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**14**

**Epigenetic Modulation of Adenosine**

*1Institute of Neuropathology, Bellvitge University Hospital-ICS,*

*Experimental Therapeutics, L'Hospitalet de Llobregat,*

Marta Barrachina1,2, Mairena Martín3, Francisco Ciruela4 and Isidre Ferrer1,2,5

*CIBERNED, Barcelona,*

*L'Hospitalet de Llobregat,*

*Ciudad Real,*

*Spain*

**A2A Receptor: A Putative Therapeutical**

**Tool for the Treatment of Parkinson's Disease**

*3Departamento de Química Inorgánica, Orgánica y Bioquímica, Facultad de Químicas, Centro Regional de Investigaciones Biomédicas, Universidad de Castilla-La Mancha,*

*(Bellvitge Biomedical Research Institute-) IDIBELL, L'Hospitalet de Llobregat, 2Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas,*

*4University of Barcelona, Unit of Pharmacology, Department of Pathology and*

*5University of Barcelona, Department of Pathology and Experimental Therapeutics,*

Adenosine is a nucleoside distributed throughout the entire organism as an intermediary metabolite. At the extracellular level, adenosine plays multiple physiologic roles, interacting with specific receptors: A1, A2A, A2B and A3 (Fredholm et al., 2001). While the A1Rs and A3Rs are coupled in an inhibitory way to adenylate cyclase through the Gi/o protein, the A2Rs are coupled in a stimulatory way to this enzymatic activity through Gs protein (Ralevic &

Adenosine levels are increased after ischemia, hypoxia, excitotoxicity, inflammation and cerebral lesions. In these situations, it is considered that high adenosine levels play a neuroprotective role (Ribeiro et al., 2002). Interestingly, adenosine regulates the release of glutamate, the main excitatory neurotransmitter of the nervous system (Sebastiao & Ribeiro, 1996). A1Rs are widely expressed in the brain and have been shown to modulate neuronal excitability by decreasing pre-synaptic release of various neurotransmitters (Fredholm & Dunwiddie, 1988). The most dramatic inhibitory actions are on the glutamatergic system (Masino et al., 2002). In the central nervous system (CNS), A1Rs are associated with neuroprotective processes (Angulo et al., 2003; Dunwiddie and Masino, 2001). Moreover, they are upregulated in human neurodegenerative diseases with abnormal protein aggregates and it is related to compensatory mechanisms (Albasanz et al., 2007, 2008; Angulo et al., 2003; Perez-Buira et al., 2007; Rodríguez et al., 2006). Regarding A2ARs, these

**1. Introduction**

Burnstock, 1998).

### **Epigenetic Modulation of Adenosine A2A Receptor: A Putative Therapeutical Tool for the Treatment of Parkinson's Disease**

Marta Barrachina1,2, Mairena Martín3, Francisco Ciruela4 and Isidre Ferrer1,2,5 *1Institute of Neuropathology, Bellvitge University Hospital-ICS, (Bellvitge Biomedical Research Institute-) IDIBELL, L'Hospitalet de Llobregat, 2Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, CIBERNED, Barcelona, 3Departamento de Química Inorgánica, Orgánica y Bioquímica, Facultad de Químicas, Centro Regional de Investigaciones Biomédicas, Universidad de Castilla-La Mancha, Ciudad Real, 4University of Barcelona, Unit of Pharmacology, Department of Pathology and Experimental Therapeutics, L'Hospitalet de Llobregat, 5University of Barcelona, Department of Pathology and Experimental Therapeutics, L'Hospitalet de Llobregat, Spain*

#### **1. Introduction**

294 Towards New Therapies for Parkinson's Disease

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mediated gene expression and tropism in mice after systemic injection. *Mol Ther*,

Adenosine is a nucleoside distributed throughout the entire organism as an intermediary metabolite. At the extracellular level, adenosine plays multiple physiologic roles, interacting with specific receptors: A1, A2A, A2B and A3 (Fredholm et al., 2001). While the A1Rs and A3Rs are coupled in an inhibitory way to adenylate cyclase through the Gi/o protein, the A2Rs are coupled in a stimulatory way to this enzymatic activity through Gs protein (Ralevic & Burnstock, 1998).

Adenosine levels are increased after ischemia, hypoxia, excitotoxicity, inflammation and cerebral lesions. In these situations, it is considered that high adenosine levels play a neuroprotective role (Ribeiro et al., 2002). Interestingly, adenosine regulates the release of glutamate, the main excitatory neurotransmitter of the nervous system (Sebastiao & Ribeiro, 1996). A1Rs are widely expressed in the brain and have been shown to modulate neuronal excitability by decreasing pre-synaptic release of various neurotransmitters (Fredholm & Dunwiddie, 1988). The most dramatic inhibitory actions are on the glutamatergic system (Masino et al., 2002). In the central nervous system (CNS), A1Rs are associated with neuroprotective processes (Angulo et al., 2003; Dunwiddie and Masino, 2001). Moreover, they are upregulated in human neurodegenerative diseases with abnormal protein aggregates and it is related to compensatory mechanisms (Albasanz et al., 2007, 2008; Angulo et al., 2003; Perez-Buira et al., 2007; Rodríguez et al., 2006). Regarding A2ARs, these

Epigenetic Modulation of Adenosine A2A Receptor:

synergistic impairment of D2R function.

**3. Clinical trials with A2AR antagonists in PD**

coadjuvant seems to allow a reduction in the L-dopa dose.

(Tomiyama et al., 2004).

et al., 2005).

A Putative Therapeutical Tool for the Treatment of Parkinson's Disease 297

Of note is the characterization of A1R-A2AR heteromers with antagonistic activities between the two receptors, preferentially at presynaptically level in glutamatergic terminals of cortico-striatal afferents to the MSNs (Ciruela et al., 2006; Quiroz et al., 2009). Under baseline conditions, reduced levels of extracellular adenosine stimulate the activity of A1Rs while glutamatergic neurotransmission is inhibited. Under conditions of neuronal excitability, the extracellular adenosine levels are increased, showing A2ARs affinity and inhibiting A1R activity, and promoting the release of glutamate, which in turn also increases activation of mGluR5 synergizing with A2ARs and thereby facilitating more glutamate release (Rodrigues

Interestingly, striatal A2ARs expression levels have been found to be increased in PD patients with dyskinesias; this upregulation is attributed to the effect of levodopa (L-dopa) treatment (Calon et al., 2004). Recently, it has been proven that high A2ARs levels in the striatum and in lymphocytes correlate with motor symptoms in PD patients who were previously either not pharmacologically treated or were treated with a wide spectrum of drugs and not restricted to only L-dopa (Varani et al., 2010). Therefore, A2AR upregulation in PD, which tonically inhibits D2R (see above), together with the low dopamine content in the striatum, a consequence of the death of dopaminergic neurons from the substantia nigra, contribute to a

It remains to be clarified whether upregulation of A2ARs levels in PD is a hallmark of the disease or is a consequence of dopaminergic terminal drop-off in the striatum. This issue has been quite controversial, as increased striatal levels of A2ARs were shown in 6 hydroxidopamine (6-OHDA)-treated rats, as a consequence of dopamine denervation (Pinna et al., 2002), and also in 6-OHDA-treated rats with intermittent L-dopa treatment

L-dopa remains the most effective treatment for symptomatic relief of PD, although its pharmacological administration over time induces motor dysfunctions such as dyskinesias

non-dopaminergic drugs that modulate dopaminergic neurotransmission. Indeed, A2AR has emerged as a potential pharmacological target in PD, as its relationship with the dopaminergic system has been clearly demonstrated (Ferré et al., 2002). There have been several clinical trials with A2AR antagonists, such as istradefylline (also known as KW-6002), confirming that their administration to PD patients reduces the "OFF" time and dyskinesias induced by L-dopa treatment (Bara-Jimenez et al., 2003; Factor et al., 2010; Hauser et al., 2003, 2008; LeWitt et al., 2008b; Mizuno et al., 2010; Stacy et al., 2008). As A2ARs expression levels are nearly exclusive of the striatum, the use of specific antagonists for these receptors could promote a specific brain-area effect (Brooks et al., 2008). Interestingly, it has recently been described how KW-6002 preferentially targets the A2AR within the postsynaptic A2AR/D2R oligomer located at the MSNs, thus potentiating D2R-mediated motor activation (Orru et al., 2011). Therefore, although the use of istradefylline as a monotheraphy in PD has not been statistically demonstrated (Fernandez et al., 2010), its administration as a

As mentioned before, there are heteromers formed by A2AR, D2R and mGluR5 in striatal GABAergic neurons or MSNs (Cabello et al., 2009). It is proposed that the increase in glutamate and adenosine extracellular levels activates A2AR and mGluR5, both synergizing

(Obeso et al., 2000). One of the strategies to reduce these is the administration of

receptors are concentrated in the striatum, modulating dopaminergic activity, but they are also present in the hippocampus and cerebral cortex, modulating the glutamate release in the brain. Adenosine activity through A2 receptors (A2ARs) can eventually give rise to neurotoxicity, neuronal damage and cellular death (de Mendoça et al., 2000). In fact, A2ARs activity is associated with the outcome of cerebral injury as well as the development of A induced synaptotoxicity (Canas et al., 2009; Cunha, 2005; Stone et al., 2009).

