**The Energy Crisis in Parkinson's Disease: A Therapeutic Target**

Mhamad Abou-Hamdan, Emilie Cornille, Michel Khrestchatisky, Max de Reggi and Bouchra Gharib *NICN, UMR6184, CNRS, Aix-Marseille University, Marseille France* 

### **1. Introduction**

272 Etiology and Pathophysiology of Parkinson's Disease

Zarow, C., Lyness, S.A., Mortimer, J.A. & Chui, H.C. (2003). Neuronal loss is greater in the

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fibers in the striatum and the substantia nigra pars reticulata in Parkinsonian model

locus coeruleus than nucleus basalis and substantia nigra in Alzheimer' and

The energy demand of the brain is very large: it accounts for 20% of the body's energy consumption, even though its weight is less than 2% of the total body mass. In the adult, brain energy comes primarily from glucose through oxidative glycolysis. The end product is acetyl-coenzyme A (acetyl-CoA), which enters the mitochondria and feeds into the tricarboxylic acid (TCA) cycle to produce energy in the form of reductants, such as NADH. The chemical energy of NADH is then used by the respiratory chain, or electron transport chain, to synthesize ATP (Fig. 1). Energy depletion is a major factor in the cascade of events culminating in dopaminergic neuronal death in Parkinson's disease (PD). There are two reasons for that. First, a frequent feature of the disease, as well as of other neurodegenerative diseases, is an alteration of glucose metabolism. Second, there is a decrease of mitochondrial respiratory chain activity due to, in particular, inhibition of the electron transport system complex I (NADH-ubiquinone oxidoreductase).

Complex I activity is redox-dependent and thiol-regulated; therefore its inhibition is associated with oxidative stress. Depletion of GSH, a major antioxidant and redox modulator, is observed in the substantia nigra of parkinsonian patients, as well as in the mouse MPTP model of the disease. Conversely, the restoration of GSH levels preserves complex I activity. Accordingly, the maintenance of cellular redox homeostasis by thiol agents protects against nigrostriatal toxicity.

In the case of the alteration of glucose metabolism, the brain has the ability to adapt its metabolism and to increase its reliance on lipids for energy production, through fatty acid βoxidation. The process involves a mitochondrial oxido-reductase superfamily with broad substrate specificity. The penultimate step of the process is catalyzed by L-3-hydroxyacyl-CoA dehydrogenase (HADH II) (EC 1.1.1.35). HADH II overexpression protects against acute brain injury and chronic neurodegeneration. By-products of fatty acid β−oxidation are the ketone bodies β-hydroxybutyrate and acetoacetate. When injected into mice or rats, or administered in the form of a ketogenic diet, ketone bodies have a protective role in a broad spectrum of cerebral injuries and diseases.

In the present report, we examine the brain energy metabolism, its alterations associated with PD and how fatty acid β-oxidation can compensate such impairment. Energy store boosting agents have potential therapeutic properties. Pantethine, the precursor of vitamin

The Energy Crisis in Parkinson's Disease: A Therapeutic Target 275

Fig. 2. Pantethine forms the active moiety of CoA. Pantetheine, the reduced form of pantethine, is first 4'-phosphorylated by pantothenate kinase and then adenylated by phosphopantetheine adenylyl transferase (CoaD) to form dephospho-CoA; finally,

in the form of acyl-CoA thioesters.

**2. Brain energy metabolism** 

dephospho-CoA is phosphorylated to coenzyme A by the enzyme dephosphocoenzyme A kinase (CoaE). Lipids are metabolized as fatty acids linked to the SH group of pantetheine,

Although the brain represents only 2% of the body weight, it receives 15% of the cardiac output and accounts for 20% of total body oxygen consumption as well as 25% of total body glucose utilization. Under physiological conditions, glycolysis is, by far, the major energetic pathway in the adult brain. Glucose is broken down in the presence of oxygen, yielding pyruvate and lactate. Evidence accumulated during the past decade supports the notion that astrocytes play a pivotal role in the regulation of brain-glucose metabolism, via the so-called neuron-astrocyte lactate shuttle (Escartin et al., 2006; Hertz, 2004; Schurr, 2006). Owing to their strategic location surrounding cerebral blood capillaries, astrocytes form the first cellular barrier encountered by glucose entering the brain parenchyma, which makes them a prevalent site of glucose uptake. Blood-borne glucose crosses the vascular endothelial cells and enters brain cells through specific hexose transporters of the glucose transporter family (GLUT) (McEwen and Reagan, 2004; Vannucci et al., 1997). When the supply of oxygen therefore does not match precisely the demand, e.g. during neuronal activation or under pathological conditions, glucose may be broken down anaerobically, to produce lactate in order to supply

