apoptosis at the
Taken together, our results show
that at the concentrations used, IU1-47 can
enhance proteasome activity and selectively
promote the degradation of specific
substrates both in vitro and in cultured cells.
concentrations tested.
In addition to
proteasome-dependent degradation and
autophagy, tau is subjected to proteolytic
cleavage by calpain in several cell types and
tissues, including the brain (51-53). Calpain
activation and subsequent tau cleavage
result in a 17-kDa tau fragment (54). A
recent study (55) tested the effects of the
parental USP14 inhibitor IU1 in primary
neurons and found that IU1 induces calpain
activation and subsequent tau cleavage. This
DISCUSSION
Toxic misfolded proteins, often of
mutant origin, are etiological agents in
neurodegenerative diseases and in many
other disorders. Given the prevalence of
diseases of this type, collectively known as
proteopathies, methods for reducing the
levels of such damaging proteins are of
7

Enhancement of proteasome function in primary neurons
potential interest. The elimination of
62, 69, 70), which in most cases remain to
be elucidated in detail.
misfolded proteins is a key function of the
ubiquitin-proteasome pathway, and it is
plausible that a significant fraction of
proteopathic proteins are proteasome
substrates, though not necessarily preferred
substrates. Thus, small molecules that
enhance proteasome activity could have
therapeutic value. Proteasome activity is
To better probe USP14 function in
cells, we have developed a significantly
improved derivative of IU1. The new
compound, IU1-47, is 10-fold more potent.
IU1-47 should prove to be an effective
research tool. The improved properties of
this
compound
may
facilitate
the
held
under
negative
control
by
identification of new functions of USP14
while helping to minimize off-target effects.
deubiquitinating enzymes, in that they
remove the ubiquitin signal needed for
effective targeting to the proteasome. Small
molecule inhibitors of USP14 and other
deubiquitinating enzymes have accordingly
been found to stimulate the degradation of
proteasome substrates (8, 10, 18, 20-22, 26,
58-62).
IU1-47 Potentiates Endogenous Protein
Degradation in Cultured Neurons
Previous work showed that IU1 can
be effective in lowering tau levels in
transiently transfected MEF cells, though the
translatability of this model system into a
more physiological context was uncertain
(8). We therefore set out in this study to
examine whether the influence of USP14
inhibition on tau levels was achievable in
neurons, whether tau overexpression—as
previously employed—was necessary for the
effect, whether mutant forms of tau
associated with disease were affected by
USP14 inhibition, and whether dosing
regimes could be found in which tau levels
were reduced without substantial effects on
neuronal viability. It will be important to
confirm that USP14 inhibitors can promote
phospho-tau clearance in not only primary
neurons but also in animal models. Studies
in animals will be facilitated by further
improvement in the properties of these
compounds.
Almost
enzymes are encoded in the human genome,
providing myriad opportunities for
100
deubiquitinating
modulating the output of the ubiquitin
pathway (63-65), although it remains
unclear what fraction of deubiquitinating
enzymes play significant roles in negative
regulation and how substrate-specific such
effects may be. In some cases, potential
applications of DUB inhibitors in cancer
chemotherapy are under investigation (66,
67).
One deubiquitinating enzyme that
has been validated as a negative regulator of
the proteasome is USP14. Its importance
was highlighted first by studies of its yeast
ortholog, Ubp6, which was discovered to be
a
potent endogenous inhibitor of the
proteasome as well as a major proteasome
component (7, 9, 15, 68). An unusual feature
of this enzyme, as compared to other DUBs,
is that its capacity to inhibit protein
degradation requires a domain on Ubp6 that
targets it to the proteasome (the Ubl domain)
(7). Thus, Ubp6 is thought to remove
ubiquitin from substrates that have already
docked at the proteasome. The identification
of a specific inhibitor of USP14, IU1,
allowed for this paradigm to be extended to
mammalian cells (8). IU1 has also been
employed to identify new functions of
USP14’s deubiquitinating activity (13, 28,
IU1-47 and Phospho-Tau Elimination
We show here by quantitative
immunoblot analysis as well as mass
spectrometry that IU1-47 induces tau
degradation in cultured primary neurons
derived from either the cortex or
hippocampus. IU1-47 treatment reduces the
levels of both total and phospho-tau
isoforms, as shown by monitoring the
differential decrease of several phospho-tau
epitopes that are associated with AD
pathology. Specifically, Ser202/Thr205 and
Ser396/Ser404, the predominant tau
phosphorylation sites in later stages of AD,
8

