FORUM ORIGINAL RESEARCH COMMUNICATION
HIF Prolyl Hydroxylase Inhibitors Prevent Neuronal Death
Induced by Mitochondrial Toxins: Therapeutic Implications
for Huntington’s Disease and Alzheimer’s Disease
Zoya Niatsetskaya,1,2Manuela Basso,1,2Rachel E. Speer,1,2Stephen J. McConoughey,1,2
Giovanni Coppola,3Thong C. Ma,1,2and Rajiv R. Ratan1,2
Mitochondrial dysfunction is a central feature of a number of acute and chronic neurodegenerative conditions,
but clinically approved therapeutic interventions are only just emerging. Here we demonstrate the potential
clinical utility of low molecular weight inhibitors of the hypoxia inducible factor prolyl-4-hydroxylases (HIF
PHDs) in preventing mitochondrial toxin-induced cell death in mouse striatal neurons that express a ‘‘knock-in’’
mutant Huntingtin allele. Protection from 3-nitropropionic acid (3-NP, a complex II inhibitor)-induced toxicity
by HIF PHD inhibition occurs without rescue of succinate dehydrogenase activity. Although HIF-1a mRNA is
dramatically induced by mutant huntingtin, HIF-1a depletion by short interfering RNAs (siRNA) does not affect
steady-state viability or protection from 3-NP-induced death by HIF PHD inhibitors in these cells. Moreover,
3-NP-induced complex II inhibition in control or mutant striatal neurons does not lead to activation of HIF-
dependent transcription. HIF PHD inhibition also protects cortical neurons from 3-NP-induced cytotoxicity.
Protection of cortical neurons by HIF PHD inhibition correlates with enhanced VEGF but not PGC-1a gene
expression. Together, these findings suggest that HIF PHD inhibitors are promising candidates for preventing
cell death in conditions such as Huntington’s disease and Alzheimer’s disease that are associated with metabolic
stress in the central nervous system. Antioxid. Redox Signal. 12, 435–443.
tors ofmanyacuteandchronic neurodegenerative conditions.
Of these, disordered energy metabolism is most closelylinked
with the pathophysiology of Huntington’s disease (HD) (6,
10). HD is a movement disorder characterized by choreiform
movements, cognitive dysfunction, and psychiatric manifes-
tations. Two converging lines of inquiry support the hy-
pothesis that mitochondrial energy metabolism may be the
glutamine repeat stretch in the protein huntingtin (mhtt).
Among its many cellular manifestations, mhtt leads to tran-
scriptional repression of many genes, including those con-
trolling adaptation to low mitochondrial energy charge such
as PPARg coactivator 1a (PGC-1a) (7, 8, 35). Indeed, recent
studies have shown that germline deletion of PGC-1a leads to
striatal degeneration similar in localization and behavioral
itochondrial dysfunction and aberrant energy
metabolism appear to be a common upstream media-
manifestations to HD (17); by contrast, PGC-1a over-
expression via lentiviral delivery in vivo prevents striatal de-
generation attributable to transgenic expression of mhtt (8). In
this context, PGC-1a is believed to coactivate genes involved
in mitochondrial proliferation and function, including a
number of antioxidant enzymes localized to mitochondria
(e.g., MnSOD and GPx) (32).
These molecular and genetic studies are amplified by a
second line of investigation using 3-NP, a toxin that selec-
tively poisons complex II (succinate dehydrogenase, SDH) of
the mitochondrial electron transport chain. Intoxication with
3-NP can induce selective CNS damage in rodents that phe-
nocopies human HD and rodent models of HD (4, 5). More-
over, agents that mitigate 3-NP-induced striatal degeneration
in vivo can also attenuate disease onset or progression in ro-
dent models of HD (3, 19).
Besides mitochondrial biogenesis and=or induction of mi-
tochondrial proteins, an alternate strategy to compensate for
1Burke-Cornell Medical Research Institute, White Plains, New York.
2Weill Medical College of Cornell University, New York, New York.
3Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, California.
