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

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 neuronal dysfunction and death.

The Energy Crisis in Parkinson's Disease: A Therapeutic Target 279

in several neurodegenerative disorders (Bordet et al., 2006; Heneka and Landreth, 2007; Woods et al., 2003). Gene-knockout experiments have revealed that, consistent with their distinct expression patterns, each PPAR subtype performs a specific function in fatty acid homeostasis. PPAR-α targets genes function together to coordinate the complex metabolic changes necessary to conserve energy during fasting and feeding in cases where catabolic

It is now well established that damage to dopaminergic neurons involves oxidative stress, microglial activation-mediated inflammation and mitochondrial impairment, which ultimately culminate in activation of an apoptotic pathway. The PPAR-γ agonist pioglitazone was shown to exert protective effects in a mouse model of PD; it attenuated the MPTP intoxication-induced glial activation and prevented dopaminergic cell loss in the substantia nigra pars compacta (Breidert et al., 2002). These findings were paralleled by another study showing that the treatment with pioglitazone may offer an opportunity for

PPAR-γ coactivator 1α (PGC-1α) is a master regulator of mitochondrial biogenesis and respiration (Lehman et al., 2000; Lin et al., 2002; Puigserver et al., 1998; Wu et al., 1999)**.** It transduces many physiological stimuli into specific metabolic programs, often by stimulating mitochondrial activity. For example, PGC-1α regulates both β−oxidation of fatty acids and gluconeogenesis in liver (Herzig et al., 2001; Puigserver et al., 2003; Rhee et al., 2003; Yoon et al., 2001). The importance of PGC-1α in these metabolic programs was further revealed through the generation of PGC-1α null mice. These mice display a reduced basal expression of many mitochondrial genes in liver, brain, skeletal muscle and heart, compared with wild-type animals (Arany et al., 2005; Leone et al., 2005; Lin et al., 2004). Moreover, it has been shown that PGC-1α knockout (KO) mice displayed neurodegenerative lesions in the brain, particularly in the striatum; the animals also showed behavioral abnormalities (Leone et al., 2005; Lin et al., 2004). At present, the cause of the brain lesions is unclear, but the lesions observed in many genetic models with altered ROS levels raise the possibility that PGC-1α plays an important role in the control of ROS in vivo (St-Pierre et al., 2006). PGC-1α and PGC-1β are powerfully induced by ROS, and these coactivators, in turn, regulate a complex and multifaceted ROS defense system. In the MPTP model, PGC-1α KO mice have a greatly increased sensitivity to damage by oxidative stress in the dopaminergic cells of the substantia nigra and hippocampal neurons. Conversely, the overexpression of PGC-1α in cultured dopaminergic neurons from embryonic rat midbrain resulted in activation of electron transport genes and protection against neuronal damage induced by mutant α-synuclein (Zheng et al., 2010). The same authors found in postmortem brain tissue samples from PD patients that the gene sets with the strongest association with PD contained nuclear genes encoding subunits of the ETC proteins. These genes all showed decreased expression in substantia nigra dopaminergic neurons even in the earliest stages of PD. Furthermore, a second gene set associated with PD and also underexpressed in the earliest stages of PD encodes enzymes involved in glucose metabolism. These results are compelling because many studies have already implicated dysfunctional mitochondria and altered energy metabolism as well as defective glucose metabolism in PD. The authors realized that these gene sets had in common the master transcriptional regulator, PGC-1α and surmised that disruption of PGC-1α expression might be a root cause of PD.

enzymes promote fatty acid β−oxidation.

treatment of PD to slow its progression (Dehmer et al., 2004).

α**-Synuclein** was the first gene associated with familial PD. It was followed by four other genes linked conclusively to autosomal recessive (parkin, PINK-1, DJ-1) or dominant (LRRK2) Parkinsonism (Bogaerts et al., 2008; Henchcliffe and Beal, 2008).

**PINK1**. Mutations in phosphatase and tensin homologue (PTEN)-induced putative kinase (PINK1) cause a rare form of autosomal recessive, PD early onset Parkinsonism following the parkin mutations (Valente et al., 2004; Winklhofer and Haass, 2010). Loss of PINK1 function increases the propensity of cells to oxidative stress-induced cell death and imbalance of calcium homeostasis. The experiments suggest that the impairment of mitochondrial calcium efflux promotes ROS formation that inhibits glucose uptake, resulting in reduced substrate delivery and respiration (Gandhi et al., 2009).

