Parkinson's disease (PD) is the most common neurodegenerative movement disorder characterized by the cardinal symptoms of muscular rigidity, resting tremor and bradykinesia [1, 2]. Underlying these motor deficits is the progressive loss of dopaminergic neurons of the substantia nigra pars compacta in addition to several other neuronal populations, the corresponding reduction of striatal dopamine levels, and the appearance of Lewy bodies and Lewy neurites in surviving neurons of the brainstem [1–3]. Lewy bodies are round eosinophilic intracytoplasmic proteinaceous inclusions composed of filamentous material, a major component of which is the protein α-synuclein . Missense mutations (A30P, E46K and A53T) and multiplications of the α-synuclein gene cause rare autosomal dominant familial forms of PD that often manifest disease in some families with an extended clinical and pathological spectrum resembling dementia with Lewy bodies (DLB) [5–9]. Thus, α-synuclein most likely plays a key role in the pathogenesis of familial and sporadic PD in addition to DLB.
It is not clear how mutations in α-synuclein, or increased levels of the wild-type α-synuclein protein due to gene multiplications, precipitate the demise of nigral dopaminergic neurons in familial PD. How α-synuclein contributes to the pathogenesis of the more common sporadic form of PD is also not known. It has been postulated that post-translational modifications of normal α-synuclein, including oxidation, nitration, phosphorylation or C-terminal truncation [10–13], could contribute to α-synuclein dysfunction and neuropathology in sporadic PD. The appearance of some of these modifications correlates well with neuronal loss and/or pathology in sporadic PD brains [11–13]. Furthermore, the exact role of Lewy bodies in dopaminergic neuronal degeneration in PD remains enigmatic. Therefore, clarifying the mechanisms underlying α-synuclein-induced dopaminergic neurodegeneration in vivo is critical to understanding the pathogenesis of familial and sporadic PD.
It is now clear from studies of transgenic mouse models that α-synuclein pathogenic mutations induce neuronal dysfunction and degeneration through a toxic gain-of-function mechanism [14–16]. A number of transgenic mice have been created that express wild-type or mutant human α-synuclein in neurons from an array of different heterologous promoter elements. These include the broadly expressing mouse prion protein, mouse Thy-1 and human platelet-derived growth factor-β promoters [17–23], or the catecholaminergic-specific rat tyrosine hydroxylase (TH) promoter [24–27]. A wide range of α-synuclein-related neurological phenotypes have been described in these mouse models that resemble some features of PD, such as behavioral motor deficits, neuronal loss, reduced striatal dopamine levels, and the formation of filamentous and non-filamentous α-synuclein-positive inclusions or aggregates. Many of these phenotypes, however, are not usually observed in the same mouse model. The most severe phenotypes tend to be observed with expression of the A53T variant whereas the wild-type protein is only ever rarely toxic to neurons [15, 17, 19]. Despite the plethora of α-synuclein mouse models that have been created, only very few of these exhibit a loss of nigral dopaminergic neurons, and in general this neuronal loss is not progressive or robust [26, 28]. Therefore, α-synuclein mouse models do not currently exist that reliably recapitulate essential features of PD especially the loss of nigral dopaminergic neurons, the neuropathological hallmark of the disease. Thus, there is a requirement for the development of new α-synuclein transgenic mouse models that can faithfully recapitulate the dysfunction of the nigrostriatal dopaminergic pathway in PD. Such models may arise through improvements in transgenic technology to increase transgene expression levels and dopaminergic neuronal specificity, as well as through the identification of more toxic α-synuclein pathogenic variants. A combination of these approaches could prove to be advantageous in developing faithful α-synuclein mouse models of PD.
Recent studies have highlighted the pathophysiological significance of C-terminally truncated α-synuclein species. Most recently, C-terminal truncation variants, including αSyn119, αSyn122 and αSyn123, have been detected in brain tissue from human PD cases and α-synuclein transgenic mice, and were found to be enriched in Lewy bodies [13, 29]. Moreover, in vitro studies demonstrate that human α-synuclein lacking the C-terminal 20 or 30 amino acids (αSyn120 or αSyn130) assemble more readily into filaments resembling the α-synuclein filaments observed in Lewy bodies [13, 29–31]. Disease-associated mutations in α-synuclein promote the accumulation of these truncated filaments compared to the wild-type protein, and C-terminally truncated species can enhance the aggregation of wild-type α-synuclein at low substoichiometric ratios suggestive of a seeding capacity [13, 29, 31]. Therefore, C-terminal truncations of α-synuclein could potentially advance disease progression or propagation through promoting the pathological aggregation and accumulation of α-synuclein. At this juncture, however, the role of C-terminally truncated α-synuclein species in the pathogenesis of PD is poorly understood. To further understand the pathophysiological significance of C-terminally truncated α-synuclein species, transgenic mice have recently been developed that express wild-type αSyn120 or A53T mutant αSyn130 truncation variants from the rat TH promoter [25, 26]. Collectively, these mice exhibit filamentous and non-filamentous α-synuclein-positive aggregates, deficits in locomotion, reduced striatal dopamine levels, and in the A53T-αSyn130 model a developmental loss of TH-positive nigral neurons and striatal nerve terminals.
Prior to the publication of these α-synuclein truncation models, we set out to develop a collection of transgenic mice capable of producing robust expression of human α-synuclein variants selectively in neurons of the nigrostriatal dopaminergic pathway. Encouraged by the promising neurodegenerative phenotype exhibited by conditional transgenic mice expressing a polyglutamine-expanded huntingtin protein , we chose to create similar conditional transgenic mice through gene targeting of a Cre-loxP-based transgene cassette at the ROSA26 genomic locus to express E46K or A53T mutant human α-synuclein, or the putative pathogenic C-terminal truncation, αSyn119, in a Cre recombinase-dependent manner. It is worth noting that transgenic mouse models expressing E46K α-synuclein do not currently exist, and that previous α-synuclein truncation models rely upon artificial truncations (αSyn120, αSyn130) [25, 26] rather than truncations associated with disease pathology (αSyn119) . Here, we combine conditional transgenic technology with new pathological variants of α-synuclein to evaluate the effects of expressing these α-synuclein variants directly in nigrostriatal pathway dopaminergic neurons.