Ventral tegmental area dopamine neurons are resistant to human mutant alpha-synuclein overexpression

Abstract

Parkinson's disease (PD) is characterized by the formation of intracytoplasmic inclusions, which contain α-synuclein (α-syn) protein. While most profound neurodegeneration is seen in the dopamine (DA) synthesizing neurons located in the ventral midbrain, it is unclear why some DA cell groups are more susceptible than others. In the midbrain, the degeneration of the substantia nigra (SN) DA neurons is severe, whereas the involvement of the ventral tegmental area (VTA) neurons is relatively spared. In the present study, we overexpressed human A53T α-syn in the VTA neurons and found that A53T toxicity did not affect their survival. There was, however, a mild functional impairment seen as altered open field locomotor activity. Overexpression of A53T in the SN, on the other hand, led to profound cell loss. These results suggest that the selective susceptibility of nigral DA neurons is at least in part associated with factor(s) involved in handling of α-syn that is not shared by the VTA neurons. Secondly, these results highlight the fact that impaired but surviving neurons can have a substantial impact on DA-dependent behavior and should therefore be considered as a critical part of animal models where novel therapeutic interventions are tested.

Introduction

In Parkinson's disease (PD), the aggregation of proteins in the cytoplasm of cells leads to formation of inclusions, termed Lewy bodies (Lewy, 1912). While many neuronal cell groups are affected in PD (Braak et al., 2004), it is predominantly a dopamine (DA) deficiency syndrome and is characterized by the loss of dopaminergic neurons in the ventral midbrain (Bernheimer et al., 1973, Kish et al., 1988, Fearnley and Lees, 1991). The vulnerability of DA neurons in PD is clear; however, it is not known why these cells are more susceptible to neurodegeneration than other systems in the brain. Furthermore, different cell groups within the dopaminergic system display different susceptibilities to cell death. Degeneration of the DA neurons in the substantia nigra (SN) is the most prominent, whereas, in the ventral tegmental area (VTA), DA neurons are less affected (Uhl et al., 1985, Hirsch et al., 1988, Kish et al., 1988, Damier et al., 1999, Braak et al., 2004). This phenomenon is also evident in animal models following the administration of toxic substances such as 6-OHDA in rats, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice and primates rotenone, and proteasome inhibitors (Grant and Clarke, 2002, German et al., 1988, German et al., 1996, Lavoie and Parent, 1991, Betarbet et al., 2000, McNaught et al., 2004). Furthermore, differences in cell susceptibility are evident in Zitter rats, which display an abnormal metabolism of H₂O₂ (Gomi et al., 1994, Grant and Clarke, 2002). Data from these studies, many of which are related to oxidative stress, clearly show that there are differences between the SN and VTA in their susceptibility to cell death induced by toxic agents.

Appearance of a familial PD phenotype in an autosomal dominant manner has been described to result from point mutations in the α-syn gene (Polymeropoulos et al., 1997, Kruger et al., 1998, Zarranz et al., 2004). In addition, recent reports showed that familial PD also occurs in cases where multiplications of the α-syn gene have been identified, leading to an overexpression of wild-type α-syn protein (Farrer et al., 2001, Singleton et al., 2003, Chartier-Harlin et al., 2004). These findings stimulated several investigators to study the function of the α-syn protein. Today, a variety of functions including synaptic modulation, neurotransmitter regulation, synaptic maintenance, neuronal regeneration and chaperone activity have been demonstrated (Murphy et al., 2000, Lee et al., 2001, Masliah et al., 2000, Perez et al., 2002, Cabin et al., 2002, Abeliovich et al., 2000, Jenco et al., 1998, Duda et al., 1999, Quilty et al., 2003, Souza et al., 2000, Ostrerova et al., 1999, Park et al., 2002).

In parallel, new animal models have been developed using recombinant viral vectors to overexpress human α-syn protein. Recombinant adeno-associated virus (rAAV) and lentiviral vectors were used to overexpress either wild-type or the mutated forms of human α-syn in the SN of rats and primates (Kirik et al., 2002, Kirik et al., 2003, Klein et al., 2002, Lo Bianco et al., 2002, Lo Bianco et al., 2004, Yamada et al., 2004, Yamada et al., 2005). In the rat model, the production of human wild-type or mutated α-syn protein is detectable at the level of cell bodies already at 1-week post-injection. At 2–3 weeks after transduction, human α-syn fills up the processes of the nigral neurons extending to the striatum. Finally, increased α-syn load leads to a progressive neurodegeneration in the SN with an onset between 3 and 5 weeks. During this period, large α-syn-positive inclusions are seen throughout the dopaminergic cells, where some cell bodies are found to be shrunken, as well as dystrophic dendrites and axonal projections are prominent. The magnitude of nigral cell loss, as measured by the number of surviving TH-positive cells at 8 weeks in rAAV2-α-syn-injected animals varies between 30% and 80% (Kirik et al., 2002). However, in these previous experiments, the overexpression of the transgenic α-syn was targeted to the SN, therefore vulnerability of the VTA neurons could not be assessed.

In the present experiment, we tested if the DA neurons located in the VTA were resistant to the overexpression of human A53T mutated α-syn.