#### **2. Human brain A2AR localization and implications in PD pathophysiology**

As mentioned in the previous section, A2ARs are G protein-coupled receptors that stimulate adenylyl cyclase through Gs proteins, promoting accumulation of intracellular cAMP (Van Calker et al., 1979). The activation of these receptors mediates multiple physiological effects of adenosine, both in the CNS and in peripheral tissues (Fredholm et al., 2001). Pharmacological activation of A2ARs promotes vasodilatation, immunosuppression, tissue protection, sleep promotion and depression (Cerqueira, 2004; El Yacoubi et al., 2003; Linden, 2001; Satoh et al., 1998).

A2ARs are widely expressed, but they are highly concentrated in spleen, thymus, leukocytes and blood platelets. A2ARs levels in immune cells play a critical role in the protection of normal tissues by attenuating inflammation and tissue damage *in vivo* (Ohta and Sitkovsky, 2001). In the CNS, A2ARs are highly expressed in the striatum (Peterfreund et al., 1996; Schiffmann et al., 1991). Most striatal neurons (95%) are GABAergic medium spiny neurons (MSNs) which can be divided into two subtypes. One subpopulation projects to the globus pallidus and contains enkephalin. The other subpopulation projects to the substantia nigra and contains substance P and dynorphin. These neurons receive inputs from glutamatergic afferents from cortical, thalamic and limbic areas and dopaminergic afferents from the substantia nigra pars compacta and the ventral tegmental area. MSNs promote two striatal efferent pathways, the "direct" and "indirect", affecting motor activation and inhibition, respectively. The MSNs of the direct pathway correspond to the subpopulation containing dynorphin and they also express dopamine D1 receptors, whereas indirect MSNs express enkephalin, dopamine D2 receptors (D2Rs) and A2ARs (Schiffmann et al., 2007). In these cells, A2ARs physically interact (oligomerize) with D2R, and this receptor-receptor interaction results in a tidy adenosine/dopamine functional interaction controlled by the A2AR/D2R oligomer. Consequently, two reciprocal antagonistic A2AR/D2R interactions have been described, namely an intermembrane interaction in which A2AR mediates the inhibition of D2R, thus modulating neuronal excitability and neurotransmitter release, and an interaction at the level of adenylyl-cyclase in which D2R inhibits A2AR-mediated protein phosphorylation and gene expression (for review see Ciruela et al., 2011). As a result of this interaction, antagonists of A2ARs have recently emerged as a leading candidate class of nondopaminergic anti-parkinsonian agents, based in part on the unique CNS distribution of A2ARs and A2AR/D2R oligomers (Fuxe et al., 2003). Moreover, the metabotropic glutamate receptors 5 (mGluR5s) are co-localized in the same GABAergic striatal output neurons and in glutamatergic nerve terminals in the striatum, and they form heteromeric complexes with A2ARs (Ferré et al., 2002; Rodrigues et al., 2005). This co-localization provides a morphological framework for the existence of multiple mGlu5/A2A/D2 receptor interactions (Cabello et al., 2009). Thus, it is proposed that the increase in glutamate and adenosine extracellular levels activates A2AR and mGluR5, both synergizing and promoting the inhibition of D2Rs (Ferré et al., 2007).

receptors are concentrated in the striatum, modulating dopaminergic activity, but they are also present in the hippocampus and cerebral cortex, modulating the glutamate release in the brain. Adenosine activity through A2 receptors (A2ARs) can eventually give rise to neurotoxicity, neuronal damage and cellular death (de Mendoça et al., 2000). In fact, A2ARs activity is associated with the outcome of cerebral injury as well as the development of A-

**2. Human brain A2AR localization and implications in PD pathophysiology**

As mentioned in the previous section, A2ARs are G protein-coupled receptors that stimulate adenylyl cyclase through Gs proteins, promoting accumulation of intracellular cAMP (Van Calker et al., 1979). The activation of these receptors mediates multiple physiological effects of adenosine, both in the CNS and in peripheral tissues (Fredholm et al., 2001). Pharmacological activation of A2ARs promotes vasodilatation, immunosuppression, tissue protection, sleep promotion and depression (Cerqueira, 2004; El Yacoubi et al., 2003; Linden,

A2ARs are widely expressed, but they are highly concentrated in spleen, thymus, leukocytes and blood platelets. A2ARs levels in immune cells play a critical role in the protection of normal tissues by attenuating inflammation and tissue damage *in vivo* (Ohta and Sitkovsky, 2001). In the CNS, A2ARs are highly expressed in the striatum (Peterfreund et al., 1996; Schiffmann et al., 1991). Most striatal neurons (95%) are GABAergic medium spiny neurons (MSNs) which can be divided into two subtypes. One subpopulation projects to the globus pallidus and contains enkephalin. The other subpopulation projects to the substantia nigra and contains substance P and dynorphin. These neurons receive inputs from glutamatergic afferents from cortical, thalamic and limbic areas and dopaminergic afferents from the substantia nigra pars compacta and the ventral tegmental area. MSNs promote two striatal efferent pathways, the "direct" and "indirect", affecting motor activation and inhibition, respectively. The MSNs of the direct pathway correspond to the subpopulation containing dynorphin and they also express dopamine D1 receptors, whereas indirect MSNs express enkephalin, dopamine D2 receptors (D2Rs) and A2ARs (Schiffmann et al., 2007). In these cells, A2ARs physically interact (oligomerize) with D2R, and this receptor-receptor interaction results in a tidy adenosine/dopamine functional interaction controlled by the A2AR/D2R oligomer. Consequently, two reciprocal antagonistic A2AR/D2R interactions have been described, namely an intermembrane interaction in which A2AR mediates the inhibition of D2R, thus modulating neuronal excitability and neurotransmitter release, and an interaction at the level of adenylyl-cyclase in which D2R inhibits A2AR-mediated protein phosphorylation and gene expression (for review see Ciruela et al., 2011). As a result of this interaction, antagonists of A2ARs have recently emerged as a leading candidate class of nondopaminergic anti-parkinsonian agents, based in part on the unique CNS distribution of A2ARs and A2AR/D2R oligomers (Fuxe et al., 2003). Moreover, the metabotropic glutamate receptors 5 (mGluR5s) are co-localized in the same GABAergic striatal output neurons and in glutamatergic nerve terminals in the striatum, and they form heteromeric complexes with A2ARs (Ferré et al., 2002; Rodrigues et al., 2005). This co-localization provides a morphological framework for the existence of multiple mGlu5/A2A/D2 receptor interactions (Cabello et al., 2009). Thus, it is proposed that the increase in glutamate and adenosine extracellular levels activates A2AR and mGluR5, both synergizing and promoting the

induced synaptotoxicity (Canas et al., 2009; Cunha, 2005; Stone et al., 2009).

2001; Satoh et al., 1998).

inhibition of D2Rs (Ferré et al., 2007).

Of note is the characterization of A1R-A2AR heteromers with antagonistic activities between the two receptors, preferentially at presynaptically level in glutamatergic terminals of cortico-striatal afferents to the MSNs (Ciruela et al., 2006; Quiroz et al., 2009). Under baseline conditions, reduced levels of extracellular adenosine stimulate the activity of A1Rs while glutamatergic neurotransmission is inhibited. Under conditions of neuronal excitability, the extracellular adenosine levels are increased, showing A2ARs affinity and inhibiting A1R activity, and promoting the release of glutamate, which in turn also increases activation of mGluR5 synergizing with A2ARs and thereby facilitating more glutamate release (Rodrigues et al., 2005).

Interestingly, striatal A2ARs expression levels have been found to be increased in PD patients with dyskinesias; this upregulation is attributed to the effect of levodopa (L-dopa) treatment (Calon et al., 2004). Recently, it has been proven that high A2ARs levels in the striatum and in lymphocytes correlate with motor symptoms in PD patients who were previously either not pharmacologically treated or were treated with a wide spectrum of drugs and not restricted to only L-dopa (Varani et al., 2010). Therefore, A2AR upregulation in PD, which tonically inhibits D2R (see above), together with the low dopamine content in the striatum, a consequence of the death of dopaminergic neurons from the substantia nigra, contribute to a synergistic impairment of D2R function.

