Original articleMorphological analysis of mitochondria for evaluating the toxicity of α-synuclein in transgenic mice and isolated preparations by atomic force microscopy
Introduction
One of the main mechanisms underlying Parkinson’s disease (PD) pathogenesis is the misfolding and aggregation of α-synuclein (α-syn), which leads to neuronal dysfunction and degeneration [1], [2]. α-Syn localizes to nuclei and subcellular organelles, including mitochondria and mitochondrion-associated endoplasmic reticulum membranes [3], [4], [5]. The N-terminus of α-syn (α-syn/N), which adopts an α-helical conformation [5], lies along the surface of the mitochondrial membrane [6] and regulates its permeability [7]. Many studies have examined the toxicity of α-syn to mitochondria in neural cell lines or primary neuronal cultures under conditions of overexpression or addition of exogenous protein to the culture medium [7], [8], [9], [10]. Changes in mitochondrial morphology such as swelling and condensation have been linked to a wide range of biological functions and pathologies [11]. For example, mitochondrial swelling is one of the most important indicators of mitochondrial permeability transition pore (mPTP) opening [12], [13], which can lead to the release of cytochrome (Cyto) C and necrotic or apoptotic cell death [14]. Quantitative analysis of morphological changes in brain mitochondria can be useful for developing neuroprotective strategies targeting mitochondria as a treatment for PD. There have been a few studies investigating the morphological changes in mitochondria induced by α-syn and α-syn/N in vitro [15], but little is known about the effect of α-syn/N on mitochondria.
Atomic force microscopy (AFM) has been widely used in medical, biological, and biophysical research [16] to investigate changes in the morphological and mechanical properties of T lymphocytes induced by aminophylline [17] and analyze mitochondria to determine the degree of myocardial injury [18], [19]. We previously described the formation of pore-like structures in the brain of rat injected with α-syn [2]. In the present study, we characterized the functional and nanostructural changes in mitochondria resulting from overexpression of full-length α-syn or α-syn/N.
Section snippets
Mouse model of α-syn overexpression
Male mice (body weight 22–25 g, 6 months) overexpressing human α-syn under the mouse Thy-1 promoter (i.e., Thy1α-syn mice, Line 15) were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and maintained on a C57BL/6N background. TG mice and WT littermates were maintained on 12:12-h light/dark cycle at 20 ± 2 °C and 60% relative humidity. The animal protocol was approved by the Animal Care and Use Committee of Capital Medical University (Beijing, China) and conformed to the National
α-Syn overexpression increases mPTP opening and decreases mitochondrial CL content in mouse brain
The western blotting results revealed low levels of VDAC1 and COX-IV in the cytosolic fraction and no β-tubulin in the mitochondrial fraction of brain homogenates from α-syn transgenic (TG) mice and their wild-type (WT) littermates (Fig. 1a). mPTP activation was detected by monitoring the fluorescence of calcein-AM in isolated mitochondria by confocal microscopy (Fig. 1b). The fluorescence intensity was 17.5% lower in TG as compared to WT mice (Fig. 1c), indicating that α-syn overexpression
Discussion
In this study, we provide evidence that the N terminus of α-syn can directly induce morphological changes to mitochondria that lead to dysfunction. We also demonstrated that quantitative analysis of these morphological changes by AFM is an effective approach for evaluating mitochondrial dysfunction in vivo and in vitro. α-Syn overexpression in mice caused mPTP opening and CL decrease, suggesting that the mitochondrial membrane was compromised. Mitochondrial dysfunction is associated with
Conflicts of interest
The authors report no conflict of interest.
Acknowledgments
This work was supported by grants from the National Key Research and Development Program of China (no. 2016YFC1306002); National Natural Science Foundation of China (no. 81371398); and Natural Science Foundation of Beijing Municipality (no. 7131001).
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