Toxin models

PD models can be toxin-induced and genetic-based, or a combination of both. Genetic models are mainly based on the discovery of PARK genes and the (over)expression, knockout and knockdown of these or other putative PD genes. Many models are available both in vivo and in vitro. All experiments in this thesis were performed in a cellular model and are based on toxin-induced neurodegeneration. Thus, the focus in the following will be on toxin-based cellular systems. The most popular parkinsonian neurotoxins are 6-OHDA, rotenone, paraquat and MPTP (Figure 4) and will be discussed in the following sections.

6-OHDA, rotenone and paraquat

6-OHDA, as first DA neurotoxin discovered, is a prime example for an oxidative stress neurotoxin. It was introduced around 40 years ago and led to the destruction of adrenergic nerve endings (Thoenen and Tranzer 1968). Still actively used in PD research both in cellular and animal models, 6-OHDA does not cross the blood brain barrier (BBB). Due to the structural similarity to DA (see Figure 4), 6-OHDA enters DA neurons, where it generates ROS and quinones and produces superoxide and hydroxyl radicals (Bove et al. 2005; Miller et

al. 2009). 6-OHDA reproduces many symptoms resembling PD, but does not induce the formation of LBs.

The insecticide, pesticide and fish poison rotenone is a highly lipophilic compound which easily crosses the BBB and demonstrates a prime example of a mitochondrial toxin. It accumulates in mitochondria and inhibits complex I of the respiratory chain. Despite the uniform distribution throughout the brain, rotenone causes selective neurodegeneration of DA neurons (Miller et al. 2009).

Figure 4: Chemical structures of dopamine, 6-OHDA, paraquat, rotenone, MPTP, MPDP⁺ and MPP⁺.

Paraquat is a widely used herbicide with structural similarity to MPP⁺ (see Figure 4). It crosses the BBB via the neutral amino acid transporter and is selectively taken up into DA neurons via the dopamine transporter (DAT). Deleterious effects are mainly mediated by redox cycling with cellular diaphorase like NOS (nitric oxide synthase) (Bove et al. 2005), giving rise to paraquat radicals and eventually to hydroxyl free radicals and superoxide anions (Miller et al. 2009). Paraquat furthermore inhibits complex I of the mitochondrial respiratory chain and causes energy depletion as well as the generation of intracellular ROS (Miller et al.

2009).

MPTP/MPP⁺

In this study, mainly the neurotoxin MPP⁺ and its parental compound MPTP are used. Effects of MPTP on humans were discovered by chance in 1982, when several drug addicts in California developed severe parkinsonian symptoms. The contaminant MPTP, a by-product of chemical synthesis of MPPP – an analogue of meperidine (Demerol) – was identified as cause (Langston et al. 1983). Already back then, a selective damage of cells in the SN was proposed by Langston and his colleagues. MPTP-induced parkinsonism is almost indistinguishable from PD in humans and non-human primates, both by clinical and neuropathological aspects (Langston and Irwin 1986). Also responses to anti-parkinsonian therapies are almost identical to those observed in PD (Przedborski 2001). Experimental models showed that MPTP is

Przedborski 2003). Once inside the brain, MPTP is metabolized to the intermediate MPDP⁺ via MAO-B (Chiba et al. 1984; Heikkila et al. 1984) in non-DA cells, mainly astrocytes (Figure 5). Subsequently and most probably by spontaneous oxidation, MPP⁺ is formed and released or exported from those cells (Przedborski 2001). The parental compound MPTP is non-toxic to DA neurons, while MPP⁺ is actively transported in DA neurons via DAT (Mayer et al. 1986), where it unrolls its full deadly potential.

Figure 5: Schematic representation of MPTP metabolism. After systemic administration, MPTP crosses the blood brain barrier and is converted to MPDP⁺ by MAO-B in glial cells. The subsequent transformation into MPP⁺ and the release into the extracellular space are not yet fully understood. MPP⁺ is then actively taken up and concentrated into DA neurons via DAT.

Once inside DA neurons, MPP⁺ interacts with cytosolic enzymes, accumulates in mitochondria and is transported into DA storage vesicles via VMAT-2 (Przedborski 2001).

The redistribution of DA from vesicles to the cytosol leads to increased oxidative stress. The main toxic function of MPP⁺ is mediated by the inhibition of mitochondrial respiratory chain complex I (Nicklas et al. 1987) and leads to ATP-depletion and a massive generation of ROS.

Energy failure and oxidative stress are not killing directly, but rather trigger cell death-related molecular pathways and apoptotic programs, ultimately leading to cellular demise. A “circular cascade of deleterious events” starts (Przedborski et al. 2004), originating from and ending with mitochondria as central player. This process finishes with the activation of the programmed cell death machinery (Figure 6). Similar mechanisms are observed in post-mortem PD patient tissue, animal and cellular toxin models of PD. Following (programmed) cell death mechanisms are relevant in the MPTP/MPP⁺ model: MPP⁺-mediated inhibition of complex I, as well as redistribution of DA from storage vesicles to the cytosol both lead to an increase in ROS production (Vila and Przedborski 2003). Intramitochondrial oxidative stress

is increased and leads to an increase in the releasable soluble pool of cytochrome c within the mitochondrial intermembrane space (Perier et al. 2005). A subsequent damage of cellular components, including DNA, activates the tumor suppressor protein p53 (Mandir et al. 2002), the JNK/c-Jun pathway (Xia et al. 2001), and stimulates poly(ADP-ribose) polymerase (PARP) activity (Mandir et al. 1999). While p53 upregulates Bax, JNK contributes to the translocation of Bax to the mitochondrial membrane. Bax, in concert with activated PARP, induces the release of cytochrome c and apoptosis-inducing factor (AIF) to the cytosol (Vila and Przedborski 2003; Przedborski et al. 2004). Cytochrome c clusters with Apaf1 and pro-caspase 9 to form the apoptosome, initiating pro-caspase 3-mediated cell death (Oettinghaus et al.

2012). In parallel, AIF contributes to caspase-independent mechanisms of cell death (Nicotra and Parvez 2002). Furthermore, reduced ATP levels, resulting from complex I inhibition, also contribute to cell death. Considering the toxic potential of MPP⁺, it is a fortunate circumstance that despite having been developed as a selective herbicide in the 1950ies (Cyberquat / Cyperquat, Gulf Oil Company), MPP⁺ never came on the market (Markey et al.

1984).

Figure 6: Schematic representation of mechanisms of MPP⁺ neurotoxicity within DA neurons. See text for details.

Following literature helped to design this overview: (Przedborski 2001; Dauer and Przedborski 2003; Vila and Przedborski 2003; Przedborski et al. 2004; Perier et al. 2007; Gorman 2008).

All mentioned models demonstrate own advantages and limitations, but none combines all PD symptoms, or can recapitulate the full complexity of of the disease. Hence, the choice of the model has to be based on the research question. At present, the MPTP toxin model of PD is seen as the best model, as it reproduce most hallmarks found in PD (Blesa et al. 2012).