ANTIOXIDANTS & REDOX SIGNALING
Volume 12, Number 4, 2010
ª Mary Ann Liebert, Inc.
towards aerobic glycolysis and away from oxidative phos-
phorylation (14). Indeed, transcriptional upregulation of
glycolytic enzymes is an essential feature of adaptation to
short supply (30). Transcriptional induction of glycolytic en-
zymes in response to metabolic challenges such as hypoxia is
mediated primarily via stabilization of the transcriptional
activator HIF-1a and the consequent induction of >100 genes
associated with adaptation to hypoxic stress (30). In addition
to glycolytic enzymes, these genes include vascular endo-
thelial growth factor (VEGF), erythropoietin, and p21waf1=cip1
Stabilization of HIF-1a in response to hypoxia is mediated
via the inhibition of a family of dioxygenases known as the
HIF prolyl hydroxylases (HIF PHDs) (12, 13). Prior studies
from our laboratory and others have demonstrated a role for
small molecule inhibitors of HIF PHDs in protecting neurons
from ischemic or oxidative injury (2, 31, 38). Another study
suggested that HIF PHD inhibition may prevent mitochon-
drial toxicity in C6 glioma cells (37). However, no studies to
date have systematically evaluated the HIF pathway in dis-
ease models of mitochondrial dysfunction such as HD;
moreover, the ability of HIF PHD inhibitors to prevent mi-
tochondrial toxicity in normal or HD associated neurons has
yet to be explored. Herein, we show that the HIF pathway is
markedly induced in immortalized striatal cells bearing a full
length huntingtin protein with a pathological number of re-
peats (111) but not in wild-type striatal neurons with 7 re-
peats. We further demonstrate that canonical low molecular
weight HIF PHD inhibitors abrogate 3-NP-induced death in
neurons. Unexpectedly, these inhibitors protect even with
marked silencing of the HIF-1a message. Altogether, these
studies add to the growing enthusiasm for HIF PHD inhibi-
may be appropriate for neurological conditions associated
with metabolic dysfunction such as HD and stroke.
Material and Methods
Clonal striatal cell lines established from E14 striatal pri-
mordia of HdhQ111=Q111(mutant) and HdhQ7=Q7(wild-type)
knock-in mouse littermates were generously provided by
Dr. Marcy E. MacDonald (Massachusetts General Hospital).
These cells were immortalized using a replication defective
retrovirus transducing the tsA58=U19 large T-antigen (33).
Striatal Q7 and Q111 cells were maintained in Dulbecco’s
modified Eagle medium (DMEM) containing 25mM D-
glucose, 1mM L-glutamine, 10% fetal bovine serum (FBS),
1mM sodium pyruvate, and 400mg=mL Geneticin (Invitro-
gen, Carlsbad, CA) and were incubated at 338C with 5% CO2.
Cells were plated in 12-well plates at a density of 5?104
cells=ml for 16h prior to viral transduction. Retroviruses
carrying siRNAs against either HIF-1a or green fluorescent
protein (GFP) were added at 5 MOI in the presence of hex-
adimethrine bromide (4mg=ml; Sigma,St.Louis, MO)for 24h.
The cells were then subjected to selection by puromycin
(4mg=ml; Sigma) as previously described (1). Cell lines ex-
pressing the siRNAs were maintained as above.
Primary cortical neurons were cultured from embryonic
E17 Sprague–Dawley rats, as previously described with slight
modification (22). Briefly, cortices were dissected, homoge-
nized, and plated in minimum essential media containing
10% FBS, and 1% penicillin=streptomycin in 96-well plates or
in 10cm dishes. Neurons were maintained at 378C and 5%
CO2. All experiments were conducted 24h after plating.
Western blot analysis
After treatment with PHD inhibitors, striatal cells were
washed in cold PBS, scraped, and pelleted by centrifugation.