**Parkin** is a multifaceted ubiquitin ligase (Moore, 2006)**.** Mutations in the *parkin* gene serve as the most common cause of young onset PD (Farrer et al., 2001; Lucking et al., 2000). Loss of function of E3 ligase activity has been suggested to result in accumulation of toxic substrates leading to autosomal recessive form of PD (Dawson, 2006). *Parkin* gene expression is upregulated in various stress examples and has a wide range of neuroprotective capacities, including protection against mitochondrial dysfunction, endoplasmatic reticulum stress, exitotoxicity, proteasome inhibition, and overexpression of α-synuclein, tau, and other proteins. Future studies of the biochemical interactions between PINK1 and parkin and identification of other components in this pathway are likely to provide insight into PD pathogenesis, and might identify new therapeutic targets (Henchcliffe and Beal, 2008).

**DJ-1** and **LRRK2**. DJ-1 has structural similarities with the stress-inducible Escherichia coli chaperone Hsp31 and mutations in the *DJ-1* gene are associated with rare cases of early onset autosomal recessive PD (Andersen, 2004). The loss of DJ-1 function leads to a striking sensitivity to the herbicide paraquat and the insecticide rotenone; this suggests that DJ-1 may have a role in the protection from oxidative stress caused by environmental toxins. It has been clearly demonstrated that, while overexpression of DJ-1 protects neurons from oxidative stress-induced damage, conversely DJ-1 deficiency renders cells more susceptible to oxidative injury. Mutations in leucin-rich repeat kinase 2 (*LRRK2* gene) cause autosomal dominant PD (Paisan-Ruiz et al., 2004; Zimprich et al., 2004). The precise physiological role of this protein is unknown but the presence of multiple functional domains suggests involvement in a wide variety of functions. A possible mechanism of LRRK2 action is at the mitochondrial level.

## **5. Lipids as an alternative carbon source for energy production**

The brain has the ability to use fatty acids as an alternative carbon source. Fatty acid βoxidation parallels glycolysis (Fig. 1). This occurs normally during embryonic development and, in adults, under conditions of inadequate glucose supply or increased energy demand, as well as under pathological conditions, such as neurodegeneration, hypoxia/ischemia and traumatic brain injury (Prins, 2008).

#### **5.1 Lipid metabolism and regulation of mitochondrial function**

Peroxisome Proliferator-Activated Receptors (PPARs) regulate development, tissue differentiation, inflammation, wound healing, mitochondrial function, lipid and glucose metabolism (Reddy et al., 2006; Reddy, 2001). PPARs are expressed in CNS neurons and astrocytes, raising the possibility of exploring the neuroprotective efficacy of PPAR agonists

α**-Synuclein** was the first gene associated with familial PD. It was followed by four other genes linked conclusively to autosomal recessive (parkin, PINK-1, DJ-1) or dominant

**PINK1**. Mutations in phosphatase and tensin homologue (PTEN)-induced putative kinase (PINK1) cause a rare form of autosomal recessive, PD early onset Parkinsonism following the parkin mutations (Valente et al., 2004; Winklhofer and Haass, 2010). Loss of PINK1 function increases the propensity of cells to oxidative stress-induced cell death and imbalance of calcium homeostasis. The experiments suggest that the impairment of mitochondrial calcium efflux promotes ROS formation that inhibits glucose uptake,

**Parkin** is a multifaceted ubiquitin ligase (Moore, 2006)**.** Mutations in the *parkin* gene serve as the most common cause of young onset PD (Farrer et al., 2001; Lucking et al., 2000). Loss of function of E3 ligase activity has been suggested to result in accumulation of toxic substrates leading to autosomal recessive form of PD (Dawson, 2006). *Parkin* gene expression is upregulated in various stress examples and has a wide range of neuroprotective capacities, including protection against mitochondrial dysfunction, endoplasmatic reticulum stress, exitotoxicity, proteasome inhibition, and overexpression of α-synuclein, tau, and other proteins. Future studies of the biochemical interactions between PINK1 and parkin and identification of other components in this pathway are likely to provide insight into PD pathogenesis, and might identify new therapeutic targets (Henchcliffe and Beal, 2008). **DJ-1** and **LRRK2**. DJ-1 has structural similarities with the stress-inducible Escherichia coli chaperone Hsp31 and mutations in the *DJ-1* gene are associated with rare cases of early onset autosomal recessive PD (Andersen, 2004). The loss of DJ-1 function leads to a striking sensitivity to the herbicide paraquat and the insecticide rotenone; this suggests that DJ-1 may have a role in the protection from oxidative stress caused by environmental toxins. It has been clearly demonstrated that, while overexpression of DJ-1 protects neurons from oxidative stress-induced damage, conversely DJ-1 deficiency renders cells more susceptible to oxidative injury. Mutations in leucin-rich repeat kinase 2 (*LRRK2* gene) cause autosomal dominant PD (Paisan-Ruiz et al., 2004; Zimprich et al., 2004). The precise physiological role of this protein is unknown but the presence of multiple functional domains suggests involvement in a wide variety of functions. A possible mechanism of LRRK2 action is at the

