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- Conflicts of Interest
The homeodomain transcription factor Pitx3 is critical for the survival of midbrain dopaminergic (mDA) neurons. Pitx3-deficient mice exhibit severe but selective developmental loss of mDA neurons, with accompanying locomotor deficits resembling those seen in Parkinson's disease (PD) models. Here, we identify specific mDA cell subpopulations that are consistently spared in adult Pitx3-hypomorphic (aphakia) mice, demonstrating that Pitx3 is not indiscriminately required by all mDA neurons for their survival. In aphakia mice, virtually all surviving mDA neurons in the substantia nigra (SN) and the majority of neurons in the adjacent ventral tegmental area (VTA) also express calbindin-D28k, a calcium-binding protein previously associated with resistance to injury in PD and in animal models. Cell-mapping studies in wild-type mice revealed that Pitx3 is primarily expressed in the ventral SN, a region particularly susceptible to MPTP and other dopaminergic neurotoxins. Furthermore, Pitx3-expressing SN cells are preferentially lost following MPTP treatment. Finally, SN mDA neurons in Pitx3 hemizygous mice show increased sensitivity when exposed to MPTP. Thus, SN mDA neurons are represented by at least two distinct subpopulations including MPTP-resistant Pitx3-autonomous, calbindin-positive neurons, and calbindin-negative Pitx-3-dependent cells that display elevated vulnerability to toxic injury, and probably correspond to the subpopulation that degenerates in PD. Impairment of Pitx3-dependent pathways therefore increases vulnerability of mDA neurons to toxic injury. Together, these data suggest a novel link between Pitx3 function and the selective pattern of mDA cell loss observed in PD.
Parkinson's disease (PD) is a progressive multisystem neurodegenerative disorder characterized primarily by locomotor deficits arising from massive loss of midbrain dopaminergic (mDA) neurons (Hornykiewicz 1966; Braak et al. 2003; Jellinger 2012). Multiple nuclei comprise the mammalian midbrain mDA system including the substantia nigra (SN) pars compacta (SNc, nucleus A9), ventral tegmental area (VTA, nucleus A10), and retrorubral field (RRF, nucleus A8). Among these DA neuronal subpopulations, the DA neurons of the ventrolateral SN are particularly susceptible in PD (Sourkes and Poirier 1965; Hornykiewicz 1966; Yamada et al. 1990; German et al. 1992). On the other hand, select DA subpopulations within and outside the midbrain tend to resist the degenerative process (Matzuk and Saper 1985; Fearnley and Lees 1991; German et al. 1992). The factors which determine why some mDA neurons are selectively vulnerable while others resist the degenerative process in PD are poorly understood.
Homeodomain transcription factors play a critical role in the specification and maintenance of mDA neurons (Wallen and Perlmann 2003; Andersson et al. 2006). We and others have previously shown that Pitx3, whose expression in the brain is restricted to mDA neurons (Semina et al. 1997; Smidt et al. 1997), plays a central role in their post-mitotic survival (Hwang et al. 2003; van den Munckhof et al. 2003; Nunes et al. 2003; Smidt et al. 2004). Aphakia (Pitx3ak/ak) mice, which harbor a functional deletion of the Pitx3 gene (Varnum and Stevens 1968; Rieger et al. 2001), exhibit profound mDA cell loss associated with massive nigrostriatal degeneration. Dopamine content in the dorsal striatum of aphakia mice is reduced by over 90% (Hwang et al. 2003; van den Munckhof et al. 2003), leading to locomotor and behavioral deficits, which are partially reversible with L-DOPA administration (van den Munckhof et al. 2003, 2006; Hwang et al. 2005; Jacobs et al. 2009; Ardayfio et al. 2010; Beeler et al. 2010). In contrast, mesolimbic dopaminergic projections are preferentially spared in both Pitx3ak/ak and Pitx3−/− mice (Kas et al. 2008), thus recapitulating a pattern of cell loss resembling that seen in patients with PD. This pattern of mDA cell loss in Pitx3-deficient mice also shows a remarkable similarity to that observed in a number of animal models of PD, suggesting that the observed dependency of these neurons on Pitx3 during developmental apoptosis may be linked to their heightened vulnerability during adulthood. For example, rodents and primates exposed to the dopaminergic neurotoxin MPTP develop a Parkinsonian syndrome, and show loss of mDA neurons in the ventrolateral SNc, which give rise to the nigrostriatal pathway. In contrast, mDA neurons in the dorsal SNc and the adjacent VTA are spared (Yamada et al. 1990; Fearnley and Lees 1991; Lavoie and Parent 1991; German et al. 1992). In addition, an increasing number of studies implicate alterations of the PITX3 gene in both sporadic and early-onset forms of PD (Bergman et al. 2010; Yu et al. 2010; Haubenberger et al. 2011). Given these parallel findings, we hypothesized that subpopulations that are susceptible to loss of Pitx3 function correspond to mDA neurons which are preferentially lost in human PD or following exposure to neurotoxins such as MPTP. Furthermore, we proposed that loss of Pitx3 function in adult mice would render mDA neurons more susceptible to toxic injury.