Nuclear extracts were prepared from the resulting pellets
using the NE-PER Nuclear and Cytoplasmic Extraction kits
(Pierce, Rockford, IL) and protein concentrations were mea-
sured using the Bradford protein assay (Sigma). Nuclear
proteins were separated by SDS-PAGE electrophoresis and
transferred onto nitrocellulose membranes. The membranes
were probed with a monoclonal antibody against HIF-1a
(Upstate, Davers, MA), which was detected using infrared-
fluorescent dye conjugated secondary antibodies (Li-cor,
Lincoln, NE). Images were acquired using the Li-cor Odyssey
quantitative Western blot system.
Q7 and Q111 cells were co-transfected with a plasmid ex-
pressing a firefly luciferase reporter gene driven by a hypoxia
responsive element from the 5’ noncoding region of the eno-
lase gene HRE promoter and a pTK-Renilla luciferase control
plasmid (Promega, Madison, WI). Luciferase activity was
assessed using a Promega dual luciferase assay and measured
with an LMAXII384luminometer (Molecular Devices, Sun-
nyvale, CA). Firefly luciferase luminescence was normalized
to Renilla luciferase luminescence according to the manufac-
Q7, Q111, or primary neurons were plated in a 96-well
black cell culture plates with clear bottoms designed for
fluorescence imaging. Striatal cells were pretreated with
50mM desferrioxamine (DFO), 50mM 3,4-dihydroxybenzoic
acid (DHB), or 2.5mM ciclopirox (CPO) for 6h and then ex-
posed to 10mM 3-NP in low glucose media for an additional
24h. Primary neurons were treated concurrently with 100mM
DFO, 20mM DHB, or 0.5mM CPO and 10mM 3-NP in low
glucose media for 48h. Fluorescence calcein and ethidium-
homodimer (LIVE=DEAD) images of striatal cells were cap-
tured using a Zeiss Axiovert microscope (Carl Zeiss Micro-
imaging, Thornwood, NY). For automated cell counting, a
fluorescence image of the entire well of each well of striatal or
primary neuron plates were captured using a flash cytometer
(Trophos, France). Cell counts of viable cells (calcein-positive)
were automatically generated from these pictures with the
Quantitative real time PCR
Two micrograms of total RNA extract from Q7, Q111, or
primary cortical neurons were reverse transcribed using Su-
perscript 3 (Invitrogen). The resulting cDNA was assayed by
436NIATSETSKAYA ET AL.
real-time PCR using TaqMan Gene Expression Assays for
HIF-1a, HIF-2a, VEGF, p21, b-actin, and GAPDH (Applied
Biosystems, Carlsbad, CA). PCR reactions were carried out
using an Applied Biosystems 7500 Fast Real Time PCR Sys-
are expressed as a fraction of the mRNA expression level in
untreated cells. Similar results were obtained when GAPDH
was used as an internal loading control and are reported
in Supplemental Fig. 1 (see www.liebertonline.com=ars).
SDH activity assay
Striatal cells were lysed with 0.1% vol=vol NP40 in 100mM
Tris-HCl (pH 7.4). Twenty microliters of the resulting lysate
was added to an assay mixture containing 100mM Tris-HCl
(pH 8.3), 0.5mM EDTA, 2mM iodonitrotetrazolium chloride,
5mM rotenone, 2mM antimycin A, 10mM sodium azide, and
20mM succinate. The rate of succinate-dependent generation
of the formazan product of iodonitrotetrazolium reduction
was taken as SDH activity as previously described (21).
All results are plotted as mean?SEM using GraphPad
Prism software. The statistical tests used for analysis are in-
To begin to query the behavior of the HIF pathway in re-
sponse to mitochondrial dysfunction in neurons, we com-
pared RNA levels for HIF-1a and two of its target genes,
VEGF and p21 in immortalized striatal neurons expressing
normal huntingtin protein (7 repeats) and striatal neurons
expressing a mutant huntingtin protein associated with ju-
venile onset HD (111 repeats). These cells were generated
from the HdhQ111 knock-in mice (STHdhQ111, which bear
STHdhQ111 striatal cells. TaqMan real-time gene expres-
sion assays for HIF-1a, VEGF, and p21 show elevated basal
expression of these genes in mutant huntingtin (Q111) cells
compared to wild-type (Q7) striatal cells. Gene expression
levels were normalized to b-actin levels and are expressed as
fold increase over Q7 cells for the same gene. Data represent
mean?SEM of three independent experiments. *p<0.05
compared to corresponding Q7, Student’s t-test.
