It has been documented that mitochondrial dysfunction, inflammation, iron dysregulation and apoptosis have a crucial role in the pathogenesis of several neurodegenerative disorders (Zecca et al. 2004; Lin and Beal 2006; Moreira et al. 2006a, 2007, 2009; Zipp and Aktas 2006). As mentioned, HIF-1 signaling pathway is involved in several physiological responses, HIF-1 activity being correlated with the modulation of various metabolic pathways. Therefore, the activation of HIF-1 signal transduction pathway and consequent expression of its protective target genes could represent an efficient neuroprotective strategy to mitigate or prevent neurodegenerative disorders. The next section is devoted to explore the potential benefits of targeting HIF signaling pathway in the context of some neurodegenerative disorders.
Cerebral ischemic stroke
Stroke is the third leading cause of death and severe long-term disability in adults. According to the World Health Organization, 15 million people suffer stroke worldwide each year (http://www.strokecenter.org/patients/stats.htm). In cerebral ischemic stroke, the most common type of stroke, the reduction in blood supply to brain tissue results in a decrease in the availability of glucose and oxygen, which are necessary for normal brain function. Additionally, cerebral ischemic stroke triggers multiple biochemical and molecular mechanisms that impairs the neurologic functions through the breakdown of cellular integrity mediated by excitotoxicity, ionic imbalance, oxidative/nitrative stress, and DNA damage (Lo et al. 2003; Rami et al. 2008).
Accumulating evidence indicates that the induction of HIF-1 provides protection against cerebral ischemic damage. Indeed, studies using models of focal and global cerebral ischemia have shown that HIF-1α protein accumulation triggers the expression of target genes involved in various adaptive responses (Semenza 2000b; Sharp and Bernaudin 2004). Moreover, previous studies also showed that the pre-treatment or post-treatment with hypoxia-mimetic agents (DFO or CoCl2) reduced cell loss in models of focal or global ischemia in vitro and in vivo (Bergeron et al. 2000; Demougeot et al. 2004a,b; Hamrick et al. 2005). Bergeron et al. (1999) reported that focal ischemia induces HIF-1 in brain. Moreover, increased expression of HIF-1 target genes as a result of HIF-1 activation by hypoxia may contribute to tissue viability in the hypoxic/ischemic penumbra by increasing glucose transport and glycolysis (Bergeron et al. 1999). Accordingly, Sharp et al. (2001) observed that after focal cerebral ischemia, mRNAs encoding HIF-1α, GLUT-1, and several glycolytic enzymes, including lactate dehydrogenase, were up-regulated in the areas around the infarction. Moreover, HIF and its target genes were induced by 7.5 h after the onset of ischemia and increased further at 19 and 24 h. Further, preconditioning with the HIF-1 inducers CoCl2 or DFO, 24 h before the stroke, decreased infarction by 75% and 56%, respectively, compared with vehicle-injected, littermate controls. Thus, HIF-1 activation could contribute to protective brain preconditioning (Sharp et al. 2001). Baranova et al. (2007) observed that neuron-specific knockdown of HIF-1 aggravated brain damage after middle cerebral artery occlusion (MCAo) and reduced survival rate of MCAo mice. Moreover, Baranova et al. (2007) demonstrated that the pharmacologic HIF-1 activators 3,4-dihydroxybenzoic acid, deferoxamine (DFO), and 2,2-dipyridyl significantly reduced ischemic injury in wild-type mice, whereas the effectiveness of these compounds was significantly attenuated in mice with neuron-specific HIF-1α knockdown. Consistent with this report, Siddiq et al. (2005) showed that hypoxia or hypoxia-mimetic agents confer neuroprotection against oxidative death in vitro and ischemic injury in vivo through the inhibition of PHDs, and consequent HIF-1α stabilization and up-regulation of HIF-1-dependent target genes. In vivo models of ischemic stroke showed that systemic DFO reduces stroke damage when administered both prior to and after MCAo (Palmer et al. 1994; Prass et al. 2002; Mu et al. 2005; Freret et al. 2006). A recent study demonstrated that DFO induced tolerance against focal cerebral ischemia in rats, and exerted protective effect on OGD cultured cortical neurons through the induction of HIF-1α and EPO both in vivo and in vitro (Li et al. 2008). Recent findings showed that intranasal administration of DFO, a non-invasive method, prevents and treats stroke damage after MCAo in rats (Hanson et al. 2009). Zhou et al. (2008) evaluated the effect of 2-methoxyestradiol (2ME2), a natural metabolite of estrogen that inhibits HIF-1α, in global cerebral ischemia in rats. As expected, the authors observed that 2ME2 treatment significantly reduced HIF-1 levels. Moreover, it was observed that 2ME2 treatment lower neuronal cell survival, which suggests that 2ME2-induced HIF-1 suppression aggravates outcomes after global ischemia in rats (Zhou et al. 2008).