It remains to be clarified whether upregulation of A2ARs levels in PD is a hallmark of the disease or is a consequence of dopaminergic terminal drop-off in the striatum. This issue has been quite controversial, as increased striatal levels of A2ARs were shown in 6 hydroxidopamine (6-OHDA)-treated rats, as a consequence of dopamine denervation (Pinna et al., 2002), and also in 6-OHDA-treated rats with intermittent L-dopa treatment (Tomiyama et al., 2004).

### **3. Clinical trials with A2AR antagonists in PD**

L-dopa remains the most effective treatment for symptomatic relief of PD, although its pharmacological administration over time induces motor dysfunctions such as dyskinesias (Obeso et al., 2000). One of the strategies to reduce these is the administration of

non-dopaminergic drugs that modulate dopaminergic neurotransmission. Indeed, A2AR has emerged as a potential pharmacological target in PD, as its relationship with the dopaminergic system has been clearly demonstrated (Ferré et al., 2002). There have been several clinical trials with A2AR antagonists, such as istradefylline (also known as KW-6002), confirming that their administration to PD patients reduces the "OFF" time and dyskinesias induced by L-dopa treatment (Bara-Jimenez et al., 2003; Factor et al., 2010; Hauser et al., 2003, 2008; LeWitt et al., 2008b; Mizuno et al., 2010; Stacy et al., 2008). As A2ARs expression levels are nearly exclusive of the striatum, the use of specific antagonists for these receptors could promote a specific brain-area effect (Brooks et al., 2008). Interestingly, it has recently been described how KW-6002 preferentially targets the A2AR within the postsynaptic A2AR/D2R oligomer located at the MSNs, thus potentiating D2R-mediated motor activation (Orru et al., 2011). Therefore, although the use of istradefylline as a monotheraphy in PD has not been statistically demonstrated (Fernandez et al., 2010), its administration as a coadjuvant seems to allow a reduction in the L-dopa dose.

As mentioned before, there are heteromers formed by A2AR, D2R and mGluR5 in striatal GABAergic neurons or MSNs (Cabello et al., 2009). It is proposed that the increase in glutamate and adenosine extracellular levels activates A2AR and mGluR5, both synergizing

Epigenetic Modulation of Adenosine A2A Receptor:

suppressed in SZ patients (Veldic et al., 2004, 2005).

gene promoter regions (Tremolizzo et al., 2002).

**5. Endogenous SAM biosynthesis cycle and alterations in PD**

methylation potential and its endogenous maintenance is very important.

A Putative Therapeutical Tool for the Treatment of Parkinson's Disease 299

Interestingly, Dnmt 1 is increased in the cortical interneurons where the GAD67 gene is

Finally, epigenetic therapies such as the use of demethylating agents are widely established in the treatment of tumors (Herranz & Esteller, 2006), but their use in neurodegeneration is poorly studied. In this line, S-adenosylmethionine (SAM) is a methyl group donor molecule necessary for DNA methylation which is reduced in AD (Linnebank et al., 2010; Morrison et al., 1996). There have been proposals to use it as a therapy for AD (Scarpa et al., 2003). Its administration to cell lines down-regulates PSEN1 and reduces -amyloid production. In contrast, deprivation of SAM up-regulates PSEN1, increasing -amyloid deposits in APP transgenic mice (Fuso et al., 2005, 2008). Interestingly, mice treated with L-methionine downregulate GAD67 and reelin levels by increasing DNA methylation of their respective

SAM is the main biological methyl donor molecule in the methionine metabolic cycle, which is involved in the methylation of DNA, and protein, lipid and polyamine synthesis. Moreover, it is a precursor of glutathione in the liver and also perhaps in the brain (Vitvitsky et al, 2006). When SAM is demethylated, it is transformed into Sadenosylhomocysteine (SAH) which in turn is hydrolysed into homocysteine (HCY) and adenosine. To prevent the accumulation of HCY, it is remethylated to form methionine (Chiang et al., 1996; Lu, 2000) (see Figure 1). The SAM/SAH ratio is also known as

L-dopa is the conventional drug used in the treatment of PD to minimize the lack of endogenous dopamine in these patients (Lewitt, 2008a; Tolosa et al., 1998). However, chronic treatment with L-dopa has been associated with hyperhomocysteinemia in plasma, peripheral tissues and brain of PD patients, as L-dopa metabolism requires Sadenosylmethionine (SAM) as a methyl donor (Lu, 2000; Müller et al., 2009a; Nutt, 2008; O'Suilleabhain et al, 2004; Zoccolella et al., 2006, 2009, 2010). Interestingly, it has been shown that L-dopa treatment in mice depletes the brain SAM content (Liu et al., 2000). In addition, elevated plasma homocysteine (Hcy) levels have been related to cognitive and motor impairment and have also been associated with the pathogenesis of other neurological diseases such as stroke and AD (Morris, 2003; Quadri et al., 2004; Seshadri et al., 2002). Interestingly, some polymorphisms described in *MTHFR* gene (methylenetetrahydrofolate reductase) have been associated with a reduction in its enzymatic activity, promoting an increase in the Hcy levels in L-dopa-treated PD patients (Frosst et al., 1995; Yasui et al., 2000). Vitamin B6 enhances the direct flow of Hcy to cysteine, the precursor of gutathione. Deficits in vitamin B6 induce oxidative stress and, in turn, enhance Hcy (Obeid et al., 2009). Therefore, several factors have been related with hyperhomocysteinemia and a concomitant reduction in the SAM levels in blood and cerebrospinal fluid of L-dopa-treated PD patients (Cheng et al., 1997). These include the following: 1. An excessive production of Sadenosylhomocysteine (SAH) when L-dopa is metabolized by catechol-O-methyltransferase (COMT), depleting the SAM levels an in turn decreasing the SAM/SAH ratio; 2. Reduced MTHFR enzymatic activity; and 3.  In this context, some clinical trials have been carried out using decarboxylase and COMT inhibitors (for instance, carbidopa and entacapone/tolcapone, respectively) or vitamin B12 and folate

and promoting the inhibition of D2Rs (Ferré et al., 2007). Interestingly, it has been demonstrated in a rat model of PD that the simultaneous inhibition of A2AR and mGluR5 synergistically reverses the parkinsonian deficits in these animals (Coccurello et al., 2004). In this line, the use of mGluR type I antagonists as a therapy for PD has been proposed (Bonsi et al., 2007). Overall, this explains why pharmaceutical companies are going after either single compounds or combinations of drugs that will simultaneously antagonize A2AR and mGluR5.

#### **4. Brain DNA methylation**

Studies in mice deficient in enzymes that control DNA methylation and the results of folatefree diets have established an important role for methylation in the development of the nervous system (Waterland & Jirtle, 2003). In fact, the manipulation of methylation and acetylation affects neuronal vulnerability in experimental models of neurodegenerative diseases (El-Maarri, 2003). It has also been proposed that memory processes are highly related with the level of neuronal methylation (Day & Sweatt, 2010; Liu et al., 2009).

DNA methylation is a normal process that occurs during mammal embryo development, and it is also implicated in X-chromosome inactivation and repression of proviral genes and endogenous transposons. This chemical modification is one of the most important epigenetic mechanisms in gene silencing in mammals. It is characterized by the methylation of cytosines that precede guanines in the well-known CpG sites. Those genome CpG-rich regions are called CpG islands (CGIs); they present a size between 200 bp and several kilobases. In general, these active gene promoter regions are not methylated, while the inactive gene promoter regions are fully methylated. Most CGIs are found in 5' UTR regions and in the first exon, although they can be found in regions distal to the transcription start site and in intronic regions. In normal tissues, CGIs are usually non-methylated, while they are methylated in tumorous cells, especially in tumor repressor genes (Illingworth & Bird, 2009; Jones, 1999).

Methylation of CpG sites is achieved by the action of DNA metyltransferases (Dnmt) which catalyze the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to DNA. The human DNA Methylome map has recently been published, annotating those genes found methylated in normal tissues and in human diseases such as Alzheimer's disease (AD) and schizophrenia (SZ) (Ballestar & Esteller, 2008). DNA methylation in the CpG sites interferes with gene expression in two ways. The first is interference with the binding of transcription factors to DNA through the methyl group. The second is caused by the binding of specific proteins, such as MeCP2, MBD1 and MBD2, to methyl CpG sites (methyl-CpG-binding proteins, MBDs). These MBDs recruit histone-modifying and chromatin-remodeling complexes to methylated sites (Portela & Esteller, 2010). The importance of these proteins is demonstrated by Rett syndrome, a disease causing severe mental dysfunction and brought on by MeCP2 mutations (Amir et al., 1999).