HIF-1a and HIF target gene mRNA are elevated in
HIF-1a in striatal cells with si-
RNA against HIF-1a. Q7 and
Q111 striatal cells were trans-
duced with a retrovirus deliv-
ering hp=GFP (siGFP) or hp=
HIF-1a (siHIF-1). (A) HIF-1a and
HIF-2a expression levels were
determined by RT-PCR. HIF-1a
expression was significantly re-
duced in siHIF cells of either
genotype whereas HIF-2a ex-
method. (B) HIF-1a protein levels
were determined byWestern blot
analysis. Under normoxic condi-
tions, HIF-1a protein is rapidly
pharmacologic stabilizer of HIF-
levels in both Q7 and Q111 cells
expressing siGFP. In contrast,
expression of siHIF prevented
the DFO-mediated increase in
HIF-1a protein. (C) The PCR
products from the RT-PCR for
HIF-2a expression assay were
resolved by agarose gel electrophoresis and detected by ethidium bromide fluorescence. Though HIF-2a levels were unde-
tectable by RT-PCR, bands corresponding to HIF-2a were present. Importantly, HIF-2a levels were not induced in cells
expressing siHIF. Data represent mean?SEM of three independent experiments. *p<0.05 compared to corresponding siGFP
expression levels, Student’s t-test.
HIF PHD INHIBITORS PROTECT AGAINST 3-NP TOXICITY437
full-length htt with an expanded polyglutamine (polyQ) tract
of 111 CAG repeats; herein referred to as Q111) and con-
trol cells from the striatum of a knock-in mouse bearing a
nonpathological polyQ expansion (STHdhQ7, herein referred
to as Q7) (8, 33). Interestingly, we found that HIF-1a mRNA
levels and levels of HIF target genes are significantly in-
creased in Q111 cells as compared to Q7 cells, raising the
possibility that HIF is induced to compensate for metabolic
compromise in HD (Fig. 1). The change in HIF message has
been observed in prior gene array studies of these cells (16)
and in arrays from brain tissue homogenates of one rodent
model of HD (analysis of R6=2 datasets) (15), but the biolog-
ical role of this change has not been previously evaluated.
to adapt to mitochondrial dysfunction, we utilized a well-
characterized retrovirus that can transduce murine cells to
that siRNA to HIF-1a significantly reduced message levels for
HIF-1a by >80% in both cell lines (Fig. 2A). To examine
whether reduction in HIF-1a message levels were associated
with reductions in HIF-1a protein, we performed immuno-
blots from lysates of Q7 or Q111 lines treated with vehicle or
the HIF PHD inhibitor desferrioxamine (DFO), which mimics
hypoxia by stabilizing HIF-1a protein levels. As expected, in
both Q7 and Q111 lines DFO induced HIF-1a protein, which
was completely suppressed by the siRNA to HIF-1a but not
the siRNA to GFP (Fig. 2B). As other HIFa isoforms may be
induced to compensate for the loss of HIF-1a, we assessed
HIF-2a mRNA levels following HIF-1a knockdown. We were
unable to detect HIF-2a message in either cell line by real-time
RT-PCR (Fig. 2A), though we were able to resolve a PCR
product corresponding to HIF-2a by agarose gel electropho-
resis,which wasnotchanged byHIF-1aknockdown (Fig.2C).
We next examined the role of HIF-1a in regulating viability
confirmed prior studies from our laboratory and others that
showed that Q111 cells are more sensitive to 3-NP as com-
pared to Q7 cells (Fig. 3B) (28). Despite these differences, we
found that HIF-1a deletion had no effect on basal viability or
Q111 cells (Fig. 3). Altogether, these findings suggest that
HIF-1a is not necessary to maintain viability of striatal neu-
rons in response to an expanded polyQ repeat in the hun-
tingtin protein or in response to the selective complex II
inhibitor 3-NP. Of note, our results also do not support a
prodeath role for HIF-1a in striatal neurons (1).