Several mechanisms have been proposed to explain HIF-1-mediated cytoprotective effects. HIF-1-induced EPO expression seems to be a crucial event to protect cells against hypoxic/ischemic injury. In vivo evidence showed that directly EPO administration into the brain reduces neurologic dysfunction in rodent models of stroke (Sadamoto et al. 1998; Sakanaka et al. 1998; Bernaudin et al. 1999; Sirén et al. 2001). Furthermore, neutralization of endogenous brain EPO potentiates ischemic brain injury, confirming a pivotal role for the endogenous EPO system in neuronal survival after ischemia (Sakanaka et al. 1998). A clinical trial conducted in 13 patients that received recombinant human EPO intravenously once daily for the first 3 days after stroke showed a reduction in the infarct size when compared with controls, this effect being associated with an improvement in clinical outcome (Ehrenreich et al. 2002). In addition, VEGF expression, another downstream gene of HIF-1, also counteracts detrimental ischemic injuries. Marti et al. (2000) demonstrated that the VEGF/VEGF receptor system, induced by hypoxia, leads to the growth of new vessels after cerebral ischemia, thereby minimizing the detrimental effects of cerebral ischemia (Marti et al. 2000). Accordingly, Jin et al. (2000) reported that VEGF also protected primary cultures of rat cerebral cortical neurons from hypoxia and glucose deprivation, being proposed that beyond its angiogenic properties, VEGF exert a direct neuroprotective effect in hypoxic-ischemic injury. There is also evidence showing that HIF-1 exerts anti-apoptotic effects through inhibition of cytochrome c release, caspase activation and poly (ADP-ribose) polymerase (PARP) cleavage, and the ability to suppress p53 activation (Li et al. 2004; Piret et al. 2004; Sasabe et al. 2005).
Additionally, brain-derived neurotrophic factor (BDNF), a member of the nerve growth factor family that promotes the survival and differentiation of neuronal tissues, has been shown to exert neuroprotective effects in response to global and focal ischemia (Schabitz et al. 1997, 2000; Larsson et al. 1999). Shi et al. (2009) constructed an adenoviral vector using five copies of HRE found in the VEGF gene to regulate the expression of BDNF gene in a hypoxic condition. Shi et al. (2009) observed that adenovirus-mediated BDNF expression regulated by HRE protects brain against ischemia-induced injury. These results support the idea that HIF-1α could act as a sensor to express therapeutic genes in a cerebral ischemic stroke context.
Recently, Guo et al. (2009) observed that HIF-1 protect cells from ischemic injury by maintaining the cellular redox status. Guo et al. (2009) found that HIF-1α knockdown in SH-SY5Y cells down-regulates key enzymes in glucose metabolism that are critical in the production of important reducing agents, decreases GSH/GSSG ratio, increases ROS levels, and induces cell death under hypoxic or OGD conditions. Additionally, HIF-1 maintains cellular redox state and prevents ischemic injury mediated by ROS production, possibly through the up-regulation of the expression of key enzymes such as GLUT-1, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase (Guo et al. 2009).
The IVS9-675C>A polymorphism of the HIF-1α gene was analyzed in patients with acute ischemic stroke and in a control group (Tupitsyna et al. 2006). Tupitsyna et al. (2006) found that this polymorphism potentiates the risk of stroke, which reinforce the idea that HIF-1 could be an effective therapeutic target for the development of pharmacological approaches to counteract stroke.
Alzheimer’s disease is a progressive and fatal neurodegenerative disorder characterized by neuronal loss, cognitive dysfunction, and dementia. The pathological hallmarks of AD are the formation of extracellular senile plaques, mainly composed of β-amyloid (Aβ) peptides and intracellular neurofibrillary tangles containing hyperphosphorylated tau protein (Selkoe 2001; Moreira et al. 2006b). Aβ peptides are generated by successive proteolysis of amyloid-β precursor protein (AβPP), a large transmembrane glycoprotein that is initially cleaved by the β-site amyloid-β precursor protein (AβPP)-cleaving enzyme 1 and subsequently by γ-secretase in the transmembrane domain (Greenfield et al. 2000).