The role of DNA methylation in the brain is an emerging field of research. Neuronal DNA methylation is modified with lifespan, and the analysis of 12 loci related with AD has revealed an age-specific epigenetic drift in the percentage of DNA methylation (Siegmund et al. 2007; Wang et al., 2008). Moreover, the degree of gene methylation varies among the different cerebral regions, and it has been reported that DNA methyltransferase 1 (Dnmt1) expression levels are different in the various cerebral regions (Ladd-Acosta et al., 2007).

and promoting the inhibition of D2Rs (Ferré et al., 2007). Interestingly, it has been demonstrated in a rat model of PD that the simultaneous inhibition of A2AR and mGluR5 synergistically reverses the parkinsonian deficits in these animals (Coccurello et al., 2004). In this line, the use of mGluR type I antagonists as a therapy for PD has been proposed (Bonsi et al., 2007). Overall, this explains why pharmaceutical companies are going after either single compounds or combinations of drugs that will simultaneously antagonize A2AR and

Studies in mice deficient in enzymes that control DNA methylation and the results of folatefree diets have established an important role for methylation in the development of the nervous system (Waterland & Jirtle, 2003). In fact, the manipulation of methylation and acetylation affects neuronal vulnerability in experimental models of neurodegenerative diseases (El-Maarri, 2003). It has also been proposed that memory processes are highly

DNA methylation is a normal process that occurs during mammal embryo development, and it is also implicated in X-chromosome inactivation and repression of proviral genes and endogenous transposons. This chemical modification is one of the most important epigenetic mechanisms in gene silencing in mammals. It is characterized by the methylation of cytosines that precede guanines in the well-known CpG sites. Those genome CpG-rich regions are called CpG islands (CGIs); they present a size between 200 bp and several kilobases. In general, these active gene promoter regions are not methylated, while the inactive gene promoter regions are fully methylated. Most CGIs are found in 5' UTR regions and in the first exon, although they can be found in regions distal to the transcription start site and in intronic regions. In normal tissues, CGIs are usually non-methylated, while they are methylated in tumorous cells, especially in tumor repressor genes (Illingworth & Bird,

Methylation of CpG sites is achieved by the action of DNA metyltransferases (Dnmt) which catalyze the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to DNA. The human DNA Methylome map has recently been published, annotating those genes found methylated in normal tissues and in human diseases such as Alzheimer's disease (AD) and schizophrenia (SZ) (Ballestar & Esteller, 2008). DNA methylation in the CpG sites interferes with gene expression in two ways. The first is interference with the binding of transcription factors to DNA through the methyl group. The second is caused by the binding of specific proteins, such as MeCP2, MBD1 and MBD2, to methyl CpG sites (methyl-CpG-binding proteins, MBDs). These MBDs recruit histone-modifying and chromatin-remodeling complexes to methylated sites (Portela & Esteller, 2010). The importance of these proteins is demonstrated by Rett syndrome, a disease causing severe mental dysfunction and brought

The role of DNA methylation in the brain is an emerging field of research. Neuronal DNA methylation is modified with lifespan, and the analysis of 12 loci related with AD has revealed an age-specific epigenetic drift in the percentage of DNA methylation (Siegmund et al. 2007; Wang et al., 2008). Moreover, the degree of gene methylation varies among the different cerebral regions, and it has been reported that DNA methyltransferase 1 (Dnmt1) expression levels are different in the various cerebral regions (Ladd-Acosta et al., 2007).

related with the level of neuronal methylation (Day & Sweatt, 2010; Liu et al., 2009).

mGluR5.

2009; Jones, 1999).

on by MeCP2 mutations (Amir et al., 1999).

**4. Brain DNA methylation**

Interestingly, Dnmt 1 is increased in the cortical interneurons where the GAD67 gene is suppressed in SZ patients (Veldic et al., 2004, 2005).

Finally, epigenetic therapies such as the use of demethylating agents are widely established in the treatment of tumors (Herranz & Esteller, 2006), but their use in neurodegeneration is poorly studied. In this line, S-adenosylmethionine (SAM) is a methyl group donor molecule necessary for DNA methylation which is reduced in AD (Linnebank et al., 2010; Morrison et al., 1996). There have been proposals to use it as a therapy for AD (Scarpa et al., 2003). Its administration to cell lines down-regulates PSEN1 and reduces -amyloid production. In contrast, deprivation of SAM up-regulates PSEN1, increasing -amyloid deposits in APP transgenic mice (Fuso et al., 2005, 2008). Interestingly, mice treated with L-methionine downregulate GAD67 and reelin levels by increasing DNA methylation of their respective gene promoter regions (Tremolizzo et al., 2002).

#### **5. Endogenous SAM biosynthesis cycle and alterations in PD**

SAM is the main biological methyl donor molecule in the methionine metabolic cycle, which is involved in the methylation of DNA, and protein, lipid and polyamine synthesis. Moreover, it is a precursor of glutathione in the liver and also perhaps in the brain (Vitvitsky et al, 2006). When SAM is demethylated, it is transformed into Sadenosylhomocysteine (SAH) which in turn is hydrolysed into homocysteine (HCY) and adenosine. To prevent the accumulation of HCY, it is remethylated to form methionine (Chiang et al., 1996; Lu, 2000) (see Figure 1). The SAM/SAH ratio is also known as methylation potential and its endogenous maintenance is very important.

L-dopa is the conventional drug used in the treatment of PD to minimize the lack of endogenous dopamine in these patients (Lewitt, 2008a; Tolosa et al., 1998). However, chronic treatment with L-dopa has been associated with hyperhomocysteinemia in plasma, peripheral tissues and brain of PD patients, as L-dopa metabolism requires Sadenosylmethionine (SAM) as a methyl donor (Lu, 2000; Müller et al., 2009a; Nutt, 2008; O'Suilleabhain et al, 2004; Zoccolella et al., 2006, 2009, 2010). Interestingly, it has been shown that L-dopa treatment in mice depletes the brain SAM content (Liu et al., 2000). In addition, elevated plasma homocysteine (Hcy) levels have been related to cognitive and motor impairment and have also been associated with the pathogenesis of other neurological diseases such as stroke and AD (Morris, 2003; Quadri et al., 2004; Seshadri et al., 2002). Interestingly, some polymorphisms described in *MTHFR* gene (methylenetetrahydrofolate reductase) have been associated with a reduction in its enzymatic activity, promoting an increase in the Hcy levels in L-dopa-treated PD patients (Frosst et al., 1995; Yasui et al., 2000). Vitamin B6 enhances the direct flow of Hcy to cysteine, the precursor of gutathione. Deficits in vitamin B6 induce oxidative stress and, in turn, enhance Hcy (Obeid et al., 2009). Therefore, several factors have been related with hyperhomocysteinemia and a concomitant reduction in the SAM levels in blood and cerebrospinal fluid of L-dopa-treated PD patients (Cheng et al., 1997). These include the following: 1. An excessive production of Sadenosylhomocysteine (SAH) when L-dopa is metabolized by catechol-O-methyltransferase (COMT), depleting the SAM levels an in turn decreasing the SAM/SAH ratio; 2. Reduced MTHFR enzymatic activity; and 3.  In this context, some clinical trials have been carried out using decarboxylase and COMT inhibitors (for instance, carbidopa and entacapone/tolcapone, respectively) or vitamin B12 and folate

Epigenetic Modulation of Adenosine A2A Receptor:

n=3; dotted lines, U87-MG, n=3).

**7. Clinical trials with SAM**

A Putative Therapeutical Tool for the Treatment of Parkinson's Disease 301

(human glioblastoma) cell lines. In this context, A2ARs levels have been reported to be upregulated in PD patients (Calon et al., 2004; Varani et al., 2010). Therefore, we postulated that SAM treatment could represent a therapeutical tool to reduce A2ARs levels in these patients. This is based on the fact that the DNA methylation profile of striatal *ADORA2A* in PD patients is lower than the one found in SH-SY5Y and U87MG cells (Figure 2B). Therefore, as A2ARs cell surface levels are reduced in these cells after SAM treatment, due to an increase in the DNA methylation profile of *ADORA2A* (Buira et al., 2010a), it is also plausible that the same mechanism of action could play a role in the striatum of PD patients.

Fig. 2. A, Scaled representation of 5' UTR region of human *ADORA2A* gene, containing 6 isoforms of non-coding exon 1 (1A-1F). Two CGIs surrounding exon 1E were recently described (Buira et al., 2010b). The translational start site (ATG) is indicated with an arrow. B, DNA methylation percentage (mean ± SD) of a locus located in the CGIs of exon 1E of 8 human post-mortem putamens of PD (black bars) and in two cell lines (black line, SH-SY5Y,

SAM has been widely used for the treatment of liver diseases, as it increases the glutathione content (Friedel et al. 1989). Interestingly, SAM has antidepressant properties and its long term tolerability is excellent, presenting few side effects (Bell et al., 1988; Bottiglieri & Hyland, 1994; Delle Chiaie et al., 2002; Kagan et al., 1990; Lipinski et al., 1984; Papakostas, 2009, 2010). Moreover, SAM administration in patients with depression and dementia, intravenously or orally, has shown that it crosses the blood-brain barrier, and as a result, it is

Impaired transmethylation potential in L-dopa-treated PD patients has been proposed (De Bonis et al., 2010). The authors argue that a possible global DNA hypomethylation in hyperhomocysteinemic PD patients could be responsible for a generalized gene expression

detected at increased levels in the cerebrospinal fluid (Bottiglieri et al., 1990).

supplementation to reduce Hcy levels in PD patients (Müller et al., 2003, 2006; Zoccolella et al., 2007).