Enhancement of proteasome function in primary neurons
are attenuated upon treatment. IU1-47 can
major role in the aging brain and in
neurodegeneration (80). Consistently,
similarly target pathological tau species such
as the P301L and P301S mutants. P301L lies
within the microtubule-binding domain of
tau, and the substitution may impair tau
binding to microtubules and promote its
hyperphosphorylation, resulting in a toxic
gain-of-function (71, 72). The htauA152T
variant is less extensively characterized, but
may be linked to an elevated incidence of
FTD–spectrum and AD diseases (46, 73-76).
The degradation of both total and some
phospho-tau species might reflect the fact
that phosphorylated tau binds less efficiently
to microtubules (77, 78). If not bound to
neurodegeneration mouse models have
shown amelioration after treatment with
autophagy-stimulating agents (81). We show
here that, in addition to stimulating the
degradation of proteasome substrates, IU1-
47 also stimulates autophagy in neuronal
cells. This additional effect of IU1-47
suggests that USP14 is an important
regulator of two key mechanisms
responsible for maintaining proteostasis in
neuronal cells: autophagy and the ubiquitin-
proteasome system.
Suppression of the UPS has proven
microtubules,
conformation that is selectively recognized
by a ubiquitin ligase or simply be more
tau
may
assume
a
to be an effective strategy in the treatment of
multiple myeloma, with proteasome
inhibitors in widespread clinical use (82).
However, enhancing the UPS might also be
a useful therapeutic strategy, given the
existence of numerous proteopathies
characterized by the expression of toxic
proteins. Among the straightforward
approaches to enhancing UPS output is the
suppression of negative regulators, and
particularly deubiquitinating enzymes. The
feasibility of approaches of this type will be
critically dependent on the substrates of the
DUB in question. In the case of USP14, we
find that lowering of a key disease-
associated target can be achieved in all cell
types tested, including neurons generated
from human-derived iPSC (Figs. 3 and 4;
Suppl. Fig. 7). Future studies will determine
the extent to which small molecule
inhibitors of USP14 can elicit broader
cytoprotective effects both in cell culture
and in vivo (17, 18).
accessible to either
a
ligase or the
proteasome. Alternatively, tau may be
ubiquitinated by a phospho-epitope specific
ligase.
K174 Mediates the IU1-47 Effect on Tau
Degradation
Recent studies have implicated
acetylation
in
tau-mediated
neurodegeneration (35, 50). In particular,
lysine 174 has been shown to play a key role
in this process and there is evidence that
K174 acetylation occurs at very early Braak
stages in AD patients. K174 also been
shown to be required for proteasome-
mediated degradation, perhaps because it is
a ubiquitination site (50). We show here that
the tauK174Q mutant did not respond to the
IU1-47 treatments in primary neurons.
These results points to the specificity of the
IU1-47 effect on tau turnover.
To evaluate the cellular effects of
USP14 inhibition more globally, it will be
important to identify additional substrates of
USP14 through proteomics and also to
further improve the potency and selectivity
of USP14 inhibitors. Such studies are
currently underway.
USP14 and Autophagy
Recent studies have shown that
inhibition of USP14 activity by IU1 (10)
induces autophagic flux in H4 cells.
Accordingly, deletion of the Usp14 gene
increases bulk turnover through both the
autophagy and proteasome pathways (26).
Autophagy is of particular importance to
maintain homeostasis in nondividing cells
such as neurons (79) and it has become
increasingly clear that autophagy plays a
9

Enhancement of proteasome function in primary neurons
EXPERIMENTAL PROCEDURES
IU1-47 showed: δ 7.64 (d, J = 8.8 Hz, 2H),
7.39 (d, J = 8.8 Hz, 2H), 6.50 (s, 1H), 3.43
(s, 2H), 2.50-2.40 (m, 4H), 2.23 (s, 3H),
1.95 (s, 3H), 1.55-1.45 (m, 4H), 1.43-1.35
(m, 2H) (Suppl. Fig. 1). LCMS (M+H+) m/z
was calculated to be 331.2; the experimental
value was 331.1 (Suppl. Fig. 2).
Synthesis of IU1-47
Compound IU1-47,
i.e.,
1-(1-(4-
Chlorophenyl)-2,5-dimethyl-1H-pyrrol-3-
yl)-2-(piperidin-1yl)ethanone (compound
4), was synthesized by Sundia Meditech
(Shanghai, China) according to the
following procedure:
LC-MS analysis of IU1-47
Stability in the Presence of USP14—IU1-47
was incubated at 2 µM in the presence or
absence of 2 µM recombinant human
USP14. Proteasome was then added at 0.5
µM to activate USP14. After 50 min at room
temperature, the reaction mixture was
applied to an LC-MS analytical system
(Agilent series 1200LC/6130MS) with a
reverse-phase pentafluorophenyl (PFP)
column (Phenomenex Luna, 100 mm x 4.60
mm, 5 µm) and a gradient solvent system
(from 10% to 100% CH3CN in 0.1% formic
acid) over 11 min. 20 microliters of sample
were injected for each analysis. The
collected LC-MS profiles were further
analyzed by extracting specific ions of m/z
at 331 for IU1-47 in the positive ion MS
mode. IU1-47 was eluted at approximately
8.0 min (Suppl. Fig. 3).
Synthesis of Compound 2
A mixture of 4-chloroaniline 1 (7.65 g, 60.0
mmol) and hexane-2,5-dione (34.2 g, 300.0
mmol) in acetic acid (40 mL) was heated at
100°C for 1 hr. The solvent was then
evaporated and the residue purified by silica
column chromatography to afford the title
compound (11.07 g, yield 89.8%).
Synthesis of Compound 3
2-Chloroacetyl chloride (6.78 g, 60.0 mmol)
was added to a suspension of AlCl3 (7.98 g,
60.0 mmol) in 1, 2-dichloroethane (50 mL)
at 0°C. The resulting mixture was stirred for
30 min, and was then added to a solution of
compound 2 (6.17 g, 30.0 mmol) in 1,2-
dichloroethane (50 mL) at 0°C. The reaction
mixture was then allowed to warm to room
temperature and stirred for 2 hr. The mixture
was poured into ice-water (20 mL) and
extracted with dichloromethane (3x15 mL).
The combined organic layers were dried
over MgSO4, filtered, and concentrated in
vacuo. The residue was purified by silica gel
chromatography to afford the title
compound (3.37 g, yield 39.9%).
Dissolving and Storing IU1-47
Anhydrous DMSO (high quality DMSO is
critical for proper solubilization) was used to
dissolve IU1-47. Powdered IU1-47 was
dissolved in 100% DMSO at ~25 mM and
the sample heated at 60°C for 6 min. This
brief heat treatment does not result in
detectable decomposition of the compound
as monitored by LC-MS. The compound
was stored at -80°C or in liquid nitrogen;
freeze-thaw cycles were avoided. The main
consideration in working with the compound
is to avoid its precipitation, which may
occur at high concentrations in tissue
culture; during thawing, if this is done
slowly; or when some dilution schemes are
used. Thawing was performed at 37°C.
Typically, IU1-47 was added to culture
media directly from the 25 mM stock. It is
recommended to quickly confirm that
cultures are free of precipitated IU1-47,
Synthesis of Compound 4 (IU1-47)
Piperidine (28 mg, 0.33 mmol) was added to
a solution of compound 3 (85 mg, 0.3 mmol)
and triethylamine (61 mg, 0.6 mmol) in
acetonitrile (10 mL). After being heated at
reflux for
1
hr, the mixture was
concentrated. The residue was re-dissolved
in dichloromethane (30 mL), washed with
saturated aqueous NaHCO3 (10 mL), dried
over MgSO4, concentrated in vacuo, and
purified by silica gel chromatography to
afford the title compound (83 mg, yield
using
a
light microscope at 10x
magnification.
1
83.8%). H NMR (400 MHz, DMSO-d6) of
10

