Chronic hypoxia, type 2 diabetes and Alzheimer's disease: a ...

Chronic hypoxia, type 2 diabetes and Alzheimer’s disease: a dangerous triad Cristina Carvalho1,2, Renato X. Santos1,2, Susana Cardoso1,2, Sónia C. Correia1,2, Paula I. Moreira1,3 1

Center for Neuroscience and Cell Biology, University of Coimbra; 2Department of Life

Sciences – Faculty of Sciences and Technology and 3Institute of Physiology – Faculty of Medicine, University of Coimbra, Coimbra, Portugal

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Abstract Type 2 diabetes is a metabolic disorder characterized by high levels of blood glucose, insulin resistance and hyperinsulinemia. These metabolic alterations may cause vascular changes resulting in a reduction of oxygen delivery to cells, a phenomenon called hypoxia. Hypoxia could be a double-sided coin, since it is able to activate pathways controlling cell survival or death. It was showed that short periods of hypoxia may be protective however chronic hypoxia activates pro-death pathways. The brain is a highly metabolic active organ requiring a continuous supply of glucose and oxygen. Because the brain consumes 20% of total oxygen consumed by the body, it is very susceptible to changes in oxygen levels. It has been postulated that chronic hypoxia is intimately associated with type 2 diabetes and potentiates neurodegenerative diseases, namely Alzheimer’s disease. This chapter aims to discuss the molecular mechanisms underlying hypoxia and its involvement in the pathologic processes of diabetes and Alzheimer’s disease.

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1- Brief Introduction The dependence of mammalian cells on a constant and abundant supply of ATP requires an adequate provision of oxygen (O2) (Vanderkooi et al., 1991). Although the brain represents 2% of the body weight, it receives 18% of the cardiac output and consumes 20% of the total O2 supply to work properly (Love et al., 1996; Faraci, 2011). This is due to its high metabolic demand and lack of appreciable O2 storage systems, such as myoglobin, that makes the brain very susceptible even to small disturbances in O2 supply (Gaehtgens and Marx, 1987; Owen and Sunram-Lea, 2011). Evidence shows that people suffering from T2DM are more vulnerable to Alzheimer’s disease (AD) compared with healthy individuals (Brands et al., 2005; Sims-Robinson et al., 2010; Baker et al., 2011). It has also been shown that 80% of AD patients exhibit either impairment in glucose tolerance or T2DM (Craft et al., 1998; Janson et al., 2004; Sims-Robinson et al., 2010). Although the causes of the neurodegenerative processes and their interactions are still under intense debate, the possible contribution of cerebrovascular alterations has been greatly supported by recent findings (Farkas and Luiten, 2001; Frontczak-Baniewicz et al., 2006). Hypoxia, a situation where O2 supply is lower than its demand, may occupy an initiating or intermediate position in the chain of events ending with cognitive failure. In this chapter we will debate discuss the mechanisms underlying hypoxia and its involvement in the pathologic processes of diabetes and Alzheimer’s.

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2- Diabetes-induced endothelial dysfunction T2DM is a metabolic disorder considered one of the main threats to human health (Zimmet et al., 2005). Almost 6% of world’s adult population suffer from this disorder that is expected to double until 2030 (Sicree et al., 2003; Wild et al., 2004). T2DM is characterized by peripheral insulin resistance and slow glucose absorption rate resulting in a state of hyperglycemia. This hyperglycemic state stimulates more insulin secretion in order to compensate the high levels of blood glucose ensuing in a secondary hyperinsulinemia (Libby and Plutzky, 2002). Hyperglycemia and insulin signalling alterations are major players in the exacerbation of oxidative stress that, among other things, affects brain endothelium. This idea is supported by animal studies showing that not only prolonged but also acute hyperglycemic episodes could be responsible for endothelial dysfunction (Taylor and Poston, 1994; Chirayath et al., 2007; Correia et al., 2009). Furthermore, a decrease in vascular density, reflecting an increase in endothelium dysfunction, was observed in diabetic animals (Bento and Pereira, 2011). Although several mechanisms could be involved in this phenomenon, the unifying theory is that oxidative stress is a central event. Glucose, the major fuel in the brain, is transported across the cell membranes by facilitated diffusion mediated by glucose transport proteins. However, when glucose exceeds the required levels, deleterious pathways are activated culminating in several metabolic and cellular abnormalities, which may compromise cellular adaptation to low O2 levels (Correia et al., 2008; 2009). In cells exposed to hyperglycemia more glucose is oxidized in the tricarboxylic acid (TCA) cycle generating high levels of electron donors, nicotinamide adenine dinucleotide reduced form (NADH) and succinate. These electron donors enhance the mitochondrial respiratory chain activity and, due to this 4