Fig. 1. SAM biosynthesis cycle

### **6. Epigenetic study of** *ADORA2A*

A2AR gene (ADORA2A) is localized to chromosome 22 (Le et al., 1996; MacCollin et al., 1994; Peterfreund et al., 1996). It consists of two coding exons (exon 2 and 3) separated by a single intron of nearly 7 Kb. The exon 1 is a non-coding exon which is located at 5′ upstream exon 2 and presents 6 tissue-specific isoforms: h1A-h1F (Yu et al., 2004) (Figure 2A). Interestingly, differential expression of these isoforms has been reported in granulocytes of patients suffering from sepsis, indicating that 5′ UTR plays an important regulatory role in A2AR expression (Kreth et al., 2008). We recently identified a functional CGI surrounding the h1E isoform, demonstrating that DNA methylation controls basal *ADORA2A* expression in several cell lines and that it is one of the molecular mechanisms responsible for A2ARs' differential expression levels in specific human brain areas, such as the putamen and the cerebellum (Buira et al., 2010a, 2010b). Interestingly, we showed that A2AR expression levels can be modulated by SAM treatment in SH-SY5Y (human neuroblastoma) and U87MG

supplementation to reduce Hcy levels in PD patients (Müller et al., 2003, 2006; Zoccolella et

A2AR gene (ADORA2A) is localized to chromosome 22 (Le et al., 1996; MacCollin et al., 1994; Peterfreund et al., 1996). It consists of two coding exons (exon 2 and 3) separated by a single intron of nearly 7 Kb. The exon 1 is a non-coding exon which is located at 5′ upstream exon 2 and presents 6 tissue-specific isoforms: h1A-h1F (Yu et al., 2004) (Figure 2A). Interestingly, differential expression of these isoforms has been reported in granulocytes of patients suffering from sepsis, indicating that 5′ UTR plays an important regulatory role in A2AR expression (Kreth et al., 2008). We recently identified a functional CGI surrounding the h1E isoform, demonstrating that DNA methylation controls basal *ADORA2A* expression in several cell lines and that it is one of the molecular mechanisms responsible for A2ARs' differential expression levels in specific human brain areas, such as the putamen and the cerebellum (Buira et al., 2010a, 2010b). Interestingly, we showed that A2AR expression levels can be modulated by SAM treatment in SH-SY5Y (human neuroblastoma) and U87MG

al., 2007).

Fig. 1. SAM biosynthesis cycle

**6. Epigenetic study of** *ADORA2A*

(human glioblastoma) cell lines. In this context, A2ARs levels have been reported to be upregulated in PD patients (Calon et al., 2004; Varani et al., 2010). Therefore, we postulated that SAM treatment could represent a therapeutical tool to reduce A2ARs levels in these patients. This is based on the fact that the DNA methylation profile of striatal *ADORA2A* in PD patients is lower than the one found in SH-SY5Y and U87MG cells (Figure 2B). Therefore, as A2ARs cell surface levels are reduced in these cells after SAM treatment, due to an increase in the DNA methylation profile of *ADORA2A* (Buira et al., 2010a), it is also plausible that the same mechanism of action could play a role in the striatum of PD patients.

Fig. 2. A, Scaled representation of 5' UTR region of human *ADORA2A* gene, containing 6 isoforms of non-coding exon 1 (1A-1F). Two CGIs surrounding exon 1E were recently described (Buira et al., 2010b). The translational start site (ATG) is indicated with an arrow. B, DNA methylation percentage (mean ± SD) of a locus located in the CGIs of exon 1E of 8 human post-mortem putamens of PD (black bars) and in two cell lines (black line, SH-SY5Y, n=3; dotted lines, U87-MG, n=3).

### **7. Clinical trials with SAM**

SAM has been widely used for the treatment of liver diseases, as it increases the glutathione content (Friedel et al. 1989). Interestingly, SAM has antidepressant properties and its long term tolerability is excellent, presenting few side effects (Bell et al., 1988; Bottiglieri & Hyland, 1994; Delle Chiaie et al., 2002; Kagan et al., 1990; Lipinski et al., 1984; Papakostas, 2009, 2010). Moreover, SAM administration in patients with depression and dementia, intravenously or orally, has shown that it crosses the blood-brain barrier, and as a result, it is detected at increased levels in the cerebrospinal fluid (Bottiglieri et al., 1990).

Impaired transmethylation potential in L-dopa-treated PD patients has been proposed (De Bonis et al., 2010). The authors argue that a possible global DNA hypomethylation in hyperhomocysteinemic PD patients could be responsible for a generalized gene expression

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dysregulation and for playing a role in the outcome of the pathology. In accordance with this, another study has shown improved cognitive function in PD patients with a higher SAM/SAH ratio and higher plasma vitamin B6 (Obeid et al., 2009). It is noteworthy that SAM treatment improved depression of PD patients in an open-label clinical trial (Di Rocco et al., 2000). In fact, vitamin dietary supplementation, including SAM, has been shown to be effective in patients with early and moderate stages of AD (Chan et al., 2008; Panza et al., 2009; Remington et al., 2009; Shea and Chan, 2008). Moreover, SAM supplementation also presented antioxidant properties in an AD animal model (Cavallaro et al., 2010). In line with this, oxidative stress is also present in early-stages of PD (Ferrer et al., 2010), which points up the benefits of SAM administration in this pathology as an adjunctive treatment.

#### **8. Conclusions and proposals for future PD interventions**

As mentioned in the introduction, it is noteworthy that inactivation of A2ARs enhances the affinity of D2Rs for dopamine, this being the probable mechanism underlying the prodopaminergic effect of A2ARs antagonists in several clinical trials with PD patients. In this chapter, we have examined the literature which, in combination with our studies based on *ADORA2A* transcriptional regulation, has led us to propose SAM treatment as an epigenetic tool to modulate the increased expression of A2ARs in PD.

It is obvious that SAM treatment presents a broad spectrum of gene targets, and not only tumor supressor genes. However, it has been reported that SAM treatment promotes a decrease in the growth of hepatocellular carcinoma cells and liver cancer in animal models, hypothesising the methylation and repression of oncogenic gene promoters by this drug (Cai et al., 1998; Pascale et al., 1992). However, the existence of several clinical trials with SAM and the reduced number of side-effects in its administration must be taken into account. Based on our studies, and bearing in mind the restrictive expression of A2ARs in the brain (mainly in 95% of striatal neurons), we postulate that SAM treatment would have a "specific" effect on A2ARs in the brain. This would be especially true in a cerebral region where it colocalizes with D2Rs, which in turn present reduced activity due to the low dopamine content in PD. Then, although SAM treatment would reduce the expression of hypomethylated genes, its effect on A2ARs might represent significant activation of D2Rs. Thus, the possible beneficial role of SAM in these patients should be examined in

randomized controlled studies, examining supplementation to L-dopa (allowing a reduction of its dose) and to A2ARs antagonists (such as istradefylline), or in triple administration with both current therapies.

#### **9. Acknowledgments**

We are very grateful to Dr. Sandra Pérez Buira, Dr. José Luis Albasanz, Guido Dentesano and Jesús Moreno for their contributions to the study of *ADORA2A* transcriptional regulation. We thank T. Yohannan for editorial help. The experimental work described in the present chapter was funded by grants from the Ministerio de Ciencia e Innovación, (PI05/1631, CP08/00095, BFU2008-00138), the European Union through the Marie-Curie Research Training Network PRAIRIES (Contract MRTN-CT-2006-035810), the Consejería de Educación y Ciencia (PCI08-0125), the Consejería de Sanidad-FISCAM (PI-2007/50 and G-2007-C/13) of the Junta de Comunidades de Castilla-La Mancha and the Fundació La Marató de TV3 (092330, 090331).

#### **10. References**

302 Towards New Therapies for Parkinson's Disease

dysregulation and for playing a role in the outcome of the pathology. In accordance with this, another study has shown improved cognitive function in PD patients with a higher SAM/SAH ratio and higher plasma vitamin B6 (Obeid et al., 2009). It is noteworthy that SAM treatment improved depression of PD patients in an open-label clinical trial (Di Rocco et al., 2000). In fact, vitamin dietary supplementation, including SAM, has been shown to be effective in patients with early and moderate stages of AD (Chan et al., 2008; Panza et al., 2009; Remington et al., 2009; Shea and Chan, 2008). Moreover, SAM supplementation also presented antioxidant properties in an AD animal model (Cavallaro et al., 2010). In line with this, oxidative stress is also present in early-stages of PD (Ferrer et al., 2010), which points

up the benefits of SAM administration in this pathology as an adjunctive treatment.