Enhancement of proteasome function in primary neurons
In Vitro Biochemical Assays, Biochemical
Reagents, and Antibodies
Human 26S proteasome and recombinant
USP14 were affinity-purified as described
previously (8). Ubiquitin, ubiquitin-7-
amido-4-methylcoumarin (Ub-AMC), and
Tris-HCl [pH 7.5], 1 mM EDTA, 1 mM
ATP, 5 mM MgCl2, 1 mM DTT, and 1
mg/mL ovalbumin). Ub-AMC was added to
a final concentration of 1 µM to initiate the
reaction and its cleavage was monitored in
real time by measuring fluorescence at
Ex365/Em460 with an Envision Plate reader
(model 2103, Perkin Elmer) equipped with
ubiquitin-vinylsulfone
(Ub-VS)
were
purchased from R&D systems (U-100H, U-
550, and U-202, respectively). IU1
derivatives were dissolved in anhydrous
DMSO (276855, Sigma) as described above
and stored at -80°C in aliquots. Freeze-thaw
cycles were avoided. The plasmid
expressing tau was kindly provided by V.M.
an
appropriate
mirror
(e.g.,
LANCE/DELFIA, 400 nm). To test the
specificity of USP14 inhibitors, each IU1
derivative was also tested against 1.5 nM of
human IsoT/USP5 in parallel. Free ubiquitin
(0.01 - 0.02 µM) was added to activate
IsoT/USP5 in the reaction (8). For
measuring IC50 values, dose-response curves
were obtained with percent inhibition for
each inhibitor being plotted. Curve fitting
was performed using graphic analysis
software (GraphPad Prism and SigmaPlot).
In Vitro Ubiquitin Conjugate Degradation
and Deubiquitination Assays
Lee
[University
of
Pennsylvania].
Ubiquitinated Sic1PY and ubiquitinated N-
terminal cyclin B1 were prepared essentially
as described (8, 39). Calpeptin (Sigma
C8999) was reconstituted in DMSO
according to the manufacturer’s protocol.
Staurosporine was used as described (83,
84) (Sigma S6942). Sources of commercial
antibodies were as follows: anti-T7-HRP
(69048-3, EMD Millipore); anti-HA-HRP
[3F10] (120113819001, Roche); anti-Tau
[TAU-5] (AHB0042, Life Technologies);
anti-actin (A5060, Sigma); anti-GAPDH
[6C5] (ab8245, Abcam); anti-GAPDH
(G9545; Sigma); anti-USP14 (A300-920A,
Bethyl); anti-tau1 [PC1C6] (MAB3420;
Human proteasomes (4 nM) were incubated
with polyubiquitinated N-terminal cyclin B1
(UbnNCB1; ~120 nM final; HA-tagged) or
polyubiquitinated
Sic1PY
(WT
or
K63RUbnSic1PY; ~240 nM final; T7-tagged)
in proteasome assay buffer (50 mM Tris-
HCl [pH 7.5], 5 mM MgCl2, and 5 mM ATP
or 5 mM ADP for proteasome purified in the
presence of ADP). N-terminally His-tagged
human USP14 was expressed from the
plasmid pET15b-USP14 (generous gift from
G. Tian) and purified using Ni-NTA agarose
resin (30210, Qiagen) according to the
EMD
Millipore);
anti-P-tau-[AT8]
(MN1020, Thermo Scientific); tau-pS396
(44752G; Life Technologies); anti-Tubulin
β-III (1967-1 Epitomics), anti-LC3B
(NB100-2220SS Novus Biologicals), anti-
caspase-3 (9662 Cell Signaling), anti-tau
(MAB361 Millipore), anti-CP-13, anti-
manufacturer's
instructions.
Purified
recombinant USP14, where indicated, was
reconstituted with proteasome for 5 min
before initiating the deubiquitination
reaction. To test IU1-47, the compound was
pre-incubated at 17 µM with USP14 for 5
min before adding proteasome. Reactions
were quenched by adding 5×SDS–PAGE
sample buffer, boiled for 5 min, and then
CP27,
and
anti-PHF1
phospho-tau
antibodies were kind gifts from P. Davies
(Albert Einstein School of Medicine, NY).
IRDye-conjugated secondary antibodies
were from LI-COR (926-32212; 926-68020;
926-68073; 926-32213).
Ub-AMC Hydrolysis Assay
subjected
to
SDS–PAGE
and
To measure the USP14 enzymatic activity,
immunoblotting analysis using anti-HA-
HRP or anti-T7-HRP antibody.
30
nM
recombinant
USP14
was
reconstituted with 2.5 nM Ub-VS-treated
human proteasomes (8) in the presence of
graded concentrations of each IU1
derivative. The reaction mixture was
prepared in Ub-AMC assay buffer (50 mM
Transient Expression of Tau in Mouse
Embryonic Fibroblasts (MEFs)
Usp14-/- and wild-type MEFs were
previously described (11). Wild-type and
11