The following step of energy production is the conversion of pyruvate to acetyl-CoA which is used by mitochondria, the power house of living cells. Mitochondria produce the reductant NADH via the tricarboxilic acid cycle; then the chemical energy of NADH is used by the electron transport chain (ETC) to synthesize ATP. Mammalian ETC is composed of least 49 individual polypeptides which constitute a series of electron carriers organized in four enzymatic complexes plus the ATP synthase machinery. Complex I (NADH ubiquinone oxidoreductase) (EC1.6.5.3) is a very large enzymatic set catalyzing the first step of the ETC (Saraste, 1999; Schultz and Chan, 2001). The enzyme oxidizes NADH, transferring electrons to Ubiquinone (Coenzyme Q, CoQ), a lipid soluble electron carrier

energy faster. This process has, however, low energetic efficiency.

B5, is one of them, with the advantage of targeting multiple pathways involved in disease process. By itself or by its constituents (Fig. 2), pantethine regulates lipid metabolism, in addition to its anti-oxidant and anti-inflammatory properties, giving rise to the promise of an original therapeutic strategy against PD.

Fig. 1. The energetic pathways. Glycolysis is the major energetic pathway in the adult brain. It is impaired in Parkinson's disease, leading to an energy crisis, which is compensated by fatty acidβ−oxidation. Solid symbols (red) indicate the injuries and alterations occurring in the processes leading to the disease; open symbols (green) indicate the protective effects of pantethine.

B5, is one of them, with the advantage of targeting multiple pathways involved in disease process. By itself or by its constituents (Fig. 2), pantethine regulates lipid metabolism, in addition to its anti-oxidant and anti-inflammatory properties, giving rise to the promise of

Fig. 1. The energetic pathways. Glycolysis is the major energetic pathway in the adult brain. It is impaired in Parkinson's disease, leading to an energy crisis, which is compensated by fatty acidβ−oxidation. Solid symbols (red) indicate the injuries and alterations occurring in the processes leading to the disease; open symbols (green) indicate the protective effects of pantethine.

an original therapeutic strategy against PD.

Fig. 2. Pantethine forms the active moiety of CoA. Pantetheine, the reduced form of pantethine, is first 4'-phosphorylated by pantothenate kinase and then adenylated by phosphopantetheine adenylyl transferase (CoaD) to form dephospho-CoA; finally, dephospho-CoA is phosphorylated to coenzyme A by the enzyme dephosphocoenzyme A kinase (CoaE). Lipids are metabolized as fatty acids linked to the SH group of pantetheine, in the form of acyl-CoA thioesters.

#### **2. Brain energy metabolism**

Although the brain represents only 2% of the body weight, it receives 15% of the cardiac output and accounts for 20% of total body oxygen consumption as well as 25% of total body glucose utilization. Under physiological conditions, glycolysis is, by far, the major energetic pathway in the adult brain. Glucose is broken down in the presence of oxygen, yielding pyruvate and lactate. Evidence accumulated during the past decade supports the notion that astrocytes play a pivotal role in the regulation of brain-glucose metabolism, via the so-called neuron-astrocyte lactate shuttle (Escartin et al., 2006; Hertz, 2004; Schurr, 2006). Owing to their strategic location surrounding cerebral blood capillaries, astrocytes form the first cellular barrier encountered by glucose entering the brain parenchyma, which makes them a prevalent site of glucose uptake. Blood-borne glucose crosses the vascular endothelial cells and enters brain cells through specific hexose transporters of the glucose transporter family (GLUT) (McEwen and Reagan, 2004; Vannucci et al., 1997). When the supply of oxygen therefore does not match precisely the demand, e.g. during neuronal activation or under pathological conditions, glucose may be broken down anaerobically, to produce lactate in order to supply energy faster. This process has, however, low energetic efficiency.

The following step of energy production is the conversion of pyruvate to acetyl-CoA which is used by mitochondria, the power house of living cells. Mitochondria produce the reductant NADH via the tricarboxilic acid cycle; then the chemical energy of NADH is used by the electron transport chain (ETC) to synthesize ATP. Mammalian ETC is composed of least 49 individual polypeptides which constitute a series of electron carriers organized in four enzymatic complexes plus the ATP synthase machinery. Complex I (NADH ubiquinone oxidoreductase) (EC1.6.5.3) is a very large enzymatic set catalyzing the first step of the ETC (Saraste, 1999; Schultz and Chan, 2001). The enzyme oxidizes NADH, transferring electrons to Ubiquinone (Coenzyme Q, CoQ), a lipid soluble electron carrier