Glucose is the main source of energy required for normal brain function. As brain neurons are incapable to synthesize or store glucose, they are dependent on glucose transport across the blood–brain barrier, which is mediated by GLUTs (Scheepers et al. 2004). Accumulating evidence shows that glucose uptake and metabolism are impaired in the brain of AD patients. Moreover, this impairment seems to be a cause, rather than a consequence, of neurodegeneration in AD (Hoyer 2004). It has been demonstrated that impaired glucose uptake and metabolism leads to hyperphosphorylation of tau protein through the down-regulation of tau O-GlcNAcylation (Liu et al. 2004; Li et al. 2006). Recently, Liu et al. (2008) reported that GLUT-1 and GLUT-3, the two major brain GLUTs responsible for glucose uptake into neurons, were significantly decreased in AD brain, which contributes to abnormal hyperphosphorylation of tau and AD neurofibrillary degeneration. In addition, Liu et al. (2008) observed that HIF-1α protein levels were decreased. These results suggest that the decrease in GLUT-1 and GLUT-3 levels may result from the down-regulation of HIF-1α in AD brain. Recent findings also revealed that the induction of HIF-1 reduces astrocyte activation by Aβ peptide (Schubert et al. 2009) (Fig. 3). It was shown that Aβ-dependent astrocyte activation leads to a long-term decrease in HIF-1α expression and a reduction in the rate of glycolysis. Moreover, glial activation and the glycolytic changes are reversed by the maintenance of HIF-1α levels under conditions that prevent the proteasomal degradation of HIF-1α (Schubert et al. 2009).
Figure 3. Protective role of hypoxia-inducible factor 1 (HIF-1) in Alzheimer’s disease (AD). Experimental evidence show that the induction of HIF-1α by mimosine, desferoxamine and clioquinol reduces glial activation promoted by β-amyloid (Aβ) peptides inhibits Aβ deposition and improves brain glucose metabolism favoring neuronal survival.
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Soucek et al. (2003) investigated the relation between HIF-1, Aβ, and glucose metabolism. The authors demonstrated that clonal nerve cell lines and primary cortical neurons that are resistant to Aβ toxicity have an enhanced flux of glucose through both the glycolytic pathway and the hexose monophosphate shunt (Fig. 3). Interestingly, the Aβ-induced changes in glucose metabolism are mediated by HIF-1 activation (Soucek et al. 2003). Indeed, preconditioning with low-dose Aβ (2 μM) or 50–100 μM mimosine, another HIF-1α inducer, rendered the neuronal cells resistant to toxicity mediated by subsequent lethal dose of Aβ (20 μM) through HIF-1α induction. In addition, activation of HIF-1 by the over-expression of a non-degradable HIF-1α prevented Aβ1-42-induced neurotoxicity, suggesting a neuroprotective role of HIF-1 in AD (Soucek et al. 2003).
Disruption of the metabolism of iron has been also associated with the pathogenesis of AD. Indeed, it has been shown an accumulation of iron in senile plaques and an altered distribution of iron transport and storage proteins in AD brains (Bishop et al. 2002). A previous in vitro study demonstrated that the pre-treatment of Aβ with the HIF-1 inducer and iron chelator DFO, attenuates neuronal toxicity, whereas its reconstitution with excess free iron restores cytotoxicity (Rottkamp et al. 2001). Furthermore, a single clinical trial conducted in AD patients subjected to intramuscular injections of DFO twice daily showed that this pharmacological activator of HIF-1 slowed cognitive decline by 50% in 2 years (Crapper McLachlan et al. 1991), suggesting that the administration of DFO may have some clinical relevance in preventing the progression of AD-associated dementia.
Copper (Cu) and Zinc (Zn) ions have also been shown to accumulate in the brains of AD patients (Atwood et al. 2000; Sayre et al. 2005; Moreira et al. 2006c). An in vivo study using APP2576 transgenic mice, an animal model of AD, revealed that oral treatment with CQ, a Cu/Zn chelator with the ability to induce HIF-1α, markedly inhibited Aβ deposition (49% decrease), in the absence of any marked neurotoxicity (Fig. 3) (Cherny et al. 2001). In a Phase II clinical trial, CQ slowed cognitive decline and decreased plasma Aβ levels in CQ-treated patients compared with those receiving the placebo (Ritchie et al. 2003).
Parkinson’s disease, the second most common neurodegenerative disorder, is characterized by a progressive and massive loss of midbrain dopaminergic (DA) neurons of the substantia nigra pars compacta (SNpc). Consequently, SNpc DA neuronal loss leads to the degeneration of neurons terminals and DA depletion in the striatum, which is required for normal motor function. One of the pathological hallmarks of PD and related synucleinopathies is the presence of intracellular inclusions called Lewy bodies, which are constituted by aggregates of the pre-synaptic soluble protein called α-synuclein (Cardoso et al. 2005). Additionally, iron levels in the SNpc have been reported to be elevated in PD patients (Sofic et al. 1988; Youdim et al. 1993). Accumulating evidence suggests that mitochondrial dysfunction, oxidative stress, neuroinflammation, excitotoxicity, and proteosomal dysfunction are also contributing factors for neuronal cell degeneration in PD (Beal 2003; Lin and Beal 2006; Nunomura et al. 2007).