To investigate whether HIF activation in Q111 cells is suf-
ficient to protect striatal neurons, we examined the effect of
pharmacological inhibition of HIF PHD inhibitors on 3-NP
induced death. HIF PHDs negatively regulate the stability of
HIF family transcription factors, and thus inhibitors of HIF
PHDs stabilize HIF-1a protein. Consistent with Fig. 2B, we
found that structurally diverse inhibitors of the HIF PHDs
that target the iron (DFO, CPO) or 2-oxoglutarate (3,4-DHB)
cofactors significantly increase HIF-1a protein (Supplemental
Fig. 2; see www.liebertonline.com=ars) and drive the ex-
pression of luciferase reporter under the control of the hyp-
oxia response element from the 5’ noncoding region of the
enolase gene (Fig. 4). We then examined the effect of 6h
pretreatment with each of the inhibitors on 24h of 3-NP ex-
posure in Q111 cells. Although there was a reduction in cell
proliferation and a small increase in cell death, each of the
does not affect viability or vulner-
ability to 3-NP toxicity in striatal
cells. (A) Basal cell counts 36h fol-
lowing an initial plating of 500
cells=well in a 96-well plate were
unchanged by HIF-1a knockdown.
(B) Cell viability was determined in
siGFP and siHIF following 3-NP
(10mM) exposure for 24h. While
Q111 cells were more sensitive to
3-NP than Q7 cells, there was no
effect of HIF-1a knockdown on vul-
nerability to 3-NP. Data represent
mean?SEM of 3–5 independent
experiments.þp<0.05 compared to
Knockdown of HIF-1a
no 3-NP, *p<0.05 compared to no 3-NP and Q7=3-NP, two-way ANOVA with Bonferroni post-test.
prolyl 4-hydroxylase inhibitors enhance the activity of a
hypoxia-response element-driven reporter in Q7 and Q111
striatal cells. Treatment with 50mM desferrioxamine (DFO),
50mM 3, 4-dihydroxybenzoic acid (DHB), or 2.5mM ciclo-
pirox (CPO) for 6h increases the activity of a HRE-driven
firefly luciferase reporter in Q7 and Q111 striatal cells. Data
represent mean?SEM of 3–12 independent experiments.
*p<0.05 comppared to corresponding genotype untreated
control,þp<0.05 compared to untreated control and corre-
sponding Q7 cells, two-way ANOVA with Bonferroni post-
4.Structurally diverse, lowmolecularweight
438NIATSETSKAYA ET AL.
inhibitors provided a significant level of neuroprotection as
measured by the LIVE=DEAD assay (Fig. 5), which was cor-
roborated by qualitative, visual observations using phase
contrast microscopy (Supplemental
To establish whether HIF PHD inhibitors act upstream or
downstream of SDH inhibition, we examined the effects of
HIF PHD inhibitors on the suppression of SDH activity by
3-NP. We found small but significant reductions in basal
SDH activity in Q111 cells compared to Q7 cells and observed
that SDH activity is rapidly depleted after treatment with 3-
NP in both cell types (Fig. 6A). This reduction in SDH activ-
ity was not associated with inhibition of the HIF PHDs as
monitored by HRE driven luciferase activity as others have
reported (not shown). Interestingly, exogenous addition of
HIF PHD inhibitors led to a reduction in steady state SDH
activity in Q7 cells, while SDH activity remained completely
lost following 3-NP inhibition in Q111 cells treated with DFO,
DHB, or CPO (Fig. 6B), suggesting that the observed protec-
tion was not simply through preserving SDH activity.