Enhancement of proteasome function in primary neurons
Usp14-/- murine embryonic fibroblasts were
conditions as their murine counterparts (see
above).
transfected with Metafectene-Pro (Biontex)
in 12-well plates according to the
manufacturer’s protocol. Forty-eight hours
after transfection with 1 µg of human-tau
pcDNA3.1 DNA, cells were treated with
IU1-47 (0, 20, 30, 40, or 50 µM) by
exchanging the medium (DMEM+FBS)
with pre-warmed medium containing IU1-
47, then incubated for 9 hr. Cells were
harvested by scraping in the presence of
PBS. Cells were then spun at 1,900 x g for 4
min at room temperature. The cell pellets
were frozen overnight at -80°C prior to
immunoblot analysis. Cell lysates were
prepared using buffer A (8 M urea, 75 mM
NaCl, 50 mM HEPES-NaOH [pH 8.2], 1
mM NaF, 1 mM β-glycerophosphate, 1 mM
Na3VO4, 10 mM Na-pyrophosphate, 1 mM
PMSF, and Protease inhibitor cocktail
Infection of Primary Neuronal Cultures
with Adeno-Associated Virus (AAV)
Rat primary neuronal cultures were
established as described above. All
experiments were performed at 4-6 DIV in
Neurobasal medium supplemented with B27
unless noted otherwise. The AAV transfer
vectors
BGH-polyA, CBA-tauP301S-WPRE-BGH-
polyA, and AAV1-CBA-wildtype-tau-
AAV1-CBA-tauA152T-WPRE-
WPRE-BGH-polyA were generated and
purified by GeneDetect (Sarasota, FL).
IU1-47 Treatment of Primary Neurons
IU1-47 treatments were performed by
replacing the culture medium with fresh
prewarmed medium containing IU1-47.
Treatments lasted 48 hr unless otherwise
indicated. Co-treatment with the calpain
inhibitor calpeptin were done under the
same conditions used for IU1-47.
Immunoblot Analysis of Primary Neurons
Cells were lysed and harvested as described
above. Lysates were then centrifuged at
14,000 x g at 4°C for 10 min. Supernatants
were collected and protein concentrations
measured using a standard BCA assay
(23227, Pierce). Analysis was carried out
using IRDye680 and 800 Infrared dye-
conjugated secondary antibodies for
quantitative immunoblot using an Odyssey
[11836170001,
Roche]).
For
immunoblotting, 10 micrograms of protein
were used.
Primary Neuronal Cultures
Murine primary neuronal cultures were
established
from
either
cortex
or
hippocampus of C57BL/6N mouse embryos
(E16, originating from Charles River
Laboratories and kind gift of the M.
Greenberg lab). Purified cells were plated at
2 x 106 cells per well onto six-well plates
coated with poly-D-lysine in Neurobasal
medium (21103-049, Life Technologies)
supplemented with serum-free B27 (17504-
044, Life Technologies), and Glutamax
(35050-079, Life Technologies). After ~3
hrs, the medium was removed and new
Neurobasal supplemented with serum-free
B27, and Glutamax was added to the cells.
All treatments were performed at 4-6 days in
vitro (DIV) in Neurobasal medium
supplemented with either serum-free B27 or
Infrared
Imaging
system
(LI-COR).
Immunoblot bands were quantified using
Odyssey software (LI-COR).
Statistical
Analysis
of
Quantitative
Immunoblotting
Data are presented as means ± SD unless
otherwise indicated. Statistical analysis was
performed
with
GraphPad
Prism.
Differences among multiple (≥3) means
with one variable were evaluated by one-
way ANOVA and the Borrefoni posthoc test
unless otherwise indicated. Differences
between two means were assessed with the
unpaired two-tailed t-test. P < 0.05 was
considered significant.
N2
supplement
(17502-048,
Life
Technologies). Rat primary neuronal
cultures were established from either the
cortex or hippocampus of Long Evans rats
(E18, originating from Charles River
Laboratories and a kind gift from the Sahin
lab [Boston Children’s Hospital]). These
cells were cultured under the same
Mass
Spectrometry
Absolute
Quantification (AQUA)
Cell lysates were prepared using buffer A.
Subsequently, cysteines were reduced with 5
12

Enhancement of proteasome function in primary neurons
mM DTT (for 25 min at 56°C) and alkylated
Human Induced Pluripotent Stem Cell
Neuron Generation and IU1-47 Treatment
Generation of an expandable neural
progenitor cell line (NPC, 8330-8) through
an iPSC intermediate from human
fibroblasts (GM8330, Corielle Institute) was
previously described (88). The resulting
NPC were terminally differentiated into
neurons by plating at a density of 8 x 105
cells/well, using a 6-well cell culture plate
(precoated with 20 µg/mL poly-ornithine
[Sigma] and 5 µg/mL laminin [Sigma] in
PBS overnight at 37°C) in neural media
(consisting of 70% DMEM (High Glucose
1×; Gibco cat# 11995), 30% Ham’s F12
with L-glutamine (Corning Cellgro), B27
(Gibco) and penicillin/streptomycin (Gibco).
The cells were fed twice weekly for six
weeks by replacement of half of the neural
media in each well. By 10 days of neural
with 14 mM iodoacetamide (for 30 min at
room temperature). A 10-µg aliquot of
protein from each sample was diluted to 1 M
urea with 50 mM HEPES-NaOH (pH 8.5)
and digested overnight at 37°C with 10
ng/µl Lys-C (125-02543, Wako). Just before
digestion, heavy isotope-labeled synthetic
peptide (Pierce) was spiked into the samples
(Peptide
sequence:
SSAKSRLQTAPVPMPDLK,
Heavy-Leu
[+7 Da]). After digestion, peptides were
acidified to a final concentration of 5%
formic acid, desalted using homemade
stage-tips as previously described (85), and
lyophilized.
Dried
peptides
were
resuspended in a solution of 5% formic acid
and 0.01% H2O2 (final concentrations),
incubated for 30 min at room temperature,
then analyzed by mass spectrometry.
Quantitative LC-MS analysis was performed
on an Exactive-Orbitrap mass spectrometer
equipped with a Thermo Fisher nanospray
source, a PAL HTC autosampler for sample
handling, and an Accela HPLC pump for
liquid chromatography separation. Skyline
(86) software was used to perform MS1
AQUA analysis.
differentiation,
the
cells
were
immunopositive for the neuronal marker β3-
tubulin (TUJ1), and had generated axonal
(SMI312-positive) and dendritic (MAP2-
positive) neuronal processes (88).
IU1-47 treatment started at week 6
of differentiation for 96 hr, the first dose
was applied at t=0 and the second dose two
days later. Cells were harvested by scraping
in the presence of PBS. Cells were then spun
at 1,300 x g for 5 min at room temperature.
Cell pellets were frozen overnight at -80°C
prior to immunoblot analysis. Cell lysates
were prepared using buffer A. For
immunoblotting, 10 micrograms of protein
were used.
RNA Isolation and Quantitative-PCR
Primary neuronal cultures were established
from murine hippocampi and treated with
IU1-47 for 48 hr. They were scraped with
1xPBS, spun down at 1,900 x g for 4 min,
and subsequently stored at -80°C. RNA
isolation was performed using the RNeasy
Mini Kit (74104, Qiagen), according to the
manufacturer’s instructions. Quantitative
PCR was performed using an ABI Prism
Primary Antibodies and Other Reagents in
Fig. 3A and Fig. 5
7900HT
Technologies)
Detection
system
to
(Life
the
The following reagents were purchased from
the indicated companies: Tau5 antibody
(Bio-Source), GAPDH antibody (Sigma),
and anti-HA (Cell Signaling). MG132 was
purchased from EMD Millipore.
according
manufacturer’s protocol. The Taqman Gene
expression assays used were: MAPT
(Mm00521988, Life Technologies) and 18S
(Hs03003631, Life Technologies). Analysis
was performed using the Comparative CT
method (87).
Primary Neuronal Cultures and Lentiviral
Infections in Fig. 3A and Fig. 5
Primary cultures were established from
cortices of Sprague-Dawley rat pups
(Charles River Laboratories) on postnatal
day 0 or 1. Purified cells were plated at
160,000 cells/ml in Neurobasal medium
supplemented with B27 (Invitrogen) on
Mouse Strains Used in this Study
Primary neurons were isolated from the
following mouse strains: 5xFAD (JAX
Stock No. 006554), 3xFAD (JAX Stock No.
034830) and C57BL/6N for wild-type mice.
13

