Neurotoxicology 71 (2019) 113–121 Contents lists available at ScienceDirect Neurotoxicology journal homepage: www.elsevier.com/locate/neuro Full Length Article Mechanistic comparison between MPTP and rotenone neurotoxicity in mice T ⁎ Sunil Bhurtel, Nikita Katila, Sunil Srivastav, Sabita Neupane, Dong-Young Choi Yeungnam University, 280 Daehak-Ro, Gyeongsan, Gyeongbuk, 38541, Republic of Korea ARTICLE INFO ABSTRACT Keywords: Animal models for Parkinson’s disease (PD) are very useful in understanding the pathogenesis of PD and MPTP screening for new therapeutic approaches. 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP) and rotenone Rotenone are common neurotoxins used for the development of experimental PD models, and both inhibit complex I of ’ Parkinson s disease mitochondria; this is thought to be an instrumental mechanism for dopaminergic neurodegeneration in PD. In Neurotrophic factors this study, we treated mice with MPTP (30 mg/kg/day) or rotenone (2.5 mg/kg/day) for 1 week and compared the neurotoxic effects of these toxins. MPTP clearly produced dopaminergic lesions in both the substantia nigra and the striatum as shown by loss of dopaminergic neurons, depletion of striatal dopamine, activation of glial cells in the nigrostriatal pathway and behavioral impairment. In contrast, rotenone treatment did not show any significant neuronal injury in the nigrostriatal pathway, but it caused neurodegeneration and glial activation only in the hippocampus. MPTP showed no such deleterious effects in the hippocampus suggesting the higher susceptibility of the hippocampus to rotenone than to MPTP. Interestingly, rotenone caused upregulation of the neurotrophic factors and their downstream PI3K-Akt pathway along with adenosine monophosphate-activated protein kinase (AMPK) activation. These results suggest that MPTP-induced dopaminergic neurotoxicity is more acute and specific in comparison to rotenone toxicity, and compensatory brain-derived neurotrophic factor (BDNF) induction and AMPK activation in the rotenone-treated brain might suppress the neuronal injury. 1. Introduction mitochondrial forms and functions (Greenamyre et al., 1999; Moon and Paek, 2015). Death of neurons occurs mostly via a reduction in mi- Parkinson’s disease (PD) is the second most common neurodegen- tochondrial complex I enzyme activity leading to ATP depletion, gen- erative disorder after Alzheimer’s disease, affecting 0.5–1% of the el- eration of ROS, and activation of caspase 3 (Greenamyre et al., 1999; derly population (Aguirre-Vidal et al., 2015). Apart from 10% of cases Martinez and Greenamyre, 2012). reported as familial PD, the rest of the cases are sporadic or idiopathic Environmental neurotoxins are utilized widely to recapitulate the with no known causes (Cannon et al., 2009; Le et al., 2014). PD is a symptoms and pathology of PD in experimental animals (Cannon et al., progressive neurodegenerative disease characterized by a profound loss 2009; Fleming et al., 2004). MPTP and rotenone are two of such well- of dopamine-producing neurons of the midbrain (specifically of the known neurotoxins. Both MPTP and rotenone have the ability to cross basal ganglia) along with death of several non-dopaminergic neurons the blood brain barrier due to their high lipophilicity (Tieu, 2011). resulting in its reclassification as a multicentric disease (Cicchetti et al., MPTP requires metabolism by the astroglial cells into its active meta- 2009; Johnson and Bobrovskaya, 2015). Some of the well-known and bolite 1-methyl-4-phenyl pyridinium (MPP+) before entering specifi- important mechanisms in the pathogenesis of PD are aging, mi- cally into dopaminergic neurons through dopamine transporters, tochondrial defects accompanied by oxidative stress, neuroinflamma- whereas rotenone needs no metabolism or transporters to enter neurons tion, disturbed proteostasis, and calcium mishandling (Andersen and (Giordano et al., 2012; Nopparat et al., 2014). These toxins inhibit the Chinta, 2016; Moon and Paek, 2015; Schapira et al., 1990). mitochondrial complex I activity thereby increasing oxidative stress Mitochondria, that are an integral part of all eukaryotic cells, are and are widely utilized in rodents and other species for mimicking involved in the production of energy via the respiratory chain, reg- experimental PD (Angelova and Abramov, 2018). ulation of cell death, calcium metabolism and production of reactive The MPTP model is the most commonly used animal model of PD, oxygen species (ROS). Deficiency of systemic mitochondrial complex I but the major limitation is the inability to produce progressive neuro- is involved in the pathogenesis of idiopathic PD as denoted by aberrant degeneration and Lewy body pathology (Le et al., 2014; Schober, ⁎ Corresponding author at: College of Pharmacy, Yeungnam University, 280 Daehak-Ro, Gyeongsan, Gyeongbuk, 38541, Republic of Korea. E-mail addresses: [email protected] (S. Bhurtel), [email protected] (N. Katila), [email protected] (S. Srivastav), [email protected] (S. Neupane), [email protected] (D.