The Energy Crisis in Parkinson's Disease: A Therapeutic Target 277

both PD platelet homogenates and mitochondria isolated from PD platelets, although the degree of its inhibition varied within a wide range. Other ETC complexes in PD platelets are also concerned: complex II (Yoshino et al., 1992), complex II + complex III (Haas et al., 1995), and complex IV (Benecke et al., 1993). Several studies showed impaired complex I, II+III, and IV activities in PD muscle (Bindoff et al., 1991; Blin et al., 1994; Cardellach et al., 1993); whereas no differences were observed in other studies (DiDonato et al., 1993; Mann et al., 1992; Reichmann et al., 1994). Similar conflicting results were reported in the ETC in PD lymphocytes (Martin et al., 1996; Yoshino et al., 1992). Such inconsistency in the experimental data may be explained by significant methodological differences in these studies, e.g. difficulties in obtaining the relevant tissue samples, individual variability of PD patients and

Complex I activity is reduced in mitochondria isolated from PD frontal cortex (Keeney et al., 2006; Parker et al., 2008); it is believed to be an early pathogenic event in PD. Ample data strongly suggest that MPTP-induced ETC disruption per se is sufficient to trigger the death of nigrostriatal neurons and to cause PD-like symptoms in animals and humans. Thus, despite some conflicting results, systemic complex I deficiency might be a predominant feature in PD pathogenesis (Banerjee et al., 2009). Accordingly, 1H and 31P magnetic resonance spectrometry demonstrated mitochondrial dysfunction in the putamen and midbrain of both early and advanced PD subjects, with a bilateral reduction of high-energy phosphates; the changes were associated with abnormally elevated lactate levels. In contrast, low-energy metabolites such as adenosine diphophosphate and inorganic phosphate were within normal ranges. These results provide strong in vivo evidence that mitochondrial dysfunction of mesostriatal neurons is a central and persistent phenomenon in the pathogenesis cascade of PD which occurs early in the course of the disease (Hattingen

Paraquat and rotenone are other neurotoxins that inhibit mitochondrial complex I activity, whereas there are substantial differences in their action (Richardson et al., 2005). Rotenone rat models of PD have been developed that reproduce essential biochemical and behavioral human PD features (Alam and Schmidt, 2002; Betarbet et al., 2000; Greenamyre et al., 2003;

Complex I activity is redox-dependent and thiol-regulated (Annepu and Ravindranath, 2000; Sriram et al., 1998). Since PD pathogenesis consistently involve reactive oxygen and nitrogen species (Bove et al., 2005; Li et al., 2003; Przedborski, 2005; Przedborski and Ischiropoulos, 2005) this may explain, at least in part, complex I inhibition. Thus, depletion of GSH, an antioxidant and redox modulator, may be one of the early events leading to the inhibition of complex I activity and loss of mitochondrial function (Chinta and Andersen,

In the past decade, a rapidly expanding list of Mendelian-inherited gene mutations has provided great insight into the mechanisms leading to loss of the nigrostriatal dopaminergic neurons in familial PD which constitutes less than 10% of the total number of PD cases. In recent years, a plethora of studies of the multifunctional consequences of these mutations point towards the disruption of mitochondrial function culminating in

**4. Mitochondrial dysfunction and the genetic causes of the disease** 

many other factors, as was suggested (Parker et al., 2008).

et al., 2009; Henchcliffe et al., 2008).

Sherer et al., 2003).

2006; Jha et al., 2000).

neuronal dysfunction and death.

embedded in the lipid bilayer of the inner mitochondrial membrane. Complex II (succinateubiquinone oxidoreductase) (EC1.3.5.1)**,** bound to the inner mitochondrial membrane, catalyzes the oxidation of succinate to fumarate with the reduction of ubiquinone to ubiquinol. Complex III (ubiquinol-cytochrome C oxidoreductase; EC 1.10.2.2) is the oligomeric protein complex that transfers electrons from ubiquinol to cytochrome C and links proton translocation to this electron transfer by the proton motive Q cycle mechanism (Trumpower, 1990). Complex IV (cytochrome C oxidase (EC 1.9.3.1) is a transmembrane protein which receives electrons from cytochrome C and transfers them to one oxygen molecule, converting molecular oxygen to water. In the whole process, a transmembrane difference of proton electrochemical potential is established that the ATP synthase then uses to synthesize ATP. Mitochondrial dysfunction results in a dwindling supply of ATP, a failure in maintaining cellular homeostasis and activation of cell death pathways.