Enhancement of proteasome function in primary neurons
poly-ornithine-coated plates. All treatments
School of Medicine, NY), V. Lee (University
of Pennsylvania), and G. Tian (Harvard
Medical School) for reagents. We also thank
J. Hanna, S. Hung, and A. Nguyen for
critical reading of the manuscript.
were performed at 7–13 DIV in Neurobasal
medium supplemented with N2 (Invitrogen)
unless noted otherwise.
Lentivirus was generated, purified, and used
for infection as described (89). Recombinant
lentivirus was produced by cotransfection of
the shuttle vector, two helper plasmids,
delta8.9 packaging vector, and VSV-G
envelope vector into 293T cells and purified
by ultracentrifugation. Viral titers were
Author information: A patent application on
this work has been filed by Harvard
University on behalf of the authors.
Author contribution: BHL performed all the
initial characterization of the compound and
all in vitro experiments. MB performed the
IU1-47 experiments in mouse and rat
primary neurons. JR performed the initial
experiments in rat primary neurons. MAP
performed the AQUA analysis shown in Fig.
3C and the experiment shown in Suppl. Fig.
5. SWM and LG generated results shown in
Fig. 5. CS assisted in the analysis shown in
Suppl. Fig. 3. SE purified Sic1PY–related
reagents and prepared ubiquitinated Sic1PY.
CC and MCS generated the human iPSC
and provided technical expertise. KMH
measured
by
p24
enzyme-linked
immunosorbent assays at the Gladstone-
UCSF Laboratory of Clinical Virology.
IU1-47 / MG-132 Treatment of Neurons
Rat primary neuronal cultures at DIV 9-12
were treated with IU1-47 (25 µM) and MG-
132 (10 µM) for 48 hrs.
In Vivo Ubiquitination Assays
Procedures were modified from a published
study (35). HEK293T cells were transfected
with expression vectors encoding FLAG-
tagged human WT tau or Mutant tau and
HA-ubiquitin. After 24 hr incubation, cells
were treated with IU1-47 (10 µM) in
Dulbecco's modified Eagle's medium and
incubated for 20 hr. MG-132 (20 µM) was
then added and incubated for 4 hr. Cells
were lysed in ubiquitination buffer (20 mM
Tris-HCl [pH 7.5], 0.1 mM EDTA, 0.2%
Triton X-100, 150 mM NaCl, and protease
inhibitor cocktail). Supernatant proteins
were immunoprecipitated with FLAG M2
agarose beads (Sigma) for 3 hr at 4°C.
Reactions were washed at least three times
with ubiquitination buffer and analyzed by
SDS-PAGE and western blotting with anti-
HA antibody.
provided
technical
assistance.
TCG
designed synthesis methods for the SAR
analysis of IU1 and oversaw quality control
of these compounds together with RWK.
SJH, SPG, RWK, and DF were responsible
for overall design and oversight of the
project. DF, MB, and BHL wrote the
manuscript.
Conflict of Interest: The authors declare
competing financial interests. Patents are
held on IU1 and IU1-47.
This work was supported by a grant from
the Tau Consortium (to D.F. and S.J.H.),
National Institutes of Health Grant R01
GM095526 and GM043601 (to D.F.), and
National Institutes of Health Grant R01
GM66492 (to R.W.K.), National Institute of
Health Grant R01 AG054214 (to L.G.).
Acknowledgments—The
authors
gratefully acknowledge the M. Greenberg
laboratory (Harvard Medical School) and
the M. Sahin laboratory (Boston Children’s
Hospital), for mouse and rat primary
neurons, respectively. We thank A. Amon
(MIT), A. M. Cuervo (Albert Einstein School
of Medicine, NY), P. Davies (Albert Einstein
14

Enhancement of proteasome function in primary neurons
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20