Here, we have determined the precise subpopulations of mDA neurons, which are lost in Pitx3ak/ak animals, and the relationship between mDA neurons containing Pitx3 and mDA neurons with known resistance to neurodegeneration in PD. Our results show that DA neurons in the ventral SNc are selectively vulnerable to developmental loss in the absence of normal Pitx3 function, with sparing of mDA neurons in the dorsal SNc. Nearly all surviving nigral neurons in Pitx3ak/ak mice are distinguished by their expression of the calcium-binding protein, calbindin-D28k (CB), a marker of resistant mDA neurons in PD and neurotoxin-based models of PD employing either MPTP or 6-hydroxydopamine (6-OHDA) (Yamada et al. 1990; Lavoie and Parent 1991; German et al. 1992). Furthermore, Pitx3- or CB-expressing mDA populations are largely complementary with only a minority subpopulation expressing both markers. Exposure to MPTP in wild-type animals results in preferential loss of Pitx3-expressing neurons in the SNc, while CB-positive/Pitx3-negative mDA neurons were relatively preserved. Reduced Pitx3 gene dosage increases the vulnerability of mDA SN neurons to MPTP, indicating that normal function of a Pitx3-dependent survival pathway is necessary for resistance to toxic injury. Together, these data suggest that multiple populations of mDA neurons, distinguished by their dependence on Pitx3 signaling and resistance to degenerative stresses, coexist in the mammalian midbrain.
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- Methods and materials
- Conflicts of Interest
The non-uniform, yet consistent, patterns of mDA neuron loss observed in PD (Kish et al. 1988; Damier et al. 1999; Halliday and McCann 2010) and numerous animal models of PD (Yamada et al. 1990; German et al. 1992; Liang et al. 1996) support the existence of multiple cell subpopulations with varying degrees of susceptibility to neurodegeneration. Identifying and characterizing these individual neuronal subpopulations and the factors which contribute to their differential vulnerability is critical toward understanding the mechanisms governing mDA degeneration in PD and related disorders. Among candidate molecules that stand out in this regard are a collection of homeodomain transcription factors whose expression is largely restricted to mDA neurons and play an important role in their specification, development, and/or maintenance. Indeed, disruption of many of these genes, namely Nurr1 (Zetterstrom et al. 1997), Engrailed 1/2 (Alberi et al. 2004; Sgado et al. 2006), Lmx1a (Ono et al. 2007; Friling et al. 2009), Lmx1b (Smidt et al. 2000; Asbreuk et al. 2002), and Pitx3 (Smidt et al. 1997; van den Munckhof et al. 2003; Nunes et al. 2003), have been shown to lead to prominent and selective cell loss in the mesencephalon. However, whereas ablation of master transcriptional molecules such as Nurr1 result in complete agenesis of mDA neurons (Zetterstrom et al. 1997), loss of Pitx3 function results in the loss of a selective, albeit major, subset of this population.
Dependence on Pitx3 segregates mDA neuron populations
Our findings indicate that the pattern of mDA neuronal loss resulting from deficient Pitx3 signaling closely resembles PD, whereby depletion is most severe in ventral SNc neurons, but considerably attenuated in dorsal SNc and VTA neurons. These results are consistent with the presence of multiple/discrete mDA subpopulations distinguished by their dependence on Pitx3 for survival. Importantly, the preservation of specific TH+ neuron subsets in Pitx3ak/ak mice indicates that not all mDA neurons require this transcription factor for developmental survival. We did not detect any further changes to this population in aged (> P700) Pitx3ak/ak mice, suggesting that Pitx3 is also dispensable for the maintenance of the surviving subpopulation throughout adulthood. On the other hand, Pitx3 appears to be critical for survival of ventral SNc neurons as well as for a minority of neurons distributed throughout the VTA. Interestingly, this same Pitx3-dependent subpopulation was also particularly susceptible to degeneration following MPTP treatment in wild-type animals, strongly implicating Pitx3 as a marker of vulnerable mDA subpopulations in this region. Consistent with their dependence on Pitx3, we observed using immunohistochemistry that Pitx3 expression is restricted primarily to ventral tier mDA neurons, and to scattered subpopulations within the VTA of wild-type animals. In addition to confirming that Pitx3-expressing mDA neurons are the main population lost in Pitx3ak/ak mice, this observation indicates that surviving Pitx3-autonomous cells are less vulnerable to stressors such as MPTP.
Our findings confirm previous reports that the vast majority of Pitx3-expressing neurons in the midbrain are dopaminergic (van den Munckhof et al. 2003; Smidt et al. 2004). In contrast, our observation that Pitx3 is differentially expressed among mDA neurons is somewhat at odds with previous reports examining localization of Pitx3 mRNA, suggesting that Pitx3 message is present in all mDA neurons (Smidt et al. 1997; Zhao et al. 2004; Maxwell et al. 2005). A plausible explanation for this perceived discrepancy is that Pitx3 expression depends on mechanisms beyond transcriptional regulation: for example, differential phosphorylation of translation-initiation factors or ribosomal proteins, micro-RNA (miRNA), and small-interfering RNA (siRNA) could result in variations in translation in dorsal-tier SNc neurons and half of VTA DA neurons. Additional studies are required to verify this. Alternatively, the higher resolution of quantitative mapping of Pitx3 and mDA populations using double immunohistochemistry compared with in situ hybridization may account for these differences.