#### **3. Energy crisis in Parkinson's disease**

PD, Huntington's disease (HD) and Alzheimer's disease (AD) are age-related diseases of diverse etiologies, but energy crisis is a common factor. The link between PD and mitochondria was first established with the identification of a deficiency in the activity of complex I in the substantia nigra of PD patients (Schapira et al., 1989) and it has been then confirmed by several authors (see reviews in (Banerjee et al., 2009; Schapira, 2006)). Complex I deficiency was noticed not only in the brain but also in the peripheral tissues of PD patients (Krige et al., 1992; Wallace et al., 1992). Whether mitochondrial dysfunction is the cause or effect of PD pathogenesis is debatable. However, it is clear that dopaminergic neurons are especially susceptible to energy deficit, meaning that mitochondrial dysfunction may underlie selective dopaminergic neuro-degeneration in PD (Beal, 2005).

The seminal discovery that MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) causes PDlike symptoms in humans (Langston et al., 1983) and the ensuing rapid unraveling of the molecular mechanism of its toxicity strongly stimulated the interest of the PD research community in the role of mitochondria in PD pathology. MPTP is metabolized to its toxic form MPP+ (1-methyl-4-phenylpyridinium ion) by mitochondrial monoamine oxidase (MAO) (Chiba et al., 1984), specifically by MAO B (Heikkila et al., 1984) and is rapidly concentrated in the mitochondria (Ramsay et al., 1986b). Once accumulated, MPP+ specifically inhibits the oxidation of NAD–linked substrates (Nicklas et al., 1985) by blocking the electron transfer through the ETC complex I (Ramsay et al., 1986a). It also inhibits the activity of a key TCA (tricarboxylic acid) enzyme KGDHC (α-ketoglutarate dehydrogenase complex) thereby impairing the ATP synthesis and inducing "**energy crisis**" *in vivo* (Mizuno et al., 1988b) and *in vitro* (Mizuno et al., 1988a). The rather selective toxicity of MPP+ to dopaminergic neurons in SN (substantia nigra) was explained by the fact that MPP+ is selectively accumulated by the dopamine uptake system involving the dopamine transporters (DAT) (Javitch et al., 1985). Platelets which also express DAT (Frankhauser et al., 2006) have been shown to accumulate MPP+ with high efficiency and suffer from mitochondrial failure as a result of MPP+ exposure (Buckman et al., 1988). Human platelets also contain high levels of MAO B and share a number of properties with aminergic neurons including receptors, uptake sites and storage granules for amine neurotransmitters. As platelets are more available for research than PD brain samples or muscle biopsy specimens, they were a natural choice for PD tissue samples in earlier studies on the status of complex I in PD. Low complex I activities have been reported in

embedded in the lipid bilayer of the inner mitochondrial membrane. Complex II (succinateubiquinone oxidoreductase) (EC1.3.5.1)**,** bound to the inner mitochondrial membrane, catalyzes the oxidation of succinate to fumarate with the reduction of ubiquinone to ubiquinol. Complex III (ubiquinol-cytochrome C oxidoreductase; EC 1.10.2.2) is the oligomeric protein complex that transfers electrons from ubiquinol to cytochrome C and links proton translocation to this electron transfer by the proton motive Q cycle mechanism (Trumpower, 1990). Complex IV (cytochrome C oxidase (EC 1.9.3.1) is a transmembrane protein which receives electrons from cytochrome C and transfers them to one oxygen molecule, converting molecular oxygen to water. In the whole process, a transmembrane difference of proton electrochemical potential is established that the ATP synthase then uses to synthesize ATP. Mitochondrial dysfunction results in a dwindling supply of ATP, a

failure in maintaining cellular homeostasis and activation of cell death pathways.

PD, Huntington's disease (HD) and Alzheimer's disease (AD) are age-related diseases of diverse etiologies, but energy crisis is a common factor. The link between PD and mitochondria was first established with the identification of a deficiency in the activity of complex I in the substantia nigra of PD patients (Schapira et al., 1989) and it has been then confirmed by several authors (see reviews in (Banerjee et al., 2009; Schapira, 2006)). Complex I deficiency was noticed not only in the brain but also in the peripheral tissues of PD patients (Krige et al., 1992; Wallace et al., 1992). Whether mitochondrial dysfunction is the cause or effect of PD pathogenesis is debatable. However, it is clear that dopaminergic neurons are especially susceptible to energy deficit, meaning that mitochondrial dysfunction may underlie