As mentioned in the introduction, it is noteworthy that inactivation of A2ARs enhances the affinity of D2Rs for dopamine, this being the probable mechanism underlying the prodopaminergic effect of A2ARs antagonists in several clinical trials with PD patients. In this chapter, we have examined the literature which, in combination with our studies based on *ADORA2A* transcriptional regulation, has led us to propose SAM treatment as an

It is obvious that SAM treatment presents a broad spectrum of gene targets, and not only tumor supressor genes. However, it has been reported that SAM treatment promotes a decrease in the growth of hepatocellular carcinoma cells and liver cancer in animal models, hypothesising the methylation and repression of oncogenic gene promoters by this drug (Cai et al., 1998; Pascale et al., 1992). However, the existence of several clinical trials with SAM and the reduced number of side-effects in its administration must be taken into account. Based on our studies, and bearing in mind the restrictive expression of A2ARs in the brain (mainly in 95% of striatal neurons), we postulate that SAM treatment would have a "specific" effect on A2ARs in the brain. This would be especially true in a cerebral region where it colocalizes with D2Rs, which in turn present reduced activity due to the low dopamine content in PD. Then, although SAM treatment would reduce the expression of hypomethylated genes, its effect on A2ARs might represent significant activation of D2Rs. Thus, the possible beneficial role of SAM in these patients should be examined in randomized controlled studies, examining supplementation to L-dopa (allowing a reduction of its dose) and to A2ARs antagonists (such as istradefylline), or in triple administration with

We are very grateful to Dr. Sandra Pérez Buira, Dr. José Luis Albasanz, Guido Dentesano and Jesús Moreno for their contributions to the study of *ADORA2A* transcriptional regulation. We thank T. Yohannan for editorial help. The experimental work described in the present chapter was funded by grants from the Ministerio de Ciencia e Innovación, (PI05/1631, CP08/00095, BFU2008-00138), the European Union through the Marie-Curie Research Training Network PRAIRIES (Contract MRTN-CT-2006-035810), the Consejería de Educación y Ciencia (PCI08-0125), the Consejería de Sanidad-FISCAM (PI-2007/50 and G-2007-C/13) of the Junta de Comunidades de Castilla-La Mancha and the Fundació La

**8. Conclusions and proposals for future PD interventions**

epigenetic tool to modulate the increased expression of A2ARs in PD.

both current therapies.

**9. Acknowledgments**

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**Part 5**

**Cell Therapies**

homocysteine levels in L-dopa-treated Parkinson's disease patients with dyskinesias. *Clinical chemistry and laboratory medicine*, Vol.44, No.7, pp. 863-866

## **Part 5**

**Cell Therapies**

312 Towards New Therapies for Parkinson's Disease

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Hyperhomocysteinemia in L-dopa treated patients with Parkinson's disease: potential implications in cognitive dysfunction and dementia? *Current medicinal*

**15**

*Italy*

**1980-2011: Parkinson's Disease**

*2Laboratory of Functional Neurochemistry, Interdepartmental*

Replenishing the depleted striatal dopamine stores with its immediate precursor, L-3,4 dihydroxyphenylalanine (L-DOPA), to mimic dopamine-mediated neurotransmission still represents the gold standard for treating Parkinson's disease (PD). This pharmacological therapy offers immediate and effective symptomatic relief, especially at the early stages of the disease; it has, however, no influence on underlying neurodegenerative processes (Dass et al., 2006) that continue to evolve with time and are paralleled by a gradual loss of drug efficacy. As the disease progresses, steady adaptation, mostly continuous increase, of dopaminergic drug dosage is necessary, thereby favoring the emergence of considerable side effects, such as dyskinesia and psychiatric disturbances. The development of new treatments, or combination of treatments, able to relief motor symptoms and also delay or even halt the loss of dopaminergic neurons, has been and remains a fundamental issue for the development of innovative clinical strategies in PD. Transplantation of dopaminesecreting cells directly providing dopamine in the striatum, in particular, has been considered an adequate substitute to pharmacotherapy. However, although efficacy of this approach has now been asserted in numerous pre-clinical studies utilizing animal models of PD, positive outcomes in clinical trials involving PD patients have been very variable and rather modest, and have been plagued by graft-induced dyskinesias. New sources for cell replacement and particularly stem cells (including induced patient-derived cells**)** may now

This chapter will briefly introduce rodent and nonhuman primate PD-like models commonly used in pre-clinical studies, which represent a fundamental platform for the preclinical evaluation of innovative interventions. We will then evidence the progresses accomplished since the first intracerebral transplantation of fetal neural tissue in PD patients describing the subsequent novel discoveries for the application of stem cell to pre-clinical PD models, and give an overview of ongoing cell-based therapeutic strategies. Thereafter, multiple issues connected to stem transplantation to efficiently contrast adverse effects of increased age will be reviewed including decrease of apoptosis related to tissue degeneration, requirement of correct graft integration in the host vascular and neural

provide advantages for future clinical therapies (Wakeman et al., 2011)**.**

**1. Introduction**

Lidia Cova1 and Marie-Therese Armentero2 *1Department of Neurology, Laboratory of Neuroscience,*

*IRCCS Istituto Auxologico Italiano, Milan*

 *Research Centre for Parkinson's Disease, IRCCS National Institute of Neurology C. Mondino, Pavia,*

**and Advance in Stem Cell Research**

### **1980-2011: Parkinson's Disease and Advance in Stem Cell Research**

Lidia Cova1 and Marie-Therese Armentero2

*1Department of Neurology, Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, Milan 2Laboratory of Functional Neurochemistry, Interdepartmental Research Centre for Parkinson's Disease, IRCCS National Institute of Neurology C. Mondino, Pavia, Italy*

#### **1. Introduction**

Replenishing the depleted striatal dopamine stores with its immediate precursor, L-3,4 dihydroxyphenylalanine (L-DOPA), to mimic dopamine-mediated neurotransmission still represents the gold standard for treating Parkinson's disease (PD). This pharmacological therapy offers immediate and effective symptomatic relief, especially at the early stages of the disease; it has, however, no influence on underlying neurodegenerative processes (Dass et al., 2006) that continue to evolve with time and are paralleled by a gradual loss of drug efficacy. As the disease progresses, steady adaptation, mostly continuous increase, of dopaminergic drug dosage is necessary, thereby favoring the emergence of considerable side effects, such as dyskinesia and psychiatric disturbances. The development of new treatments, or combination of treatments, able to relief motor symptoms and also delay or even halt the loss of dopaminergic neurons, has been and remains a fundamental issue for the development of innovative clinical strategies in PD. Transplantation of dopaminesecreting cells directly providing dopamine in the striatum, in particular, has been considered an adequate substitute to pharmacotherapy. However, although efficacy of this approach has now been asserted in numerous pre-clinical studies utilizing animal models of PD, positive outcomes in clinical trials involving PD patients have been very variable and rather modest, and have been plagued by graft-induced dyskinesias. New sources for cell replacement and particularly stem cells (including induced patient-derived cells**)** may now provide advantages for future clinical therapies (Wakeman et al., 2011)**.**

This chapter will briefly introduce rodent and nonhuman primate PD-like models commonly used in pre-clinical studies, which represent a fundamental platform for the preclinical evaluation of innovative interventions. We will then evidence the progresses accomplished since the first intracerebral transplantation of fetal neural tissue in PD patients describing the subsequent novel discoveries for the application of stem cell to pre-clinical PD models, and give an overview of ongoing cell-based therapeutic strategies. Thereafter, multiple issues connected to stem transplantation to efficiently contrast adverse effects of increased age will be reviewed including decrease of apoptosis related to tissue degeneration, requirement of correct graft integration in the host vascular and neural

1980-2011: Parkinson's Disease and Advance in Stem Cell Research 317

primates. Toxins can be given systemically or intra-cerebrally, depending on the type of toxin used and animal specie involved, and mimic the selective degeneration of nigrostriatal