Selective vulnerability in Pitx3+ mDA neurons
Our data point to a heightened susceptibility of Pitx3-dependent mDA neurons to developmental and neurotoxic insults. How this is mediated remains unclear. Interestingly, the majority of CB-expressing mDA neurons in the SNc and the VTA of wild-type mice appear to be Pitx3 autonomous as evidenced by their relative sparing in Pitx3ak/ak mice. Previous reports have shown that this group, which comprises the majority of mDA neurons in the dorsal SNc and a subpopulation of mDA neurons in the VTA in rodents, is relatively resistant to injury in PD (Yamada et al. 1990; Lavoie and Parent 1991; German et al. 1992; Liang et al. 1996). Similarly, CB-expressing mDA neurons are also spared in rodents following exposure to neurotoxins such as MPTP and 6-OHDA. Calcium-binding proteins, such as CB and CR, may provide neuroprotection by buffering intracellular calcium and attenuating the generation of harmful reactive oxygen species (Dauer and Przedborski 2003; Dawson and Dawson 2003; Surmeier et al. 2011). High intracellular levels of calcium may also mediate dopamine toxicity in neurons (Mosharov et al. 2009). The observation that another calcium-binding protein, calretinin, also colocalizes to resistant mDA subpopulations (Liang et al. 1996) further suggests that calcium homeostasis may be a major contributing factor. Along these lines, it has also recently been shown that the Cav1.3 class of voltage-gated L-type calcium channels expressed by SNc neurons renders them particularly susceptible to both MPTP and 6-OHDA exposure, and that blockade by the calcium-channel antagonist isradipine is neuroprotective (Surmeier et al. 2011). CB-expressing neurons in the VTA and dorsal SNc are also overwhelmingly spared in weaver mutant mice containing a mutation of the Girk2 inward-rectifier channel (Gaspar et al. 1994). Furthermore, single-nucleotide polymorphisms in the calbindin-1 gene have been associated with increased risk of PD in a Japanese population (Mizuta et al. 2008), adding to the argument that the buffering capacity of calcium-binding proteins are protective.
Nonetheless, it is noteworthy that previous studies failed to demonstrate detectable changes in mDA neuron survival in CB-null mice challenged with either MPTP or in the presence of the weaver (GIRK2) mutation (Airaksinen et al. 1997), suggesting that additional factors such as differences in target innervations and firing rate (Gerfen et al. 1987; Liss et al. 1999; Brown et al. 2009) may also play a determining role. For example, important regulators of dopamine, including dopamine transporter and vesicular monoamine transporter 2, are also differentially expressed between ventral and dorsal SNc regions (Haber et al. 1995; Sanghera et al. 1997).
The striking resemblance between the pattern of mDA neuron loss seen in Pitx3ak/ak mice, DA-toxin-induced neurodegeneration, and human PD (Yamada et al. 1990; Lavoie and Parent 1991; German et al. 1992) suggests a defect dependent on the Pitx3 pathway may underlie the vulnerability of these mDA subpopulations. During embryonic development, survival of mDA neurons is influenced by Pitx3 via transcriptional regulation of the enzyme aldehyde dehydrogenase 2, an enzyme responsible for retinoid production (Jacobs et al. 2007, 2011). Furthermore, Pitx3 appears to regulate key components of the dopamine metabolic pathway such as vesicular monoamine transporter 2 and Vip by forming a transcriptional complex with Nurr1 (Jacobs et al. 2009), another mDA transcription factor enriched in mDA populations. Pitx3 also participates in the feed-forward regulation of brain-derived neurotrophic factor and glial-derived neurotrophic factor (Peng et al. 2011), both of which are essential to mDA neuron survival. Significant reductions in Pitx3 expression within the CNS (Smidt et al. 1997) and peripheral tissues (Liu et al. 2011) have been reported in human PD, further supporting the view that impaired Pitx3-dependent functions are responsible for the loss of mDA neurons. Interestingly, genetic aberrations at the PITX3 locus or polymorphisms within promoter elements are also associated with increased risk of PD (Kim et al. 2007; Bergman et al. 2010; Haubenberger et al. 2011). Together with our observation that Pitx3 hemizygous mice are more susceptible to toxic challenge, it is likely that reduction in Pitx3 signaling pathway represents an upstream event leading to the impairment of dopamine metabolism and possibly other pathways. While it remains to be determined whether loss of Pitx3 function directly influences mature mDA neurons, our findings suggest that Pitx3 dependency in mDA neurons is closely linked to susceptibility to degeneration, and highlights the need for additional studies on the role of this transcription factor in both physiology and disease.