enhancement, the voltage gradient across the mitochondrial membrane increases until a critical threshold is reached exacerbating superoxide anion (O2-) production and activating the stress metabolic pathways (Brownlee, 2005) such as the polyol and protein kinase C (PKC) pathways and advanced glycation end products (AGEs) formation. The activation of the polyol pathway leads to concomitant decreases in reduced nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione (GSH) (Brownlee, 2001) increasing the sensitivity of cells to oxidative stress. Furthermore, high levels of glucose inside the cells also increase the synthesis of diacylglycerol (DAG), which is a critical activating co-factor of the classic isoform of protein kinase C (PKC) (DeRubertis and Craven, 1994; Xia et al., 1994; Feener et al., 1996; Koya et al., 1997). When PKC is activated, it exerts a variety of effects on gene expression decreasing the levels of endothelial nitric oxide synthase (eNOS) and increasing the levels of the vasoconstrictor endothelin 1 (ET1) and transforming growth factor-α (TGF-α). These alterations have several consequences including blood flow anomalies, capillary occlusion and increased vascular permeability (Kuboki et al., 2000). Under a hyperglycemic state there is also the formation of AGEs, which has also been implicated in the pathogenesis of the major microvascular complications of diabetes mellitus (Basta et al., 2004; Cooper, 2004; Stitt et al., 2004). Endothelial cells are highly susceptible to oxidative damage, which compromises their activity, and it usually precedes the development of morphological alterations during the progression of diabetes (Potenza et al., 2009). These alterations lead to a decrease in blood flow with a concomitant decrease in O2 delivery to the cells of different organs, especially brain the brain, reaching a state of hypoxia.

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3- Alzheimer’s Disease and Type 2 diabetes, is there a relationship? AD is a progressive neurodegenerative disease characterized by the existence of aggregates of intraneuronal hyperphosphorylated tau and extracellular deposits of amyloid (Aβ) protein (Shwartz et al., 2010) leading to a progressive cognitive dysfunction and inability to perform daily activities (Park et al., 2011). Evidence from the literature shows that older people with T2DM have a higher risk of cognitive dysfunction or dementia (Stewart et al., 1999; Park et al., 2011) especially AD (Li and Hölscher 2007; Sims-Robinson et al., 2010). In fact, studies in spontaneous animal models of T2DM showed the presence of pronounced neurodegenerative alterations, consisting in neuronal loss, gliosis, synaptic loss and high levels of hyperphosphorylated tau protein (Park et al., 2011). Although the impact of diabetes in central nervous system is not fully understood, it is known that depends on the age of onset, degree of glycemic control and duration of diabetes (Sims-Robinson et al., 2010). There are several potential mechanisms linking T2DM and AD, such as insulin resistance, mitochondrial dysfunction, oxidative stress, formation of AGE’s and toxicity of glucose to neurons through osmotic insults (Umegaki 2010; Santos et al., 2010; Correia et al., 2011). However, less attention was given to the role of brain microvascular alterations on the development and progression of the disease. As described before, glucose toxicity, AGE’s and oxidative stress could induce endothelium dysfunction and, subsequently, a state of hypoxia, rendering the microenvironment more susceptible to AD pathological changes (Schwartz et al., 2010). The next section is devoted to discuss cellular and molecular aspects of hypoxia and its involvement in AD pathogenesis.

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4- Hypoxia: the double sided coin When endothelium is compromised, there is a decrease in O2 availability, which is crucial for the homeostasis of several cellular processes. Under hypoxic conditions most biological systems elaborate a variety of mechanisms for O2 sensing and homeostasis maintenance (Low et al., 1993; Anju et al., 2011). Cells present a biological example of multivalent responses to the same stimulus. When a drop in O2 levels occurs, hypoxiainducible factor-1 (HIF-1), the key transcriptional activator involved in cellular adaptation to hypoxic stress, is recruited (Fig. 1). HIF-1 is composed by two subunits, α and β, which are tightly regulated by a family of proline and asparagine hydroxylases that utilize dioxygen, ferrous iron (Fe2+) and 2-oxoglutarate to catalyse the hydroxylation of specific residues on HIF-1α subunits (Mazure et al., 2010). During hypoxia, this repression is removed and HIF-1 pathway is rapidly activated (Taylor, 2008). The activation of this pathway starts with the dimerization of HIF-1α and HIF-1β subunits. This dimer then interacts with the hypoxia response element (HRE) present in the DNA through the basic helix-loop-helix and PER-ARNT-SIM domains localized in the N-terminal of both HIF-1α and HIF-1β subunits (Mazure et al., 2010). HIF-1 works as a master transcriptional regulator of hypoxia-dependent gene expression and its transcriptional activation is a stress response developed through evolution to allow cells to avoid a bioenergetic crisis in low O2 levels, stimulating a fundamental genomic reprogramming that confers protection and survival (Fig. 1) (Carvalho et al., 2009; Ara et al., 2011). However, whether and to what extent the HIF-1 system participates in the disease process remains to be elucidated. There is a wide array of physiological responses to hypoxia, ranging from mechanisms that increase cell survival to those inducing cell cycle arrest or even apoptosis (Acker 7

