**6.1 Potential therapeutic compounds**

**Coenzyme Q (CoQ)** is a component of the electron transport chain where it accepts electrons from complexes I and II (Beal, 2004). Its administration increases mitochondrial uncoupling protein (UCP) expression in the nervous system of primates, and this is associated with marked neuroprotection in both *in vitro* and *in vivo* models of MPTP, paraquat and rotenone-induced mitochondrial dysfunction. CoQ10 exerts significant protection against MPTP-induced dopamine depletion and loss of tyrosine hydroxylase immunoreactive neurons in aged mice (Shults et al., 2002).

**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 (Matthews et al., 1999).

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 (Bharat et al., 2002).

**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; Virmani et al., 1995).

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

#### **6.2 Ketone bodies**

280 Etiology and Pathophysiology of Parkinson's Disease

As in the case of glycolysis, the end product of fatty acid β-oxidation is acetyl-CoA (Fig. 1) which feeds into the TCA cycle to produce energy in the form of NADH and FADH2. The enzymes involved in fatty acid β-oxidation all act on acyl-CoA, i.e. fatty acids linked to CoA as thioesters. They belong to an oxido-reductase superfamily with broad substrate specificity. The enzymes are subdivided into several groups, according to the substrate chain length. Each of the constituent steps of the pathway comprises three consecutive enzymatic reactions catalyzed by a trifunctional enzymatic complex: 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase. The reactions yield acetyl-CoA plus a chain-shortened acyl-CoA (at each step, the fatty acid chain loses

In the present review, we focus attention on the penultimate step of the process, i.e. acyl-CoA dehydrogenation. It involves four classes of enzymes: short-chain acyl-CoA dehydrogenase (SCAD, active with C4 and C6), medium-chain acyl-CoA dehydrogenase (MCAD, active with C4 to C12), long-chain acyl-CoA dehydrogenase (LCAD, active with C8 to C20) and very-long-chain acyl-CoA dehydrogenase (VLCAD, active with C12 to C24). SCAD, MCAD and LCAD are homotetramers located in the mitochondrial matrix. VLCAD, however, is a homodimer and is located in the inner mitochondrial membrane. VLCAD is a

Short chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) also known as L-3-hydroxyacyl-CoA dehydrogenase/amyloid-peptide binding alcohol dehydrogenase (HADH II/ABAD) is predominantly a mitochondrial enzyme (Furuta et al., 1997; Kobayashi et al., 1996) that belongs to the short-chain dehydrogenase/reductase superfamily (Jornvall et al., 1995). It acts on a wide spectrum of substrates, including steroids, cholic acids, and fatty acids, with a preference for short chain methyl-branched acyl-CoAs. Abnormally low or high levels of SCHAD in certain brain regions may contribute to the pathogenesis of some neural disorders. The human SCHAD gene and its protein product are therefore potential targets for intervention in conditions such as PD, AD and an X-linked mental retardation, which may arise from the impaired degradation of branched fatty acid chains and of isoleucine

The levels of SCHAD/HADH II are significantly reduced in the ventral midbrain of both PD patients and of a PD mouse model generated by MPTP injection (Przedborski et al., 2004). Conversely, transgenic mice with increased expression of human HADH II/ABAD are significantly more resistant to MPTP; overexpression of the enzyme mitigates MPTPinduced impairment of oxidative phosphorylation and ATP production (Tieu et al., 2004). This observation suggests that a new way to prevent PD may be to increase expression of

**Coenzyme Q (CoQ)** is a component of the electron transport chain where it accepts electrons from complexes I and II (Beal, 2004). Its administration increases mitochondrial uncoupling protein (UCP) expression in the nervous system of primates, and this is associated with marked neuroprotection in both *in vitro* and *in vivo* models of MPTP, paraquat and rotenone-induced mitochondrial dysfunction. CoQ10 exerts significant

**5.2 Fatty acid** β**-oxidation** 

two carbons) (Eaton et al., 2000).

constituent of the trifunctional protein described above.

(Yang et al., 2005; Zschocke et al., 2000).

SCHAD/HADH II in the midbrain.