FIGURE LEGENDS
FIGURE 1. IU1-47, a specific inhibitor of USP14 with improved potency. A, summary of
SAR analysis of IU1 derivatives (see also Suppl. Tables 1 and 2). B, chemical structures of IU1-
47 and related compounds. IU1-47 combines structural features of IU1-2 and IU1-33. C, dose-
response curves for small-molecule inhibition of the Ub-AMC hydrolysis activity of proteasome-
bound USP14 (top left) and IsoT/USP5 (bottom left), a closely related deubiquitinating enzyme
(curves were fitted using nonlinear regression). Values correspond to the average of two
duplicates. A representative example of n>3 independent experiments is shown. A table of IC50
values of IU1 and its more potent derivatives is given at right. D, effect of IU1-47 (17 µM) on
Ub-AMC hydrolysis activity of recombinant USP14 (2 µM) in the absence of proteasome. Error
bars represent SD.
FIGURE 2. IU1-47 antagonizes USP14 deubiquitinating activity and stimulates substrate
degradation in vitro. A, USP14-mediated deconjugation of polyubiquitinated T7-Sic1PY by
human proteasome (4 nM) purified in the presence of ADP (Ptsm[ADP]), and with the addition
of recombinant wild-type USP14 (80 nM). In these assays ADP was further supplemented to 5
mM to suppress substrate degradation. Samples to which IU1-47 (17 µM) was added are
indicated; control samples received the vehicle DMSO. Ubn-Sic1PY conjugates in this experiment
were prepared using K63R ubiquitin. B, degradation of polyubiquitinated T7-Sic1PY by USP14-
free human proteasome (4 nM), and with the addition of recombinant wild-type USP14 (80 nM)
in the presence or absence of IU1-47 (25 µM). This experiment was carried out in the presence of
ATP (5 mM). Ubn-Sic1PY adducts were substantially rescued from degradation by the proteasome
when USP14 was added. This effect is counteracted by IU1-47. A representative example of two
independent experiments is shown.
FIGURE 3. IU1-47 treatment reduces the level of endogenous wild-type tau in murine
primary neurons. A, rat primary cortical neurons were infected with lentiviral vector expressing
human wild-type tau for four days. Cultures were then treated with either IU1-47 (25 µM) alone
or in combination with MG132 (10 µM) for 48 hrs. Lysates were prepared, and proteins resolved
by SDS-PAGE, followed by immunoblot analysis at left to detect total tau (tau5 antibody).
GAPDH was used as a loading control. The proteins molecular weights are indicated. Right,
quantification of IU1-47 mediated tau reduction (n=4; treatment from two independent
experiments). Asterisks denote p<0.01 (one-way ANOVA, Tukey-Kramer posthoc analyses). B,
murine hippocampal primary neurons (DIV4) were incubated with graded doses of IU1-47 for 48
hr. Lysates were prepared, resolved by SDS-PAGE, and immunoblotted with antibodies against
endogenous total tau (tau5), unphosphorylated tau (tau1), and multiple forms of phosphorylated
tau (pS396/S404, pS396, and pS202). GAPDH was used as a loading control. Proteins of interest
were visualized with IRDye-conjugated secondary antibodies using an Odyssey imaging system
(left). The proteins molecular weights are indicated. 37 KDa band is shown as it recognizes
monomeric tau species detected with the tau5 antibody. Quantification of total tau (as detected by
tau5 antibody) and of the phospho-tau species pS396/404 (as detected by PHF1 antibody) and
pS202 is shown for three independent experiments. Error bars represent SD. Asterisks denote p
values < 0.05; double asterisks denote p values < 0.01 of differences from appropriate DMSO
controls (right panel). C, quantification by AQUA analysis of total tau level from whole cell
lysates of murine primary neurons treated with graded amounts of IU1-47. Elution profiles of
heavy and light peptides resolved by LC-MS are shown with their retention time and mass errors
(ppm). Peak heights are given in arbitrary units; intensity values have been divided by 106 for
21

simplicity of presentation (left). Quantification was derived from the area under the curves, and
values are given as a percentage of tau present as compared with lysate from DMSO-treated cells
(right). D, quantitative PCR showing the level of tau transcript normalized to 18S rRNA and
relative to DMSO-treated control after 48 hr treatment with increasing concentrations of IU1-47
in mouse cortical primary neurons. Values correspond to the average of four replicates. Error bars
represent SD. E, Viability of murine cortical primary neurons (DIV6) after 48 hr treatment with
IU1-47 was assayed using Toxilight bioassay (Lonza), which measures adenylate kinase activity
released into the medium upon cell death. No treatment Medium alone and treatment with 1 µM
Staurosporine were indicated as (M) and (S) respectively. Staurosporine, a nonselective kinase
inhibitor that induces rapid programmed cell death in neurons was used as a positive control for
dead cells (84,85). The graph shows neuronal viability upon IU1-47 treatment from three
independent experiments. Values shown are averages, and error bars correspond to SD.
Measurements for medium alone and controls for dead cells were done in two of the three
independent experiments. F, murine cortical primary neurons were treated with 12.5 µM IU1-C, a
structural analog of IU1-47 that does not inhibit USP14, for 48 hr. Lysates were prepared and
resolved by SDS-PAGE, probed with antibodies against total tau (tau5) and GAPDH (loading
control), then visualized as above (left). Quantification of total tau indicates that there is no
decrease in tau levels during the course of IU1-C treatment (n=3; right). Error bars represent SD.
FIGURE 4. IU1-47 treatment reduces the level of pathological tau in primary neurons. A,
rat cortical primary neurons were infected with AAV-tauP301S, which expresses the human tau
mutant P301S. After 5 days, IU1-47 was added at the indicated concentrations. Following an
additional two days of culture, lysates were prepared and proteins resolved as described above.
Human tau was visualized by immunoblot using an antibody specific for human tau (CP-27), and
GAPDH was probed as a loading control. The proteins molecular weights are indicated. B, rat
cortical primary neurons were infected with AAV-tauA152T, which expresses human tau variant
A152T, for 5 days prior to IU1-47 treatment for 48 hr at the indicated concentrations. Lysates
were prepared and resolved as described above. Human tau was visualized by immunoblot using
an antibody specific for human tau (CP-27), and GAPDH was probed as a loading control. C,
murine cortical primary neurons (DIV5) isolated from APPSwe/P301L transgenic animals were
incubated with the indicated doses of IU1-47 for 48 hr, then harvested and processed as above.
Immunoblots developed with IRDye-conjugated secondary antibodies show the level of total tau
and of several specific phospho-tau species upon IU1-47 treatment. Phospho-epitopes are
indicated in parentheses. The top three blots are from one gel, the bottom four from another (left).
The USP14 blot shows that no change in USP14 protein level was observed upon IU1-47
treatment. Values in the graphs represent an average of two biological replicates representing
cortical neurons from littermates. Error bars represent SEM (right). Monoclonal antibodies are
listed below the matching epitope in the bar graph. D, murine cortical primary neurons (DIV6)
isolated from 5XFAD mice were incubated with the indicated concentrations of IU1-47 for 48
hours. Cells were harvested, and lysates prepared and resolved as described above. A
representative immunoblot showing the levels of total tau (as detected by tau5 antibody) in
cortical neurons derived from one of two independently processed embryos upon treatment with
IU1-47 (left). Secondary antibodies were IRDye-conjugated. Decrease in phosphorylated tau
pS396/S404 was also visualized with IRDye-conjugated secondary antibodies upon IU1-47
treatment. Quantification of total tau from independent samples is shown (right). E, NPC
terminally differentiated into neurons for six weeks were treated with increasing concentrations of
IU1-47. Lysates were prepared, resolved by SDS-PAGE, and immunoblotted with antibodies
against phosphorylated tau (pS396/S404). GAPDH was used as a loading control. βΙΙΙ−tubulin
22