In the early 1990s, Ben-Shachar et al. (1991) demonstrated that intracerebroventricular-injection of DFO protects against dopaminergic neurodegeneration induced by 6-hydroxydopamine in rats. Moreover, DFO pre-treatment prevents the manifestation of dopaminergic-related behavioral responses, such as spontaneous movements in a novel environment and rearing (Ben-Shachar et al. 1991). In addition, it was demonstrated that DFO also prevents neurotoxicity in the MPTP-mouse model of PD (Lan and Jiang 1997). Using this mouse model, Kaur et al. (2003) also demonstrated that animals treated for a period of 8 weeks with CQ exhibited a 30% decrease in iron levels in the substantia nigra and were protected against motor dysfunction compared with control animals. Moreover, CQ pre-treatment significantly attenuates the increase in oxidative stress markers and glutathione depletion induced by MPTP (Kaur et al. 2003).
A recent study demonstrated that 3,4-dihydroxybenzoate, a pharmacological inhibitor of PHDs, protects against in vitro and in vivo MPTP-induced neurotoxicity possible through the induction of HIF-1α (Lee et al. 2009). Indeed, Lee et al. (2009) observed that 3,4-dihydroxybenzoate treatment is capable to stabilize HIF-1α transcription factor, leading to the up-regulation of several proteins involved in iron efflux (ferroportin and heme oxygenase-1), DA synthesis (tyrosine hydroxylase) and mitochondrial integrity and bioenergetics (manganese superoxide dismutase, pyruvate dehydrogenase, and peroxisome proliferator-activated receptor-γ coactivator-1α), cell components that are compromised in PD. Therefore, these data reinforce the idea that the pharmacological induction of HIF-1α could have neuroprotective effects, in this specific case with a beneficial impact on DA synthesis, iron homeostasis, antioxidant defenses and mitochondrial dysfunction (Fig. 4).
Figure 4. Protective role of hypoxia-inducible factor 1 (HIF-1) in Parkinson’s disease (PD). Clioquinol, desferoxamine and 3,4-dihydroxybenzoate, pharmacological inhibitiors of prolyl hydroxylase enzymes (PHDs), promote neuroprotection through the induction of HIF-1α. The induction of HIF-1α improves dopamine (DA) synthesis, iron homeostasis and antioxidant defenses and prevents mitochondrial dysfunction. Additionally, HIF-1α induction ameliorates motor functions in mouse models of PD.
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Huntington’s disease, an autosomal dominant disease, is a severe movement disorder characterized by cognitive decline and early death. HD results from an abnormal expanded polyglutamine repeat in the huntingtin (htt) protein, which leads to the degeneration of the neurons of the striatum and cortex (Ross et al. 1997).
Mitochondrial dysfunction in neurons and glia cells appears to be intimately involved in the pathogenesis of HD. Panov et al. (2002) demonstrated that lymphoblast mitochondria from HD patients have a lower membrane potential and depolarize at lower calcium loads than mitochondria from controls. Moreover, Panov et al. (2002) observed that brain mitochondria from transgenic mice expressing full-length mutant htt also showed similar mitochondrial calcium defects, which preceded the onset of pathological or behavioral abnormalities by months. In addition, the brains of 12-week-old transgenic mouse model of HD (R6/2) demonstrated a significant reduction in aconitase and mitochondrial complex IV activities in the striatum and a decrease in complex IV activity in the cerebral cortex (Tabrizi et al. 2000). It has been shown that chronic administration of 3-nitropropionic acid, a mitochondrial toxin that irreversibly inhibits the succinate dehydrogenase, in rats produces selective striatal lesions that replicate many of the histological and neurochemical features of HD (Beal et al. 1993). It was demonstrated that the preconditioning of rat C6 astroglial cells with the HIF-1 inducers, CoCl2, mimosine, and DFO exerted substantial cytoprotective effects against metabolic insults induced by 3-nitropropionic acid exposure (Yang et al. 2005). Moreover, it was observed that application of cadmium chloride capable of neutralizing cobalt-induced HIF-1 activation, HIF-specific oligodeoxynucleotide decoy, and antisense phosphorothioate oligodeoxynucleotide against HIF-1α abolished the protective effect induced by CoCl2 preconditioning (Yang et al. 2005). These findings suggest that induction of HIF-1 and its downstream genes could mediate neuroprotective events in neurodegenerative disorders, such as HD, in which mitochondrial dysfunction plays a prominent role (Yang et al. 2005). Finally, Nguyen et al. (2005) demonstrated the beneficial effects of CQ treatment to reduce cell death and mutant protein accumulation using in vitro models of HD. Moreover, CQ treatment of transgenic HD mice R6/2 improved behavioral and pathologic phenotypes, decreased htt aggregates accumulation and striatal atrophy, improved rotarod performance, reduced weight loss, normalized blood glucose and insulin levels, and extended lifespan (Nguyen et al. 2005).