**6.1 Potential therapeutic compounds** 

**6. Therapeutic strategies to target the energy crisis** 

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

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

The Energy Crisis in Parkinson's Disease: A Therapeutic Target 283

novel mitochondrial targets such as PGC-1α and the sirtuin family of enzymes that are known to modulate aging, mitochondrial biogenesis, metabolic homeostasis and mitochondria-dependent cell death. These observations hold promise for future development of neuroprotective strategies in PD by targeting mitochondrial dysfunction. However it is important to remember that PD is a multi-factorial disease and mitochondrial dysfunction may only be a part of this complex process. Future research should thus focus on developing neuroprotective strategies by targeting multiple pathways involved in the disease process. Therapeutic approaches targeting both mitochondrial dysfunction and oxidative damage in neurodegenerative diseases and aging have great promise (Beal, 2005). Pantethine provides an example of molecule able to restore mitochondrial function while displaying antioxidant and anti-inflammatory properties. Perhaps, this safe and effective compound of natural origin merits consideration for broader use against pathologies such as

Fig. 3. Attenuation of microglial reaction by pantethine treatment in MPTP-injected mice.

compared to saline ones. No immunostaining was observed in control animals.

Fig. 4. Prevention of BBB leakage by pantethine treatment in MPTP-injected mice.

Evans blue was administered intravenously on day 16 after MPTP injection. The figure shows brains of saline mice with a diffuse dye leakage, and some intensively stained areas. In contrast, brains of pantethine-treated mice looked normal, with limited stained areas.

The figure shows macrophage antigen-1 (Mac-1) immunostaining in SNpc, two days after MPTP injection. Microglial reaction was drastically reduced in pantethine-treated mice as

PD.

development of PD-like motor abnormalities in mice (Tieu et al., 2003). Tieu's studies provide *in vivo* and *in vitro* evidence that DβHB protects not by alleviating MPTP-related complex I inhibition, but by enhancing oxidative phosphorylation via a mechanism dependent on mitochondrial complex II (succinate-ubiquinone oxidoreductase).

Therefore, modulation of DβHB levels may be a neuroprotective strategy for the treatment of neurodegenerative diseases such as PD. However, the long-term effects of the chronic use of DβHB on the cellular metabolism, and especially on mitochondrial function, are not known. DβHB has been administered orally for several months to two 6-months-old infants with hyperinsulinemic hypoglycemia (Plecko et al., 2002). The high dosage (up to 32 g/d) seemed to be tolerated by these patients. In addition, ketogenic diets have been used in humans as a treatment for refractory epilepsy. In general, patients tolerate the ketogenic diet well with mild side effects (Freeman et al., 2006). However, long-term ketone therapy will have to take into consideration possible adverse effects.

### **6.3 Pantethine**

A promising emerging therapeutic strategy involves fatty acids combined with pantethine. CoA is central in these fields, as illustrated by pantothenate kinase-associated neurodegeneration (PKAN). Pantothenate kinase catalyzes pantetheine phosphorylation to 4'-phosphopantetheine, the first step of CoA synthesis (Fig. 2). PKAN, due to insufficient kinase activity, occurs in early adulthood and its symptoms, such as dystonia, rigidity and tremor, recall PD and it may lead to PD in late adulthood. PKAN results in a decrease of CoA levels associated with mitochondrial dysfunction. These defects can be reversed by pantethine, the oxidized form of pantetheine. Dietary pantethine increased CoA synthesis, improved mitochondrial function, rescued brain degeneration, enhanced locomotor abilities, and increased lifespan in a Drosophila model of PKAN (Rana et al., 2010). Moreover, pantethine circumvented the impairment of fatty acid β-oxidation in rat liver mitochondria and microvessels of the brain (Morisaki et al., 1983). It has been shown recently that pantethine mitigated MPTP neurotoxicity in the mouse via the enhancement of fatty acid β−oxidation, leading to increased levels of circulating ketone bodies and improved mitochondrial function (Cornille et al., 2010). In addition, pantethine attenuates MPTPinduced neuroinflammation, as shown by reduced expression of macrophage antigen-1 (MAC-1), a critical trigger of microglial activation associated with neurodegeneration (Pei et al., 2007) (Fig. 3). Ultimately, pantethine protects from MPTP-induced blood-brain barrier (BBB) leakage (Fig. 4) and significantly attenuates the clinical scores (Fig. 5).