The classical systemic model is based on the injection of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), a toxin that selectively affects dopaminergic neurons and first recognized in the mid-1980's as the cause of marked parkinsonism in young drug users of Northern California (Langston et al., 1983). MPTP crosses the blood brain barrier and is transformed in its active metabolite, 1-methyl-4-phenylpyridinium ion (MPP+) that is then actively taken up by dopaminergic neurons of the substantia nigra pars compacta (SNc). Systemic administration of MPTP to nonhuman primates induces a parkinsonian phenotype closely resembling the human pathology. MPTP-treated monkeys have an excellent antisymptomatic response to dopaminergic drug treatment and develop motor complications linked to long term L-DOPA therapy (Kim et al., 2009). Concordantly, several pharmacological drugs currently applied to treated PD patients (i.e. dopamine agonists, amantadine, etc..) have been developed in this MPTP model (Fox et al., 2006). Alternative cell replacement strategies using various cell sources have been successfully performed indicating the technical feasibility of the model for future studies (Redmond et al., 2010; Serra et al., 2008). The large related costs and the difficulty to reliably standardize acute toxin administration to replicate most of the underlying mechanisms of a chronic progressive disorder form still represent major drawbacks and limit the use of this PD model in large scale studies (Fox and Brotchie, 2010). MPTP can also be systemically administered to mice but not rat, which are resistant, and several different experimental

The prototypical intracerebral model is based on the local injection of 6-hydroxydopamine (6-OHDA) and was the first PD animal model ever generated (Ungerstedt, 1968). Six-OHDA is a hydroxylated analogue of dopamine and, similarly to MPTP, is actively taken up by dopaminergic neurons. The neurotoxin can be injected in the SNc, or into the medial forebrain bundle (mfb) that conveys efferent fibers from the nigral cell bodies to the striatum (as shown in Figure 1) and causes massive and rapid anterograde degeneration of the nigrostriatal pathway. This procedure induces large nigral cell loss and striatal dopamine depletion (90-100%). The neurotoxin can also be injected into the striatum; in this case rapid damage to striatal dopaminergic terminals is observed followed by a progressive loss of SNc neurons (50-70% neuronal loss), which are secondarily affected through a "dying back" mechanism. This procedure has a slower time course compared to the intra-mfb injection and provides a progressive model of nigrostriatal degeneration, more similar to the gradual evolution of the neurodegenerative process of human PD. Importantly, injection of 6-OHDA is commonly carried out unilaterally, with the contralateral hemisphere serving as control, because of the high mortality rate associated with bilateral injection. The rat 6-OHDA model is commonly used in neuroprotective studies, both involving administration of novel pharmaceutical compounds or cell transplantation because it is i) cost-effective, ii) highly reproducible and iii) opened to articulate behavioural analyses (Redmond et al., 2010; Serra

Numerous transgenic mouse models, that reproduce monogenic mutations observed in familiar forms of PD, have also been developed over the years. These models have not been discussed in this chapter as they typically display very low degree or even no nigrostriatal degeneration (for review see (Dawson et al., 2010)) and are not commonly used in stem cell

neurons characteristic of the human disorder.

paradigms have been developed and used over time.

et al., 2008).

transplantation experiments.

circuits, reduction of diffuse inflammatory response/oxidative stress and correct release of key growth factors. In addition we will discuss the emergence of novel biotechnologies that will, most likely, help unravel the complex interrelationship between transplanted stem cells and the host environment and will favor the development of novel therapeutic procedures readily applicable in PD patients.

#### **2. Animal models: A necessary pre-clinical tool for the development of innovative cellular therapy in PD**

Animal models represent a fundamental step in the attempt to elucidate gene-environment interactions and to define pathogenic mechanisms involved in the aetiology and progression of the neurodegenerative diseases. Most of the current knowledge on pathophysiology of PD originates from studies conducted on animal models of the disease, since animals and humans share several anatomical features (as shown in Figure 1). In addition, they represent the first essential pre-clinical platform for the evaluation of any targeted therapeutic intervention. Notably, subsequent clinical trials in small human cohorts remain essential for the development of efficient therapies able also to alleviate disability related non-motor symptoms (Meissner et al., 2011) which are currently underestimated in animal models (Dunnett and Lelos, 2010).

Fig. 1. Anatomical comparison between mouse and human brains

The comparison between rodent (A) and human brain (B) enlightens their anatomical similarities and physiology. The connections between striatum, SNc, and cerebral cortex in the human brain are indicated by dotted black lines and may correlate the simultaneous degeneration of these areas during disease onset and progression. Moreover, dopaminergic innervations of the striatum/SVZ (black dotted lines) could also explain reduction of adult brain neurogenesis both in PD patients and animal models.

PD models have been classically based on the administration of neurotoxins able to replicate some of the pathological and phenotypic features of the human disease both in rodents and

circuits, reduction of diffuse inflammatory response/oxidative stress and correct release of key growth factors. In addition we will discuss the emergence of novel biotechnologies that will, most likely, help unravel the complex interrelationship between transplanted stem cells and the host environment and will favor the development of novel therapeutic procedures

Animal models represent a fundamental step in the attempt to elucidate gene-environment interactions and to define pathogenic mechanisms involved in the aetiology and progression of the neurodegenerative diseases. Most of the current knowledge on pathophysiology of PD originates from studies conducted on animal models of the disease, since animals and humans share several anatomical features (as shown in Figure 1). In addition, they represent the first essential pre-clinical platform for the evaluation of any targeted therapeutic intervention. Notably, subsequent clinical trials in small human cohorts remain essential for the development of efficient therapies able also to alleviate disability related non-motor symptoms (Meissner et al., 2011) which are currently underestimated in animal models

**2. Animal models: A necessary pre-clinical tool for the development of**

Fig. 1. Anatomical comparison between mouse and human brains

brain neurogenesis both in PD patients and animal models.

The comparison between rodent (A) and human brain (B) enlightens their anatomical similarities and physiology. The connections between striatum, SNc, and cerebral cortex in the human brain are indicated by dotted black lines and may correlate the simultaneous degeneration of these areas during disease onset and progression. Moreover, dopaminergic innervations of the striatum/SVZ (black dotted lines) could also explain reduction of adult

PD models have been classically based on the administration of neurotoxins able to replicate some of the pathological and phenotypic features of the human disease both in rodents and

readily applicable in PD patients.

(Dunnett and Lelos, 2010).

**innovative cellular therapy in PD**

primates. Toxins can be given systemically or intra-cerebrally, depending on the type of toxin used and animal specie involved, and mimic the selective degeneration of nigrostriatal neurons characteristic of the human disorder.

The classical systemic model is based on the injection of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), a toxin that selectively affects dopaminergic neurons and first recognized in the mid-1980's as the cause of marked parkinsonism in young drug users of Northern California (Langston et al., 1983). MPTP crosses the blood brain barrier and is transformed in its active metabolite, 1-methyl-4-phenylpyridinium ion (MPP+) that is then actively taken up by dopaminergic neurons of the substantia nigra pars compacta (SNc). Systemic administration of MPTP to nonhuman primates induces a parkinsonian phenotype closely resembling the human pathology. MPTP-treated monkeys have an excellent antisymptomatic response to dopaminergic drug treatment and develop motor complications linked to long term L-DOPA therapy (Kim et al., 2009). Concordantly, several pharmacological drugs currently applied to treated PD patients (i.e. dopamine agonists, amantadine, etc..) have been developed in this MPTP model (Fox et al., 2006). Alternative cell replacement strategies using various cell sources have been successfully performed indicating the technical feasibility of the model for future studies (Redmond et al., 2010; Serra et al., 2008). The large related costs and the difficulty to reliably standardize acute toxin administration to replicate most of the underlying mechanisms of a chronic progressive disorder form still represent major drawbacks and limit the use of this PD model in large scale studies (Fox and Brotchie, 2010). MPTP can also be systemically administered to mice but not rat, which are resistant, and several different experimental paradigms have been developed and used over time.

The prototypical intracerebral model is based on the local injection of 6-hydroxydopamine (6-OHDA) and was the first PD animal model ever generated (Ungerstedt, 1968). Six-OHDA is a hydroxylated analogue of dopamine and, similarly to MPTP, is actively taken up by dopaminergic neurons. The neurotoxin can be injected in the SNc, or into the medial forebrain bundle (mfb) that conveys efferent fibers from the nigral cell bodies to the striatum (as shown in Figure 1) and causes massive and rapid anterograde degeneration of the nigrostriatal pathway. This procedure induces large nigral cell loss and striatal dopamine depletion (90-100%). The neurotoxin can also be injected into the striatum; in this case rapid damage to striatal dopaminergic terminals is observed followed by a progressive loss of SNc neurons (50-70% neuronal loss), which are secondarily affected through a "dying back" mechanism. This procedure has a slower time course compared to the intra-mfb injection and provides a progressive model of nigrostriatal degeneration, more similar to the gradual evolution of the neurodegenerative process of human PD. Importantly, injection of 6-OHDA is commonly carried out unilaterally, with the contralateral hemisphere serving as control, because of the high mortality rate associated with bilateral injection. The rat 6-OHDA model is commonly used in neuroprotective studies, both involving administration of novel pharmaceutical compounds or cell transplantation because it is i) cost-effective, ii) highly reproducible and iii) opened to articulate behavioural analyses (Redmond et al., 2010; Serra et al., 2008).