(LRRK2) Parkinsonism (Bogaerts et al., 2008; Henchcliffe and Beal, 2008).

resulting in reduced substrate delivery and respiration (Gandhi et al., 2009).

**5. Lipids as an alternative carbon source for energy production** 

**5.1 Lipid metabolism and regulation of mitochondrial function** 

The brain has the ability to use fatty acids as an alternative carbon source. Fatty acid βoxidation parallels glycolysis (Fig. 1). This occurs normally during embryonic development and, in adults, under conditions of inadequate glucose supply or increased energy demand, as well as under pathological conditions, such as neurodegeneration, hypoxia/ischemia and

Peroxisome Proliferator-Activated Receptors (PPARs) regulate development, tissue differentiation, inflammation, wound healing, mitochondrial function, lipid and glucose metabolism (Reddy et al., 2006; Reddy, 2001). PPARs are expressed in CNS neurons and astrocytes, raising the possibility of exploring the neuroprotective efficacy of PPAR agonists

mitochondrial level.

traumatic brain injury (Prins, 2008).

in several neurodegenerative disorders (Bordet et al., 2006; Heneka and Landreth, 2007; Woods et al., 2003). Gene-knockout experiments have revealed that, consistent with their distinct expression patterns, each PPAR subtype performs a specific function in fatty acid homeostasis. PPAR-α targets genes function together to coordinate the complex metabolic changes necessary to conserve energy during fasting and feeding in cases where catabolic enzymes promote fatty acid β−oxidation.

It is now well established that damage to dopaminergic neurons involves oxidative stress, microglial activation-mediated inflammation and mitochondrial impairment, which ultimately culminate in activation of an apoptotic pathway. The PPAR-γ agonist pioglitazone was shown to exert protective effects in a mouse model of PD; it attenuated the MPTP intoxication-induced glial activation and prevented dopaminergic cell loss in the substantia nigra pars compacta (Breidert et al., 2002). These findings were paralleled by another study showing that the treatment with pioglitazone may offer an opportunity for treatment of PD to slow its progression (Dehmer et al., 2004).

PPAR-γ coactivator 1α (PGC-1α) is a master regulator of mitochondrial biogenesis and respiration (Lehman et al., 2000; Lin et al., 2002; Puigserver et al., 1998; Wu et al., 1999)**.** It transduces many physiological stimuli into specific metabolic programs, often by stimulating mitochondrial activity. For example, PGC-1α regulates both β−oxidation of fatty acids and gluconeogenesis in liver (Herzig et al., 2001; Puigserver et al., 2003; Rhee et al., 2003; Yoon et al., 2001). The importance of PGC-1α in these metabolic programs was further revealed through the generation of PGC-1α null mice. These mice display a reduced basal expression of many mitochondrial genes in liver, brain, skeletal muscle and heart, compared with wild-type animals (Arany et al., 2005; Leone et al., 2005; Lin et al., 2004). Moreover, it has been shown that PGC-1α knockout (KO) mice displayed neurodegenerative lesions in the brain, particularly in the striatum; the animals also showed behavioral abnormalities (Leone et al., 2005; Lin et al., 2004). At present, the cause of the brain lesions is unclear, but the lesions observed in many genetic models with altered ROS levels raise the possibility that PGC-1α plays an important role in the control of ROS in vivo (St-Pierre et al., 2006). PGC-1α and PGC-1β are powerfully induced by ROS, and these coactivators, in turn, regulate a complex and multifaceted ROS defense system. In the MPTP model, PGC-1α KO mice have a greatly increased sensitivity to damage by oxidative stress in the dopaminergic cells of the substantia nigra and hippocampal neurons. Conversely, the overexpression of PGC-1α in cultured dopaminergic neurons from embryonic rat midbrain resulted in activation of electron transport genes and protection against neuronal damage induced by mutant α-synuclein (Zheng et al., 2010). The same authors found in postmortem brain tissue samples from PD patients that the gene sets with the strongest association with PD contained nuclear genes encoding subunits of the ETC proteins. These genes all showed decreased expression in substantia nigra dopaminergic neurons even in the earliest stages of PD. Furthermore, a second gene set associated with PD and also underexpressed in the earliest stages of PD encodes enzymes involved in glucose metabolism. These results are compelling because many studies have already implicated dysfunctional mitochondria and altered energy metabolism as well as defective glucose metabolism in PD. The authors realized that these gene sets had in common the master transcriptional regulator, PGC-1α and surmised that disruption of PGC-1α expression might be a root cause of PD.

