The main motor symptoms of PD are caused by a very selective loss of DA neurons in the SN, i.e. DA neurons of the nigrostriatal pathway. Other DA pathways in the brain are the mesolimbic, mesocortic and tuberoinfundibular pathway (Figure 32A). While in healthy individuals nigral DA neurons project to the striatum (putamen and caudate nucleus), the nigrostriatal pathway degenerates in PD (Figure 32B). A strong loss of DA neurons projecting to the putamen is observed, whereas loss of projections to the caudate is modest (Dauer and Przedborski 2003). Two questions arise looking at neuronal loss in PD. Why do only / mainly DA neurons die? And after a more detailed look: why do not all DA neurons, but only subpopulations die? Those questions cannot be fully answered using a system with only one cell type, but lessons learned from LUHMES and findings from other groups offer some explanations.
During the characterization phase of LUHMES, a relatively robust neuronal fate determination was found. We showed that LUHMES develop a mature neuronal phenotype, independent of the addition of differentiation factors to the medium. Addition of cAMP and GDNF only had influence on the “strength” and “completeness” of the DA phenotype. Parts
of the DA synthesis and storage machinery (e.g. DAT, VMAT-2) were present both in differentiations with (+/+) and without (-/-) cAMP/GDNF. Significant differences were found in markers directly related to DA synthesis. Both TH and Aromatic L-amino acid decarboxylase (AADC; DOPA-Decarboxylase), key enzymes in DA synthesis, were absent in -/- cells. This explains the lack of dopamine using this differentiation procedure. We found that expression of TH and DA synthesis were strictly dependent on cAMP. The partial pre-determination of LUHMES can be modulated in a certain range by the differentiation conditions. This enables studies on the contribution of DA to neurodegeneration and research projects that need non-DA neuronal cells. In initial experiments, we demonstrated that +/+
differentiated LUHMES are sensitive to low concentrations of MPP+. Blockage of DAT by Mazindol or GBR 12909 completely prevented MPP+-toxicity and demonstrated the selective uptake of MPP+ by this transpoter. A comparison of undifferentiated and mature +/+ and -/- cells showed that a functional DAT (uptake of 3H-MPP+) is present in both differentiations, but not in undifferentiated/proliferating cells. We showed that the neurotransmitter DA also contributes to MPP+-toxicity, since lack of DA (-/- differentiation) resulted in a much weaker effect of MPP+. Also inhibitors of TH activity attenuated MPP+-triggered cell death. Since DA is at least in part necessary for sensitivity to low concentrations of MPP+, the neurotransmitter seems to contribute to stress-induced neurodegeneration.
Figure 32: DA pathways in the brain. A: Midbrain DA neurons are located in the SN and the ventral tegmental area (VTA). Their axons project to different brain regions. In PD, the nigrostriatal pathway (red), projecting to the striatum (caudate nucleus and putamen) is affected while other DA pathways are not or only slightly affected. Adapted from (Arias-Carrion et al. 2010). B: Schematic representation of the nigrostriatal pathway (red) in normal (left) and PD brain (right). Cell bodies are located in the SN (arrows) and project to the striatum (putamen and caudate nucleus). In PD, a reduction of DA neurons in the SN and a loss of their projections to the putamen (dashed red line), and a more modest loss of those cells that project to the caudate (thin red line). Adapted from (Dauer and Przedborski 2003).
DA is very reactive, its oxidation increases the production of ROS and contributes to elevated stress levels (Berman and Hastings 1999). Oxidized DA is able to react with and damage
lipids, proteins and DNA/RNA (Sulzer 2007). Under healthy conditions, cellular defense mechanisms protect from DA-toxicity. The sequestration in storage vesicles via VMAT-2 (vesicular monoamine transporter 2) and anti-oxidative molecules prevent the formation of toxic DA species. Cellular events like mitochondrial dysfunction and subsequent loss of ATP can lead to redistribution of DA to the cytosol, massively increasing oxidative stress and thereby inducing a selective loss of those cells (Sulzer 2007).
Not all DA neurons are affected in PD. Cell loss in PD is not uniform, instead some DA neuron groups are more vulnerable to cell death than others. Thus, the neurotransmitter as source of additional cellular stress is surely not the single cause for DA neuron degeneration.
The ventral mesencephalon harbors two main groups of DA neurons, that are differentially affected (Figure 32A). A9 DA neurons in the SN project to the dorsolateral striatum to control motor functions. A10 DA neurons in the ventral tegmentum (VTA) project to the ventromedial striatum and limbic and cortical regions, and are involved in reward and emotional behavior (McRitchie et al. 1996; Chung et al. 2005). Both in PD patients and in toxin-models, nigrostriatal A9 DA neurons degenerate, while neighboring A10 mesolimbic and mesocortical DA neurons are much less affected (Hirsch et al. 1988; Dauer and Przedborski 2003; Chung et al. 2005; Braak and Del Tredici 2008).
Molecular differences like differentially expressed proteins in those subpopulations may be the cause for variances in susceptibility. Some proteins, as e.g. the G-protein-coupled inward rectifying K+ channel (GIRK2 = KCNJ6) are exclusively expressed in vulnerable A9 neurons.
This marker was also found to be expressed in LUHMES cells. Also elevated levels of some pro-apoptotic proteins, like caspase 7 and Bim are found in A9 neurons, and might contribute to the selective vulnerability (Chung et al. 2005). Furthermore, voltage-dependent L-type calcium channels, which have a special role in SNpc cells in their autonomous pacemaking activity and Ca2+-dyshomeostasis are discussed as cause for the selective vulnerability (Chan et al. 2009). The recruitment of those channels during autonomous pacemaking activity creates oxidative stress in SN, but not in VTA DA neurons (Guzman et al. 2010; Schon and Przedborski 2011).
Also typical for degenerating neurons is the presence of neuromelanin, which is capable of binding and releasing high amounts of iron. Neuromelanin positive neurons of the SN were shown to be more susceptible to cell death than neuromelanin negative cells from the VTA (Hirsch et al. 1988; German et al. 1989). During PD pathogenesis, iron can be released from the neuromelanin storage. The co-occurrence of DA, neuromelanin and elevated iron levels contributes to the high vulnerability of SN DA neurons to oxidative stress (Fasano et al.
2006). According to post-mortem studies, oxidative stress and iron content are enhanced in association with neuronal death. Iron-mediated toxicity is generally executed by ROS and/or reactive nitrogen species (RNS). Iron is found in the human body as reduced Fe2+ or oxidized Fe3+. While in healthy subjects the Fe3+:Fe2+ ratio is 2:1, PD patients show an inverted ratio of 1:2. This shift towards the more toxic form may lead to an increased production of H2O2 -derived hydroxyl-radicals via Fenton reaction, contributing to the execution of neuronal cell demise (Sian-Hülsmann et al. 2011). An involvement of iron in oxidative stress-mediated toxicity was also observed in LUHMES cells. MPP+-toxicity was fully prevented by the chelation of free iron (see chapter 6.4.1, Oxidative stress).
Since LUHMES were derived from human mesencephalic tissue and also markers of A9 DA neurons have been found, they represent an adequate model to study the cell type specifically lost in PD.