levels were also assayed as a marker for differentiation and to assess cytoskeletal integrity.
Proteins of interest were visualized with IRDye-conjugated secondary antibodies using an
Odyssey imaging system. Similar results were obtained using an antibody against tau-pS202 (not
shown). Quantification of phospho-tau (pS396/404) as detected by PHF1 (right) is shown for
three independent experiments for the indicated time points or for two independent experiments.
Error bars represent SD for n=3 (DMSO, 10 and 30 µM IU1-47) and SEM for n=2 (3 µM IU1-
47). Asterisks denote p values < 0.05.
FIGURE 5. Lysine174 of tau is required for IU1-47 mediated degradation. A, Primary
neurons were infected AAV vectors that express either wild-type human tau or htauK174Q. Four
days after infection, the cultures were treated with either 25 µM IU1-47 or DMSO for 48 hr.
Cells were harvested, and lysates prepared and resolved by SDS-PAGE. Total human tau was
visualized by immunoblot using the tau5 antibody. GAPDH was probed as a loading control.
Right, Quantification of IU1-47 mediated tau reduction is shown. htau wild-type treated with
DMSO (n=6), hTau wild-type treated with IU1-47 (n=7), hTauK174Q treated with DMSO (n=6),
htauK174Q treated with IU1-47 (n=7) from four independent experiments. Asterisks denote p <
0.01, Mann-Whitney non-parametric test. Values are means ± SEM. B, HEK293 cells were
transfected with vectors expressing either FLAG-tagged human wild-type or mutant tau (K174Q
or K274Q) and HA-ubiquitin. Cells were then treated with IU1-47 (10 µM) for 20 hours and
subsequently treated with MG-132 (20 µM) for 4 hours. Cells were lysed with ubiquitination
buffer and supernatant proteins were immunoprecipitated with FLAG M2 agarose beads for three
hours. Reactions were resolved by SDS-PAGE and immunoblotted with anti-HA antibody. Tau5
antibody was used to detect total tau at the (bottom left). Right panel shows the quantification of
poly-ubiquitinated tau normalized to total tau. Wild-type tau (n=7) TauK174Q (n=7) from 5
independent experiments. TauK274Q (n=5) from two independent experiments. Asterisks denote
p<0.01 (one-way ANOVA, Tukey-Kramer posthoc analyses).
FIGURE 6. USP14 inhibition elicits an increase in autophagic flux in primary neurons. A,
Rat cortical primary neurons (DIV4) were incubated with graded doses of IU1-47 for 48 hr. Cells
were harvested, and lysates prepared and resolved by SDS-PAGE and immunoblotted with
antibodies against the Microtubule-associated protein 1B-light chain 3 (LC3), endogenous total
tau (tau5), multiple forms of phosphorylated tau (pS396/S404 and pS202/pT205), caspase3 (full-
length and cleaved forms). βIII-tubulin was used as a marker for structural integrity. GAPDH
used as a loading control. Proteins of interest were visualized with IRDye-conjugated secondary
antibodies using an Odyssey imaging system. A representative sample of three independent
experiments is shown. Right panel shows the quantification of LC3-II expression. Each data point
represents average from two independent samples. Error bars represent SD from n=3
independent experiments. Asterisks denote p values < 0.05. Double asterisks denote p values <
0.01 of differences from appropriate DMSO controls (one-way ANOVA, Tukey-Kramer posthoc
analyses). The images were produced from three independent gels to avoid stripping the
membrane. B, Rat cortical primary neurons DIV3 were treated with 12.5 µM IU1-47 for 48 hr in
combination with the calpain inhibitor calpeptin (2 and 10 µM). Lysates were prepared and
proteins resolved by SDS-PAGE followed by immunoblot analysis using monoclonal tau
antibody (a.a.210-241, clone tau5) to detect full length tau (Tau-FL) and if present, the 17 kDa
Tau fragment resulting from calpain-mediated tau cleavage (one asterisk denotes unspecific band,
two asterisks denotes the expected 17 kDa fragment position in the blot above). GAPDH was
used as a loading control. The blot above is representative of two independent experiments.
23

