Overexpression of human α-synuclein causes dopamine neuron death in primary human mesencephalic culture

Abstract

Mutations in the α-synuclein gene have been linked to rare cases of familial Parkinson’s disease (PD). α-Synuclein is a major component of Lewy bodies (LB), a pathological hallmark of PD. Transgenic mice and Drosophila expressing either wild-type or mutant human α-synuclein develop motor deficits, LB-like inclusions in some neurons, and neuronal degeneration. However, the relationship between abnormal aggregates of α-synuclein and human dopamine (DA) neuron degeneration remains unclear. In this report, we have investigated the influence of α-synuclein expression on DA neurons in primary culture of embryonic human mesencephalon. Two days after culture, human DA cells were transduced with wild-type or mutant human (Ala₅₃Thr) α-synuclein adenoviruses and maintained for 5 days. Overexpression of mutant and wild-type human α-synuclein resulted in 49% (P<0.01) and 27% (P<0.05) loss of DA neurons, respectively, while not affecting viability of other cells in the culture. Overexpression of rat α-synuclein or GFP (green fluorescent protein) had no effect on DA neuron survival. Cytoplasmic inclusions of α-synuclein were detected immunohistochemically in DA cells transduced with mutant human α-synuclein, but not wild-type α-synuclein. These results show that overexpression of human α-synuclein, particularly the mutant form, can cause human DA neuron death, suggesting that α-synuclein may have a primary role in the pathogenesis of PD.

Introduction

Parkinson’s disease (PD) is a common neurodegenerative disorder that affects about 1% of the population older than age 60. Clinical symptoms include rigidity, bradykinesia, postural instability and resting tremor [13], [32]. Neuropathologic examination reveals degeneration of dopamine (DA) neurons in the substantia nigra pars compacta with intraneuronal cytoplasmic inclusions termed Lewy bodies (LB) [14], [25]. Recently, two missense mutations in the α-synuclein gene have been found in rare familial PD patients (Ala₅₃Thr and Ala₃₀Pro) [2], [24], [34]. Moreover, wild-type α-synuclein protein has been shown to be a major component of LB in sporadic PD, in dementia with Lewy body disease, multiple system atrophy and other neurodegenerative diseases [3], [21], [37], [38], [39]. These data suggest that α-synuclein may be involved in the pathogenesis of PD and other LB-related neurodegenerative diseases [6], [10].

α-Synuclein is abundantly expressed in brain with localization in presynaptic nerve terminals [18], [20], [27], [33]. α-Synuclein can aggregate and form filamentous fibrils when present in high concentration [7], [8], [16], with heavy metals [31], or under condition of oxidative stress [17]. The mutant forms of α-synuclein aggregate much more rapidly than wild-type protein in vitro [5], [12], [28], [42]. The normal function of α-synuclein remains uncertain. α-Synuclein may play a role in neuronal plasticity and regulation of dopamine uptake [1], [4], [11]. Studies from transgenic mice and Drosophila have shown that expression of either wild-type or mutant human α-synuclein recapitulates many features of PD, including loss of dopamine in striatum, motor impairments, formation of α-synuclein-positive inclusions and neuronal degeneration [15], [22], [26], [40].

To understand the relationship between α-synuclein overexpression and DA neuron death, we have previously reported that overexpression of mutant human α-synuclein caused death of rat DA neurons in primary culture [44]. Because only humans develop PD, we hypothesized that human DA neurons might differ in their susceptibility to α-synuclein toxicity. In this study, we have examined the influence of α-synuclein overexpression on human DA neurons in primary cultures of human embryonic mesencephalon. We have found that overexpression of human α-synuclein caused DA cell death and led to the formation of α-synuclein positive inclusions.