To examine whether HIF-1a is necessary for protection by
HIF PHD inhibitors in striatal Q111 cells, we again utilized a
retrovirus expressing an siRNA to GFP or HIF-1a. As ex-
pected, siHIF reduced the activation of a luciferase reporter
driven bythe hypoxia response element inQ111cells whereas
a retrovirus expressing siGFP had no effect (Fig. 7A). How-
ever, siHIF did not affect the protection induced by HIF PHD
inhibitors (Fig. 7B). Together these studies demonstrate that
pretreated with the indicated PHD inhibitors for 6h and then exposed to 3-NP (10mM) for an additional 24h, after which cell
survival was assessed by LIVE=DEAD assay. (A–E) Images of Q111 cells following viability assay: green fluorescence (calcein)
indicates live cells while red fluorescence (ethidium homodimer) labels the nuclei of dead cells. (A) untreated control cells, (B)
3-NP (10mM), (C) 50mM desferrioxamine (DFO)þ3-NP, (D) 50mM 3,4-dihydroxybenzoic acid (DHB)þ3-NP, (E) 2.5mM
ciclopirox (CPO)þ3-NP, (F) quantitation of cell survival following pretreatment and 3-NP exposure. Data represent
mean?SEM of four independent experiments. All cells treated with 3-NP were significantly different than no 3-NP treat-
ment, *p<0.01 compared to control Q111 cells treated with 3-NP only,þp<0.001 compared to corresponding Q7 cells, two-
way ANOVA with Bonferroni post-test.
Prolyl 4-hydroxylase inhibitors abrogate 3-NP toxicity in Q111 striatal cells. Q7 and Q111 Striatal cells were
HIF PHD INHIBITORS PROTECT AGAINST 3-NP TOXICITY439
HIF PHD inhibitors abrogate 3-NP toxicity independent of
HIF-1a and downstream of SDH inhibition.
It is formally possible that the protection we observe in
the expression of a temperature sensitive T-antigen. To ex-
clude this possibility, we examined the ability of DFO, DHB,
and CPO to prevent 3-NP toxicity in postmitotic cortical
neurons (1–2 days in vitro) cultured from E17 rat embryos. As
expected, all three compounds showed neuroprotection as
8A); and unlike dividing striatal cells, no reduction in cell
number was evident in primary cortical neurons (Fig. 8A).
Further, these protective concentrations of HIF PHD inhibi-
tors induced VEGF expression, a HIF-1a target gene, in these
neurons (Fig. 8B). These data also suggest that HIF PHD in-
hibitors may be generally protective to neurons under con-
ditions of metabolic stress independent of mhtt expression.
Recent studies have suggested a connection between HIF-1a
and PGC-1a, and given the role of PGC-1a as a target for mhtt
toxicity, we determined whether HIF PHD inhibitors induce
PGC-1a message. Indeed, while DFO and DHB induced the
expected increases in VEGF message in wild-type cortical
neurons, there was no effect on PGC-1a message in these cells
(Fig. 8B), suggesting that HIF PHD inhibitors act indepen-
dently of PGC-1a rescue to prevent 3-NP toxicity.
Despite its prominent role in metabolic adaptation, no
studies have formally evaluated the role of hypoxia inducible
factors and their upstream regulators, the HIF PHDs, in
compensating for mitochondrial dysfunction in Huntington’s
disease and other neurological conditions. Here we demon-
strate that three structurally diverse, low molecular weight
inhibitors of the HIF PHDs prevent toxicity from 3-NP in
striatal neuronal cells that accurately express a mutant hun-
tingtin allele with 111 polyQ repeats that leads to abnormal-
ities in ATP=ADP ratios (Fig. 9) (20). DFO and CPO bind iron
with high affinity and are predicted to inhibit HIF PHDs by
binding to iron in the active sites of these 2-oxoglutarate and
oxygen-dependent dioxgenases (9, 34). By contrast, DHB ap-
pears to inhibit the HIF PHDs, stabilize HIF-1a, and drive
HIF-dependent expression by iron-independent mechanisms
(18). Indeed, prior studies from our laboratory showed that
20–50mM DHB (unlike DFO and CPO) does not affect intra-
cellular calcein fluorescence, IRP binding in an RNA gel shift
assay, or total iron levels as measured by inductively coupled
mass spectrometry, in neurons (31). Thus, HIF PHDs, and not
iron, are the common established target for these compounds
(Fig. 9). Metal chelators have been shown to prolong survival
and diminish pathology in an R6=2 model of HD but the
HIF-1a inhibits PHD inhibitor-
mediated induction of HRE-
reporter activity but does not
abrogate their protection against
3-NP toxicity. HRE-luciferase ac-
tivity was measured in Q111 cells
following treatment with PHD
inhibitors for 6h. (A) PHD in-
hibitors do not induce HRE-
luciferase activity in Q111 striatal
cells expressing siHIF, indicating
significant attenuation of the HIF
confer similar protection to Q111
compared to cells expressing siGFP. Data represent mean?SEM of three independent experiments. *p<0.05 compared to
corresponding untreated control cells, two-way ANOVA with Bonferroni post-test. In (B), all PHD inhibitor-treated cells are
significantly different from corresponding 3-NP only cells, two-way ANOVA with Bonferoni post-test. Student’s t-test
p values are shown comparing survival between siGFP and siHIF cells.