and Acker, 2005). Previous studies reported that, in an initial phase, HIF-1 activates a survival pathway that involve the expression of angiogenic and vasodilator genes like vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS) and erythropoietin (EPO) (Fig. 1) (Bernaudin et al., 1999; Brines et al., 2000; Jin et al., 2000; 2002; Aminova et al., 2005). Nevertheless, it was already demonstrated that sustained and prolonged activation of the HIF-1 pathway may lead to a transition from neuroprotective to cell death responses. The long-lasting activation includes responses with adverse effects on cell function by inducing cell-cycle-arrest-specific and proapoptotic proteins such as defective chorion-1 (DEC-1), Bcl2/adenovirus E1B 19kDinteracting protein-3 (BNIP3), its orthologue Nip3-like protein X (NIX) and cyclin G2 expression (Fig.1). Furthermore, direct stabilization through the pro-apoptotic protein p53 has been suggested in studies demonstrating physical and functional interactions between HIF-1α and p53 (Acker and Plate, 2002). Hypoxia also leads to an inhibition of oxidative phosphorylation system (Herlein et al., 2005) becoming glycolysis the only pathway to produce ATP, which is less effective in terms of energy production. Indeed, animal studies showed that during prolonged hypoxia, several vital organs suffer a decline in energy levels causing permanent membrane damage and loss of cellular ion homeostasis (Boutilier, 2004).

4.1- Mitochondria in HIF-1 regulation Mitochondria are subcellular organelles that are essential in the process of cellular energy production (Dykens, 1997; Green and Kroemer, 2004). However, mitochondria

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are also the major producers of reactive oxygen species (ROS) (Gutterman 2005; Sun et al., 2010). The metabolism of glucose through the TCA cycle generates the electron donors, NADH and succinate that donate electrons to mitochondrial complexes I and II, respectively. Electrons from these complexes are transferred to coenzyme Q, complex III, cytochrome c, complex IV and finally to O2 that is reduced to water. The electron transport system is organized in this way in order to regulate ATP production. During this process, the oxidation of NADH and succinate, a process that is coupled with the phosphorylation of ADP to ATP in a cascade of multienzyme complexes embedded in the inner mitochondrial membrane, leads to a continuous production of ROS (Hoye et al., 2008). Superoxide (O2●-), nitric oxide (NO), hydroxyl radical (HO-), peroxynitrite (ONOO-), and hydrogen peroxide (H2O2) are the prevalent ROS produced in the cells (Goetz and Luch, 2008). Mitochondrial phosphorylation and electron transport and iron metabolism are down regulated under hypoxic conditions (Mazure et al., 2010). Microscopy studies showed a shift of mitochondria from a normoxic tubular to a hypoxic enlarged phenotype with an intact membrane but with rearrangements in their cristae (Zhang et al., 2007; Rohwer and Cramer, 2011). As previously discussed, a drop in tissue O2 levels to the point where O2 demand exceeds supply leads rapidly to a metabolic crisis and represents a severe threat to ongoing physiological function and, ultimately, viability (Taylor, 2008). Cellular and molecular pathways underlying hypoxic neurotoxicity and cell death are multifaceted and complex involving several cellular responses, including oxidative stress, altered

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ionic homeostasis, mitochondrial dysfunction, and activation of apoptotic cascades (Jayalakshmi et al., 2005; Maiti et al., 2006; Hota et al., 2007). Initial studies in exploring the mechanism by which mitochondria regulate the stability of HIF-1α demonstrated that ROS levels, produced at mitochondrial complex III, increase in hypoxic conditions (Bell and Chandel, 2007). This increase in ROS production seems required and sufficient to stabilize HIF-1α protein avoiding its degradation by the proteasome (Chandel et al., 2000; Hirota and Semenza, 2001; Bell and Chandel, 2007). However, this topic remains controversial in view of the fact that other reports have shown a general ROS decrease under hypoxic conditions (Görlach et al., 2003) and further documented that a functioning mitochondrial respiratory chain may not be necessary for HIF-1α regulation (Srinivas et al., 2001; Vaux et al., 2001; Acker and Acker, 2005). In turn, HIF-1α could interfere with mitochondria by modifying their metabolic function and ultrastructure and the expression of the mitochondrial complex IV proteins (Mazure et al., 2010). An increase in mitofusin 1 was observed in LS174 cells under a hypoxic stimulus suggesting that hypoxia may affect mitochondrial fusion (Mazure et al., 2010). It was also observed an increase in mitophagy mediated by HIF-1α-dependent BNIP3, which disrupts Beclin-Bcl2 complex decreasing the number of mitochondria and, consequently, ROS production leading to an increase in cell survival (Zhang et al., 2008). However, this is a controversial issue since several studies demonstrated that BNIP3 leads to an increase in apoptosis, necrosis and autophagy potentiating cell death and, through the blockage of Bcl2 anti-apoptotic activity, induction of the permeability transition pore and activation of Bax (Bristow et al., 2011).

