To begin to query the behavior of the HIF pathway in response to mitochondrial dysfunction in neurons, we compared RNA levels for HIF-1α 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 juvenile onset HD (111 repeats). These cells were generated from the HdhQ111
knock-in mice (STHdhQ111
, which bear full-length htt with an expanded polyglutamine (polyQ) tract of 111 CAG repeats; herein referred to as Q111) and control cells from the striatum of a knock-in mouse bearing a nonpathological polyQ expansion (STHdhQ7
, herein referred to as Q7) (8
). Interestingly, we found that HIF-1α mRNA levels and levels of HIF target genes are significantly increased in Q111 cells as compared to Q7 cells, raising the possibility that HIF is induced to compensate for metabolic compromise in HD (). 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 biological role of this change has not been previously evaluated.
FIG. 1. HIF-1α and HIF target gene mRNA are elevated in STHdhQ111 striatal cells. TaqMan real-time gene expression assays for HIF-1α, VEGF, and p21 show elevated basal expression of these genes in mutant huntingtin (Q111) cells compared to wild-type (more ...)
To explore whether the increase in HIF-1α allows Q111 cells to adapt to mitochondrial dysfunction, we utilized a well-characterized retrovirus that can transduce murine cells to express an siRNA to GFP or HIF-1α (1
). As expected, we found that siRNA to HIF-1α significantly reduced message levels for HIF-1α by >80% in both cell lines (). To examine whether reduction in HIF-1α message levels were associated with reductions in HIF-1α protein, we performed immunoblots from lysates of Q7 or Q111 lines treated with vehicle or the HIF PHD inhibitor desferrioxamine (DFO), which mimics hypoxia by stabilizing HIF-1α protein levels. As expected, in both Q7 and Q111 lines DFO induced HIF-1α protein, which was completely suppressed by the siRNA to HIF-1α but not the siRNA to GFP (). As other HIFα isoforms may be induced to compensate for the loss of HIF-1α, we assessed HIF-2α mRNA levels following HIF-1α knockdown. We were unable to detect HIF-2α message in either cell line by real-time RT-PCR (), though we were able to resolve a PCR product corresponding to HIF-2α by agarose gel electrophoresis, which was not changed by HIF-1α knockdown ().
FIG. 2. Stable silencing of HIF-1α in striatal cells with si-RNA against HIF-1α. Q7 and Q111 striatal cells were transduced with a retrovirus delivering hp/GFP (siGFP) or hp/HIF-1α (siHIF-1). (A) HIF-1α and HIF-2α expression (more ...)
We next examined the role of HIF-1α in regulating viability in response to the selective complex II inhibitor, 3-NP. We first confirmed prior studies from our laboratory and others that showed that Q111 cells are more sensitive to 3-NP as compared to Q7 cells () (28
). Despite these differences, we found that HIF-1α deletion had no effect on basal viability or sensitivity to 3-NP (10
) in control Q7 or disease associated Q111 cells (). Altogether, these findings suggest that HIF-1α is not necessary to maintain viability of striatal neurons in response to an expanded polyQ repeat in the huntingtin 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-1α in striatal neurons (1
FIG. 3. Knockdown of HIF-1α does not affect viability or vulnerability to 3-NP toxicity in striatal cells. (A) Basal cell counts 36h following an initial plating of 500 cells/well in a 96-well plate were unchanged by HIF-1α knockdown. (more ...)
To investigate whether HIF activation in Q111 cells is sufficient 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-1α protein. Consistent with , 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-1α protein (Supplemental Fig. 2
; see www.liebertonline.com/ars
) and drive the expression of luciferase reporter under the control of the hypoxia response element from the 5’ noncoding region of the enolase gene (). We then examined the effect of 6
h pretreatment with each of the inhibitors on 24
h of 3-NP exposure in Q111 cells. Although there was a reduction in cell proliferation and a small increase in cell death, each of the inhibitors provided a significant level of neuroprotection as measured by the LIVE/DEAD assay (), which was corroborated by qualitative, visual observations using phase contrast microscopy (Supplemental Fig. 3
; see www.liebertonline.com/ars
FIG. 4. Structurally diverse, low molecular weight prolyl 4-hydroxylase inhibitors enhance the activity of a hypoxia-response element-driven reporter in Q7 and Q111 striatal cells. Treatment with 50μM desferrioxamine (DFO), 50μ (more ...)
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 (). This reduction in SDH activity 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 (), suggesting that the observed protection was not simply through preserving SDH activity.
FIG. 6. SDH activity following 3-NP and PHD inhibitors. Striatal cells were treated with 3-NP or 3-NP+PHD inhibitors for the indicated times, lysed, and assayed for SDH activity. (A) Q111 cells exhibited lower basal SDH activity, which was rapidly (more ...)
To examine whether HIF-1α is necessary for protection by HIF PHD inhibitors in striatal Q111 cells, we again utilized a retrovirus expressing an siRNA to GFP or HIF-1α. As expected, siHIF reduced the activation of a luciferase reporter driven by the hypoxia response element in Q111 cells whereas a retrovirus expressing siGFP had no effect (). However, siHIF did not affect the protection induced by HIF PHD inhibitors (). Together these studies demonstrate that HIF PHD inhibitors abrogate 3-NP toxicity independent of HIF-1α and downstream of SDH inhibition.
FIG. 7. Knockdown of HIF-1α inhibits PHD inhibitor-mediated induction of HRE-reporter activity but does not abrogate their protection against 3-NP toxicity. HRE-luciferase activity was measured in Q111 cells following treatment with PHD inhibitors for (more ...)
It is formally possible that the protection we observe in Q111 cells is facilitated by the immortalization of these cells by the expression of a temperature sensitive T-antigen. To exclude 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 measured by calcein uptake and automated cell counting (); and unlike dividing striatal cells, no reduction in cell number was evident in primary cortical neurons (). Further, these protective concentrations of HIF PHD inhibitors induced VEGF expression, a HIF-1α target gene, in these neurons (). These data also suggest that HIF PHD inhibitors may be generally protective to neurons under conditions of metabolic stress independent of mhtt expression. Recent studies have suggested a connection between HIF-1α and PGC-1α, and given the role of PGC-1α as a target for mhtt toxicity, we determined whether HIF PHD inhibitors induce PGC-1α message. Indeed, while DFO and DHB induced the expected increases in VEGF message in wild-type cortical neurons, there was no effect on PGC-1α message in these cells (), suggesting that HIF PHD inhibitors act independently of PGC-1α rescue to prevent 3-NP toxicity.
FIG. 8. PHD inhibitors abrogate 3-NP toxicity and do not induce PGC-1α 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 (more ...)