PHD inhibitors. Striatal cells were treated with
times, lysed, and assayed for SDH activity. (A)
Q111 cells exhibited lower basal SDH activity,
whichwasrapidly abolishedby3-NP treatment
in both cell types. SDH activity was undetect-
able in Q111 cells by 15min of exposure. (B)
Q111 cells were co-treated with 3-NP and PHD
inhibitors for 15min and then assayed for SDH
activity. While PHD inhibitors decreased basal
SDH activity, they did not prevent the complete
loss of SDH activity caused by 3-NP exposure.
Data represent mean?SEM of three indepen-
dent experiments. *p<0.01 compared to corre-
sponding untreated control cells,
compared to corresponding Q7 cells, two-way
ANOVA with Bonferroni post-test.
SDH activity following 3-NP and
440NIATSETSKAYA ET AL.
mechanism of protection in this model remains obscure (25).
Our data argue that metal chelators may have benefit in HD
by inhibiting one or all of the HIF PHDs (three HIF PHD
isoforms are expressed in brain) (36). Of course, our data do
not allow us to exclude the possibility that HIF-independent,
2-oxoglutarate-dependent dioxygenases may be relevant for
protection in HD or in response to mitochondrial toxins. In-
deed, the fact that HIF-1a is not required for protection by
DFO, DHB, or CPO is congruent with this possibility. Future
studies using siRNAs to each isoform or conditional deletion
inhibition in protection from mitochondrial dysfunction in
HD and other disorders.
An interesting and surprising finding is the dramatic ele-
vation in HIF-1a message in Q111 versus Q7 cells. This change
in message does not appear to be associated with an increase
in HIF-1a protein (Fig. 2B) or HIF-dependent reporter ex-
pression (data not shown), although we do see associated
elevation of two known HIF-dependent genes, VEGF and p21
(Fig.1). Most attention has focused on HIF-1a protein stability
or increased message translation as a mechanism for en-
hancing nuclear HIF activity. Recent studies have shown that
the versatile, nuclear threonine=serine kinase homeodomain
interacting protein kinase 2 (HIPK2) can negatively regulate
transcription of HIF-1a (23, 24). These studies raise the inter-
esting possibility that mutant huntingtin negatively regulates
HIPK2. Under basal conditions, HIPK2 co-represses a host of
genes including HIF-1a; thus loss of HIPK2 activity related to
mutant huntingtin could lead to de-repression of HIF-1a,
leading to the observed changes in message in Q111 cells.
Despite the dramatic increase in HIF-1a in Q111 cells, HIF-1a
does not appear to be necessary for steady-state viability or
HIF PHD inhibitor-induced neuroprotection. Both HIF-1a
and HIF-2a are hypoxia-responsive HIFa subunit isoforms
encoded bydistinct gene lociand showsome overlapin tissue
localization and target gene expression, though both subunits
may also have distinct transcriptional targets (27). Notably,
the expression of glycolysis-related genes seems to be regu-
lated primarily by HIF-1a (11). Thus along with nearly un-
detectable HIF-2a levels, it is unlikely that the dissociation of
silencing HIF-1a and protection is related to redundancy
provided by HIF-2a (Fig. 2A and C).