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4.2. Is hypoxia associated to T2DM a main road to AD? It is known that T2DM induces brain cortical microvasculature damage, associated with decreased vascular density that increases the risk of AD development (Bento and Pereira 2011; Schwarts et al., 2010). A decreased cerebral blood flow, lower metabolic rates of glucose and O2 and a compromised structural integrity of the cerebral vasculature with special attention to microvessels are representative degenerative features of the vascular system of a diabetic brain (Farkas and Luiten, 2001; SimsRobinson et al., 2010). Indeed, recent evidence suggests a profound implication of endothelial dysfunction in AD-related cerebral hypoperfusion and AD pathophysiology (Lange-Asschenfeldt and Kodja, 2008; Austin et al., 2010). Changes in synaptic efficacy occur very early during hypoxia and may, indeed, be the first response of the neurons to the ischemic insult (Aoyagi et al., 1998; Fleidervish et al., 2001). Studies have suggested that hypoxia can induce apoptosis, dependent on the transcriptional activation of apoptotic factors (Harris, 2002). In addition, disruption of brain endothelium compromise the blood brain barrier (BBB) function, with alterations in Aβ transport (Fig. 2) out of the brain that usually occurs through three pathways; a) across the capillary endothelium by receptor-mediated active transport, b) via solute diffusion and the bulk flow of cerebrospinal fluid, and c) by active transport across the choroid plexus epithelium (Altman and Rutledge 2010; Silverberg et al., 2010). Therefore, alterations in brain endothelium that may result from diabetes-induced vascular damage, could also lead to an increase in Aβ accumulation in the brain (Fig. 2) (Silverberg et al., 2010). Sun and collaborators (2006) demonstrated that hypoxia markedly increased Aβ deposition and neuritic plaque formation and potentiated memory deficits in Swedish mutant amyloid  precursor protein (APP) transgenic mice confirming that hypoxia can 11

facilitate AD pathogenesis (Sun et al., 2006). Moreover, it was showed that hypoxia significantly increased -site APP cleaving enzyme (BACE1) gene transcription through an early up-regulation dependent on the release of mitochondrial ROS and a late up-regulation due to the overexpression and activation of HIF-1α, resulting in increased BACE1 activity and Aβ production (Sun et al. 2006; Zhang et al. 2007; Gluglielmotto et al., 2009). This evidence supports the idea that hypoxia increases ROS production potentiating brain damage and AD development.

Conclusion The prevalence of T2DM is increasing in the developing world with a concomitant increase in several complications including neurodegenerative events. Cerebral hypoxia, resulting from diabetes-associated endothelial dysfunction, is emerging as a link between T2DM and AD, due to the decreased delivery of O2, nutrients and hormones to brain cells, which contribute to endothelial and neuronal cell degeneration and death. Furthermore, alterations in the brain vasculature potentiate A deposition in the brain. These findings corroborate the idea that T2DM is a risk factor for AD.

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Legends Figure 1 - Hypoxic response to diabetic alterations. Under diabetic conditions, the increases in blood glucose levels and insulin alterations lead to an increase in reactive oxygen species (ROS) production that underlie vascular changes and blood flow anomalies, with a concomitant decrease in oxygen (O2) delivery to cells. The decrease in O2 levels leads to hypoxia-inducible factor-1 (HIF-1) stabilization and migration to the nucleus where it binds to hypoxia response element (HRE) increasing the expression of proteins involved in cell survival (VEGF- vascular endothelial growth factor; iNOSinducible nitric oxide synthase; EPO- erythropoietin) and death (DEC-1- defective chorion-1; NIX- Nip3–like protein X), while the role of Bcl-2 and nineteen-kilodalton interacting protein (BNIP3) role remains unsolved. Figure 2 – Endothelial dysfunction decreases efflux of A protein (Aβ) from the brain to blood. In normal conditions Aβ is removed from the brain to blood by a receptormediated active transport. However, when brain endothelium is compromised, this transport fails and Aβ deposits in vessels wall leading to the formation of senile plaques, one of the major features of Alzheimer’s disease (AD). ECs- endothelial cells; RBCsred blood cells.

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Figure 1

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Figure 2

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