Numerous transgenic mouse models, that reproduce monogenic mutations observed in familiar forms of PD, have also been developed over the years. These models have not been discussed in this chapter as they typically display very low degree or even no nigrostriatal degeneration (for review see (Dawson et al., 2010)) and are not commonly used in stem cell transplantation experiments.

1980-2011: Parkinson's Disease and Advance in Stem Cell Research 319

progeny, have been successfully transplanted in animal models of PD granting significant behavioral improvements. Ideally, however, new therapies should not only aim at replenishing the depleted dopamine store, but should also allow rescue of neuronal

Innovative therapeutic strategy should also take advantage and enhance the plastic property of the adult Central Nervous System (CNS) to regenerate affected brain areas through the activation of endogenous neurogenesis following cell transplantation. To reach these targets, other known intrinsic properties of SCs have already been exploited. Indeed, the potential of SCs to restore injured tissues is not only related to their direct differentiation but also to their capacity to produce and release trophic factors that may in turn inhibit apoptosis, promote angiogenesis and even direct stimulation of host regeneration (Lindvall and Kokaia, 2009; Lindvall and Kokaia, 2010). Neuroprotection can also be enhanced by transplanting SCs engineered to express either tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine synthesis, or neurotrophic factors, such as glial cell-line derived neurotrophic factor (GDNF), the most potent survival factor for degenerating dopaminergic neurons. Grafting of engineered SCs in this case provides a substantial reservoir allowing the unlimited supply of the required substance without the requirement of invasive injection pumps, as detailed below. Several SC types have already been transplanted in PD

animal models yielding interesting but variable results (Wakeman et al., 2011).

SCs can be efficiently derived from early stage embryos (pluripotent SCs with unlimited potential to differentiate) or from committed resident tissues (multipotent SCs with restricted potential to differentiate). Interestingly almost all organs arising from endodermal, mesodermal and ectodermal germ layers can originate both fetal and adult SCs (i.e. amniotic/cord blood and tissue derived SCs). Regardless of their origin SCs could physiologically repair damaged tissues after intense injuries also promoting angiogenesis and neurogenesis processes, essential for CNS development and regeneration (Lindvall and

Embryonic Stem cells (ES) are derived from the inner cell mass of pre-implantation embryos and are a source of pluripotent cells, as they are able to differentiate into all adult cell types. Once established, the pluripotent ES cells can be maintained under defined culture conditions, but can also be induced to terminally differentiate into a specific lineage (Bibikova et al., 2008). ES cells may potentially give rise to an infinite number of dopaminergic neurons that may be subsequently transplanted in depleted brain areas either in animal models of PD or even in patients (Lindvall and Kokaia, 2009). A particular emphasis has been posted on the validation of reliable methods for differentiation of ES cells towards midbrain dopaminergic neurons with a high survival index following transplantation (Hwang et al., 2010). Several protocols, characterized by presence of different feeder cell layers (i.e. bone marrow stromal cells, (Perrier et al., 2004) or astrocytes (Roy et al., 2006)) coupled to morphogen/growth factor exposition, have been developed. Transplantation of these *in vitro*-differentiated cells has produced variable results and often gave rise to tissue overgrowth with formation of neuroepithelial tumors, probably linked to the presence of residual immature cells in the original grafts. To overcome uncontrolled proliferation within transplants, cell-sorting protocols have been applied to specifically isolate pure populations of ES-derived dopaminergic neurons. These procedures, however, selectively impaired neuron survival indicating that fundamental factors were lost in the

terminals and soma both in striatum and SNc.

Kokaia, 2010).

**4.1 Embryonic stem cells**

negative fraction (Friling et al., 2009).

#### **3. Neural tissue transplantation**

In 1987, the first clinical trials involving transplantation of human fetal mesencephalic tissues or xenotransplantation of fetal pig neural cells in humans were performed with the clear scope of replacing dopaminergic neurons lost during PD progression. Since then and overall, contradictory results have been observed, even among patient groups treated within the same centers (Bjorklund et al., 2003). Along with the observed poor graft survival (around 10%; (Hagell and Brundin, 2001), a substantial subset of patients (15-56%) developed dyskinesias (Freed et al., 2001; Hagell and Brundin, 2001; Olanow et al., 2003), while the presence of Lewy bodies in long-term implants, recently reported, suggests the possibility of a *host-to-graft* disease propagation (Kordower et al., 2008; Li et al., 2008; Mendez et al., 2008). Functional improvements, represented by reduction of symptoms (30%–40% improvement of the unified Parkinson's disease rating scale, motor score in the drug-free phase) have been clearly observed (Hagell and Brundin, 2001) even in the long term (Mendez et al., 2008; Piccini et al., 1999). Positive outcomes were dampened by enhanced microglial activation and disruption of blood brain barrier linked to surgical procedures (Olanow et al., 2003). Failures were also related to the scarcity, as well as heterogeneous composition of the donor tissue itself (Carlsson et al., 2009; Freed et al., 2001). Nonetheless, these first trials supported the feasibility of transplantation procedures as an alternative therapeutic approach in PD (Brundin et al., 2010; Olanow et al., 2003). Moreover, these first trials have enlightened critical parameters mandatory for successful tranplantations including: a) purity of cell preparation; b) correct localization of the graft in the host brain c) preference for reduced age of donor/host cells (whenever possible), d) limited extension of brain injury at the time of transplantation (early pathological phases), and e) appropriate time of grafting (in relation to disease onset) to maximize survival of endogenous dopaminergic neurons (Lindvall and Kokaia, 2009). Altogether, these observations have encouraged the search for alternative cell sources that need to be efficient, safe, and ethically acceptable (additional details on this debated/controversial topic can be found in (Kimmelman et al., 2009)).

#### **4. Stem cell therapy**

The scientific progresses in biological and cellular technologies have allowed a better conception of the mechanisms involved in cell development, and in particular of factors/conditions ruling Stem Cells (SCs) proliferation and differentiation. Essentially, SCs are undifferentiated multipotent cells capable of both self-renewal and generation of several differentiated functional cell types to preserve tissue homeostasis throughout the entire life span of an organism. Multiple properties of SCs, including their the ability to potentially generate an unlimited number of dopaminergic neurons under physiological conditions, make them attractive candidates for regenerative therapy (Dass et al., 2006).

Therefore, SCs have been increasingly recognized as a valuable replacement and/or supporting tool for PD wherein a well-characterized cell type is mainly affected. Cell therapy may be performed using either autologous (ideal from an immunologic perspective) or allogeneic tissue-specific differentiated cells. Transplantation of healthy SCs that have been collected, expanded and eventually pre-differentiated *in vitro* have been originally proposed as a feasible appealing neural-replacement strategy. To date, several fetal and adult SC lineages have been directly differentiated into multiple cellular types, including neural cells and dopaminergic phenotypes. These naïve SCs, or their induced neuronal progeny, have been successfully transplanted in animal models of PD granting significant behavioral improvements. Ideally, however, new therapies should not only aim at replenishing the depleted dopamine store, but should also allow rescue of neuronal terminals and soma both in striatum and SNc.

Innovative therapeutic strategy should also take advantage and enhance the plastic property of the adult Central Nervous System (CNS) to regenerate affected brain areas through the activation of endogenous neurogenesis following cell transplantation. To reach these targets, other known intrinsic properties of SCs have already been exploited. Indeed, the potential of SCs to restore injured tissues is not only related to their direct differentiation but also to their capacity to produce and release trophic factors that may in turn inhibit apoptosis, promote angiogenesis and even direct stimulation of host regeneration (Lindvall and Kokaia, 2009; Lindvall and Kokaia, 2010). Neuroprotection can also be enhanced by transplanting SCs engineered to express either tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine synthesis, or neurotrophic factors, such as glial cell-line derived neurotrophic factor (GDNF), the most potent survival factor for degenerating dopaminergic neurons. Grafting of engineered SCs in this case provides a substantial reservoir allowing the unlimited supply of the required substance without the requirement of invasive injection pumps, as detailed below. Several SC types have already been transplanted in PD animal models yielding interesting but variable results (Wakeman et al., 2011).

SCs can be efficiently derived from early stage embryos (pluripotent SCs with unlimited potential to differentiate) or from committed resident tissues (multipotent SCs with restricted potential to differentiate). Interestingly almost all organs arising from endodermal, mesodermal and ectodermal germ layers can originate both fetal and adult SCs (i.e. amniotic/cord blood and tissue derived SCs). Regardless of their origin SCs could physiologically repair damaged tissues after intense injuries also promoting angiogenesis and neurogenesis processes, essential for CNS development and regeneration (Lindvall and Kokaia, 2010).

#### **4.1 Embryonic stem cells**

318 Towards New Therapies for Parkinson's Disease