Enhancement of proteasome function in primary neurons
Figure 1
B
A
O
O
C
N
N
F
N
IU1
A
F
N
B
IU1
Basic amine is required and
ter=ary amine is favored
Electron withdrawing
group at 4-posi=on favored
Spacing is important
Cl
IU1-33
IU1-2
N
Alkyl group is important
Keto group is necessary
Dimethyl is necessary and
imidazole subs=tu=on is tolerated
O
N
Cl
N
IU1-47
C
100
80
60
40
20
0
IU1
IU1-47
D
IC50 (µM)
USP14
120
80
40
0
IsoT /
USP14
Compound
IsoT
IU1
5.5
1.7
1.1
0.6
100
18.2
32.4
30
0.01
0.1
1
10
100
1000
IU1-2
IU1-33
IU1-47
55
33
20
[Compound] (µM)
100
80
60
40
20
0
33.3
USP14 +
IU1-47
USP14
0.01
0.1
1
10
100 1000
[Compound] (µM)
F.1

Enhancement of proteasome function in primary neurons
Figure 2
A
B
Ptsm +
Ptsm (ADP) Ptsm (ADP) +
USP14 +
Ptsm +
+ USP14
USP14 +
IU1-47
Ptsm
(ADP)
USP14
IU1-47
Ptsm
5
Time
(min)
Time
(min)
0
5
10
0
5
10
0
10
0
2
4
0
2
4
0
2
4
250
150
250
150
100
75
100
75
Ubn-Sic1PY
K63R-Ubn-
Sic1PY
50
37
50
37
Sic1PY
Sic1PY
F.2

Figure 3
Enhancement of proteasome function in primary neurons
ns
1.2
1.2
1.0
1.0
**
A
IU1-47
MG-132
total tau (tau5)
+
+
+
-
0.8
0.8
-
-
0 6
0.6
65
37
0.4
0.4
GAPDH
0.2
0.
2
Len@-hTau (WT)
0
IU1-047
-
-
+
-
+
+
MG-132
Wild type
B
65
Total tau
IU1-47 (µM)
P-tau (S396/S404)
P-tau (S202)
100
80
60
40
20
0
55
37
total tau (tau5)
*
*
*
**
*
*
unP-tau (tau1)
P-tau (S396/S404)
P-tau (S396)
*
P-tau (S202)
GAPDH
0
2.5
5
7.5
IU1-47 (µM)
10
12.5
C
3.0
100
80
60
40
20
0
24.4
+2.2 ppm
Heavy Pep=de
Light Pep=de
2.5
2.0
1.5
1.0
0.5
0.0
24.4
+1.8 ppm
Reten2@4on Time (2m5 in)
23
26
IU1-47 (µM)
0
2.5
5
7.5
10
12.5
D
E
100
75
50
25
0
3
2
1
0
0
2.5
5
7.5
10 12.5
IU1-47 (µM)
0
2.5
5
7.5 10 12.5 M
S
IU1-47 (µM)
F
-
+
+
+
IU1-C
200
150
100
50
55
total tau (tau5)
37
GAPDH
0
IU1-C
-
+
-
F.3

Enhancement of proteasome function in primary neurons
Figure 4
A
B
AAV-tau-A152T
IU1-47
AAV-tau-P301S
IU1-47
65
37
65
hTau (CP-27)
hTau (CP-27)
37
GAPDH
GAPDH
C
tau-P301L/APPSwe
IU1-47
101000
Total tau
P-tau (S396)
total tau (tau5)
55
37
50
50
P-tau (S396)
5 10
total
tau
0
0
0
5
10
0
IU1-47 (µM)
P-tau (S396)
GAPDH
100
100
P-tau (S396/404)-(PHF1)
P-tau (S202/T205)-(AT8)
P-tau (S202/T205)
P-tau (S396/S404)
50
0
50
0
USP14
GAPDH
0
5
10
0
5
10
IU1-47 (µM)
D
5XFAD
150
IU1-47
100
55
100
50
total tau (tau5)
P-tau (S396/S404)
50
β-ac@n
0
0
GAPDH
0
5
10
12.5
IU1-47 (µM)
E
150
100
iPSC-derived neurons
IU1-47
100
*
P-tau (S396/404)
55
37
*
5050
βIII-tubulin
GAPDH
0
0
0
3
10
30
IU1-47 (µM)
F.4

Enhancement of proteasome function in primary neurons
Figure 5
1.2
A
DMSO
IU1-47
1.0
0.8
0.6
DMSO IU1-47
DDMMSSOO IU1-47
65
37
total tau (tau5)
GAPDH
0.4
0.2
Len@-hTau (WT)
Len@-hTau (K174Q)
0
WT
K174Q
B
WT K174Q
WT K274Q
1.5
250
150
100
1.0
0.5
0
65
Input
total tau
WT
K174Q K274Q
F.5

Enhancement of proteasome function in primary neurons
Figure 6
A
D
a
t a
1
0
12.5
25
IU1-47 (µM)
P-tau (AT8)
*
8 8
55
55
**
6
6
total tau (tau5)
4
4
P-tau (pS396/404)
55
53
2
2
βIII-tubulin
0
0
GAPDH
37
)
)
O
5
5
.
DMSO
12.5
25
IU1-47 (µM)
2
S
2
1
(
M
LC3-I
(
7
16
14
30
D
4
7
-
4
1
-
LC3-II
U
1
I
U
I
Caspase-3
15
Cleaved caspase 3
B
_
_
_
_
_
2
_
10
_
IU1-47 (µM)
Calpain Inhibitor (µM)
+
_
+
+
+
+
_
2
10 10
10
50
37
tau-FL (full length)
20
15
*
tau-Δ (cleaved Tau)
**
10
GAPDH
F.6

An inhibitor of the proteasomal deubiquitinating enzyme USP14 induces tau
elimination in cultured neurons
Monica Boselli, Byung-Hoon Lee, Jessica Robert, Miguel A Prado, Sang-won Min, Chialin
Cheng, M Catarina Silva, Changhyun Seong, Suzanne Elsasser, Ketki M Hatle, Timothy C.
Gahman, Steven P. Gygi, Stephen J. Haggarty, Li Gan, Randall W. King and Daniel Finley
J. Biol. Chem. published online September 26, 2017
Access the most updated version of this article at doi: 10.1074/jbc.M117.815126
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