Another unexpected finding of the current study was our
reporter activity in response to 3-NP treatment. Prior studies
have shown that pharmacological or molecular suppression
buildup of succinate and competitive inhibition of HIF PHDs
(29). These events would result in HIF stabilization and in-
duction of genes that compensate for hypoxia. The model has
4-hydroxylases. A series of structurally diverse proyl-
4-hydroxylase inhibitors target distinct cofactors necessary
for enzyme function. These compounds are known to chelate
iron (DFO and CPO) or block 2-oxoglutarate binding (DHB)
in the active site of the enzyme and are protective under
conditions of oxidative and metabolic stress. Further, the
neuroprotective properties of these compounds may be in-
dependent of their ability to stabilize HIF-1a. (For inter-
pretation of the references to color in this figure legend, the
reader is referred to the web version of this article at
Structurally diverse compounds inhibit proyl-
gate 3-NP toxicity and do not
induce PGC-1a in embryonic
cortical neuronal cultures. (A)
Cortical neuron cultures from E17
rats were co-treated with PHD
inhibitors and=or 3-NP (10mM)
for 48h and viability was assessed
(100mM), DHB (20mM), and CPO
(0.5mM) protected primary neu-
rons against 3-NP toxicity. (B)
Cortical neuron cultures were
treated with PHD inhibitors for
24h and assayed the expression
of PGC-1a and VEGF mRNA by
were unchanged by PHD inhibi-
tors while VEGF, a HIF target
gene, was significantly induced with these agents. Data represent mean?SEM of 3–6 independent experiments. *P<0.05
compared to corresponding control cells treated with 3-NP only (A) or untreated control cells (B),þP<0.05 compared to
corresponding untreated control cells, two-way ANOVA with Bonferroni post-test.
PHD inhibitors abro-
HIF PHD INHIBITORS PROTECT AGAINST 3-NP TOXICITY 441
been confirmed for tumor cells, but little data support the
notion that SDH inhibition leads to stabilization of HIF or
activation of HIF-dependent gene expression in neurons ei-
ther via succinate or alternatively, reactive oxygen species.
Future studies that utilize siRNAs to SDH subunits in post-
mitotic neurons should address these open questions. At this
time we cannot exclude the possibility that off-target effects of
3-NP mask the succinate-induced inhibition of HIF PHDs and
activation of the HIF pathway.
Our findings set the table for a study of PHD inhibitors in
fly or rodent models of HD to determine whether these drugs
will also be effective in vivo. In our laboratory, HIF PHD in-
hibition in vitro is a good predictor of neuroprotection in an-
imal models of stroke (26, 31), suggesting that these drugs
may be beneficial for many neurological disorders that in-
volve metabolic stress, including stroke and Alzheimer’s
disease. However, much work is yet to be done to fully un-
derstand the target and mechanism of action of these drugs.
Additionally, our findings suggest that at the very least, HIF
PHD inhibition is a good screen to identify agents that could
prevent mitochondrial dysfunction and oxidative stress in
This workwassupported bya National Institutes ofHealth
PO1 AG014930 grant and New York State Center of Research
Excellence Grant to RRR (CO19772).
Author Disclosure Statement
No completing financial interests exist.
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Address correspondence to:
Rajiv R. Ratan, M.D., Ph.D.
Burke–Cornell Medical Research Institute
785 Mamaroneck Avenue
White Plains, New York 10605
Date of first submission to ARS Central, July 30, 2009; date of
acceptance, August 6, 2009.
FBS¼fetal bovine serum
GFP¼green fluorescent protein
HIF¼hypoxia inducible factor
kinase homeodomain interacting
protein kinase 2
HRE¼hypoxia responsive element
htt¼wild-type huntingtin protein
mhtt¼mutant huntingtin protein
PGC-1a¼PPARg coactivator 1a
Q7¼STHdhQ7striatal cell line
Q111¼STHdhQ111striatal cell line
siRNA¼short interfering RNA
VEGF¼vascular endothelial growth factor
HIF PHD INHIBITORS PROTECT AGAINST 3-NP TOXICITY443
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