The Energy Crisis in Parkinson's Disease: A Therapeutic Target 281

protection against MPTP-induced dopamine depletion and loss of tyrosine hydroxylase

**Creatine** is a nitrogenous guanidine compound that helps to supply energy to muscle and nerve cells (Adhihetty and Beal, 2008). Chronic administration of creatine protects against MPTP-induced dopamine loss and improves the survival of neurons in the substantia nigra

α**-Lipoic acid** (ALA) is a disulfide compound found in mitochondria as a coenzyme for pyruvate dehydrogenase and α-ketoglutarate dehydrogenase. Dietary supplementation with α-lipoic acid in old rats improved ambulatory activity, decreased oxidative damage, and improved mitochondrial function (Hagen et al., 1999). *In vitro*, pre-treatment of PC12 cells with R-lipoic acid acts to prevent depletion of GSH content and preserves the mitochondrial complex I activity which normally is impaired as a consequence of GSH loss

**Carnitine** transports long-chain fatty acid into the mitochondrial matrix for subsequent β−oxidation (Di Lisa et al., 1985; Rosenthal et al., 1992). Carnitine also facilitates the removal of short-chain and medium chain fatty acids that accumulate in the mitochondria during normal and abnormal metabolism. Carnitine and acetyl-L-carnitine attenuate neuronal damage produced by 3-nitropropionic acid, rotenone, and MPTP *in vitro* (Snyder et al., 1990;

**Nicotinamide** is a precursor of NADH, a substrate for Complex I (NADH-ubiquinone oxidoreductase). It is also an inhibitor of poly-ADP-ribose polymerase, which is activated by DNA damage and in turn depletes both NADH and ATP. Activation of poly-ADP-ribose polymerase plays a role in neuronal injury induced by both ischemia and MPTP (Eliasson et

If the amounts of acetyl-CoA generated by fatty acid β-oxidation challenge the processing capacity of the TCA cycle or if activity in the TCA cycle is low, acetyl-CoA is used in the biosynthesis of ketone bodies via β−hydroxy-metylglutaryl-CoA (HMG-CoA) synthesis. The main ketone bodies are D-β-hydroxybutyrate (DβHB) and acetoacetate (ACA). They are produced by hepatocytes and are transported to the tissues, including the brain; astrocytes

DβHB and ACA have a protective role in a broad spectrum of cerebral injuries and diseases and they preserve neuronal cell integrity and stability *in vitro*. The experimental approaches used are intravenous infusion of mice or rats with DβHB (Prins, 2008) or administration of a ketogenic diet (Maalouf et al., 2009). Exogenous ketone bodies have been employed successfully in both rapidly developing pathologies (glutamate excitotoxicity, hypoxia/ischemia) and neurodegenerative conditions (PD, AD) [for review see(Prins, 2008)]. In vitro DβHB prevents neuronal damage induced by glucose deprivation (Izumi et al., 1998). Following MPP+ exposure, administration of 4 mmol/L of DβHB increased the survival of cultured neurons (Kashiwaya et al., 2000). In the rotenone *in vitro* model, application of 8 mmol/L of DβHB improved mitochondrial membrane potential and reduced cytochrome C release by mouse neuronal cultures (Imamura et al., 2006); under the same conditions, cell survival was increased by 60% in human neuroblastoma cell culture. *In vivo*, 24 h infusion of DβHB, using mini-osmotic pumps, protected SNpc dopaminergic neurons against MPTP in a dose-dependent and stereospecific manner and prevented the

are also ketogenic, although to a lesser extent (Guzman and Blazquez, 2001).

immunoreactive neurons in aged mice (Shults et al., 2002).

(Matthews et al., 1999).

(Bharat et al., 2002).

Virmani et al., 1995).

**6.2 Ketone bodies** 

al., 1997; Mandir et al., 1999).