The seminal discovery that MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) causes PDlike symptoms in humans (Langston et al., 1983) and the ensuing rapid unraveling of the molecular mechanism of its toxicity strongly stimulated the interest of the PD research community in the role of mitochondria in PD pathology. MPTP is metabolized to its toxic form MPP+ (1-methyl-4-phenylpyridinium ion) by mitochondrial monoamine oxidase (MAO) (Chiba et al., 1984), specifically by MAO B (Heikkila et al., 1984) and is rapidly concentrated in the mitochondria (Ramsay et al., 1986b). Once accumulated, MPP+ specifically inhibits the oxidation of NAD–linked substrates (Nicklas et al., 1985) by blocking the electron transfer through the ETC complex I (Ramsay et al., 1986a). It also inhibits the activity of a key TCA (tricarboxylic acid) enzyme KGDHC (α-ketoglutarate dehydrogenase complex) thereby impairing the ATP synthesis and inducing "**energy crisis**" *in vivo* (Mizuno et al., 1988b) and *in vitro* (Mizuno et al., 1988a). The rather selective toxicity of MPP+ to dopaminergic neurons in SN (substantia nigra) was explained by the fact that MPP+ is selectively accumulated by the dopamine uptake system involving the dopamine transporters (DAT) (Javitch et al., 1985). Platelets which also express DAT (Frankhauser et al., 2006) have been shown to accumulate MPP+ with high efficiency and suffer from mitochondrial failure as a result of MPP+ exposure (Buckman et al., 1988). Human platelets also contain high levels of MAO B and share a number of properties with aminergic neurons including receptors, uptake sites and storage granules for amine neurotransmitters. As platelets are more available for research than PD brain samples or muscle biopsy specimens, they were a natural choice for PD tissue samples in earlier studies on the status of complex I in PD. Low complex I activities have been reported in

**3. Energy crisis in Parkinson's disease** 

selective dopaminergic neuro-degeneration in PD (Beal, 2005).

both PD platelet homogenates and mitochondria isolated from PD platelets, although the degree of its inhibition varied within a wide range. Other ETC complexes in PD platelets are also concerned: complex II (Yoshino et al., 1992), complex II + complex III (Haas et al., 1995), and complex IV (Benecke et al., 1993). Several studies showed impaired complex I, II+III, and IV activities in PD muscle (Bindoff et al., 1991; Blin et al., 1994; Cardellach et al., 1993); whereas no differences were observed in other studies (DiDonato et al., 1993; Mann et al., 1992; Reichmann et al., 1994). Similar conflicting results were reported in the ETC in PD lymphocytes (Martin et al., 1996; Yoshino et al., 1992). Such inconsistency in the experimental data may be explained by significant methodological differences in these studies, e.g. difficulties in obtaining the relevant tissue samples, individual variability of PD patients and many other factors, as was suggested (Parker et al., 2008).

Complex I activity is reduced in mitochondria isolated from PD frontal cortex (Keeney et al., 2006; Parker et al., 2008); it is believed to be an early pathogenic event in PD. Ample data strongly suggest that MPTP-induced ETC disruption per se is sufficient to trigger the death of nigrostriatal neurons and to cause PD-like symptoms in animals and humans. Thus, despite some conflicting results, systemic complex I deficiency might be a predominant feature in PD pathogenesis (Banerjee et al., 2009). Accordingly, 1H and 31P magnetic resonance spectrometry demonstrated mitochondrial dysfunction in the putamen and midbrain of both early and advanced PD subjects, with a bilateral reduction of high-energy phosphates; the changes were associated with abnormally elevated lactate levels. In contrast, low-energy metabolites such as adenosine diphophosphate and inorganic phosphate were within normal ranges. These results provide strong in vivo evidence that mitochondrial dysfunction of mesostriatal neurons is a central and persistent phenomenon in the pathogenesis cascade of PD which occurs early in the course of the disease (Hattingen et al., 2009; Henchcliffe et al., 2008).

Paraquat and rotenone are other neurotoxins that inhibit mitochondrial complex I activity, whereas there are substantial differences in their action (Richardson et al., 2005). Rotenone rat models of PD have been developed that reproduce essential biochemical and behavioral human PD features (Alam and Schmidt, 2002; Betarbet et al., 2000; Greenamyre et al., 2003; Sherer et al., 2003).

Complex I activity is redox-dependent and thiol-regulated (Annepu and Ravindranath, 2000; Sriram et al., 1998). Since PD pathogenesis consistently involve reactive oxygen and nitrogen species (Bove et al., 2005; Li et al., 2003; Przedborski, 2005; Przedborski and Ischiropoulos, 2005) this may explain, at least in part, complex I inhibition. Thus, depletion of GSH, an antioxidant and redox modulator, may be one of the early events leading to the inhibition of complex I activity and loss of mitochondrial function (Chinta and Andersen, 2006; Jha et al., 2000).