-Y. Choi). https://doi.org/10.1016/j.neuro.2018.12.009 Received 27 September 2018; Received in revised form 24 December 2018; Accepted 28 December 2018 Available online 31 December 2018 0161-813X/ © 2019 Elsevier B.V. All rights reserved. S. Bhurtel et al. Neurotoxicology 71 (2019) 113–121 2004). Rotenone on the other hand has been shown to induce phos- 2.3. Western blot analysis phorylation and aggregation of α-synuclein, Lewy body-like pathology, DJ-1 acidification and translocation, proteasomal dysfunction and ni- Brain tissue samples were homogenized with ice-cold NP-40 lysis gral iron accumulation which are cardinal pathologic changes in the buffer [0.15 M NaCl, 1% NP-40, 0.05 M Tris (pH 8.0), and 1% protease brains of patients with PD (Cannon et al., 2009; Duty and Jenner, 2011; inhibitor cocktail (Thermo Fisher Scientific Inc.)]. The tissue homo- Johnson and Bobrovskaya, 2015). Despite its great potential, the main genates were centrifuged at 4 °C for 20 min at 13,000 g, and the su- problem with this toxin has been its systemic toxicity leading to high pernatant was transferred to a fresh tube. Protein concentration was morbidity and mortality. Also, lower doses either fail or cause highly determined using a BCA protein assay kit (Thermo Fisher Scientific variable loss of dopaminergic neurons (Vingill et al., 2017). Some of the Inc.). Equal amount of protein (30∼40 μg) was mixed with loading other limitations of this model are the location and distribution of the buffer (0.125 M Tris−HCl, pH 6.8, 20% glycerol, 4% sodium dodecyl lesions within the striatum, the magnitude of the lesions and neuro- sulfate (SDS), 10% mercaptoethanol, and 0.002% bromophenol blue) chemical deficits (Wrangel et al., 2015). These issues have been over- and boiled for 7 min to induce denaturation of proteins. The samples come to some extent by using different routes of administration were separated on 12% SDS-polyacrylamide gel by electrophoresis and (Johnson and Bobrovskaya, 2015). transblotted to polyvinylidene difluoride membranes (Millipore In this investigation, we analyzed the difference between the sub- Corporation, Temecula, CA). Membranes were blocked using 5% chronic effects of MPTP and rotenone on the behavioral, neurochem- skimmed milk in Tris-buffered saline (0.1% Tween 20, TBST), for 1 h. ical, immunohistological, and biochemical parameters. This study dis- To detect phospho-proteins, the membrane was blocked in 5% bovine plays that MPTP neurotoxicity is more specific and acute than rotenone serum albumin in TBST. Then, the membrane was incubated overnight toxicity, and induction of neurotrophic factor and AMP-activated pro- at 4 °C with specific primary antibodies against BDNF (1:2000; Santa tein kinase (AMPK) activation may be related to the minimal effects of Cruz), GDNF (1:2000, Abcam), AMPK (1:2000; Cell Signaling rotenone on the biochemical physiology of the brain. Intriguingly, ro- Technology), p-AMPK (1:2000; Cell Signaling Technology), phosphati- tenone induced degeneration of the hippocampal neurons to some ex- dylinositol-3 kinase (PI3K; 1:2000, Cell Signaling Technology), p-PI3K tent. (1:2000, Cell Signaling Technology), protein kinase B (Akt; 1:2000; Cell Signaling Technology), p-Akt (1:2000; Cell Signaling Technology), β- actin (1:10,000; Ab-Frontier), and GAPDH (1:10,000; Ab-Frontier). 2. Materials and methods After being washed thrice with TBST, the membrane was incubated with a secondary antibody for 1 h at room temperature and again wa- 2.1. Animals and treatment of toxins shed thrice with TBST. Finally, the membrane was incubated with en- hanced chemiluminescence reagents (Thermo Scientific Inc.) and ex- Adult (10-week-old) male C57BL/6 mice (20–25 g, Japan SLC, Inc.) posed in a luminescence image analyzer (Fusion Solo, VilberLourmat, were housed in microisolator cages on a 12 h light/dark cycle with free France) to detect the immunoreactive complex. The density of each blot access to food and water. After 1 week of acclimatization, the mice were was assessed using GelQuant.Net software. divided into different groups. Animal experiments were performed according to the protocol ap- 2.4. Behavioral tests proved by the IACUC of Yeungnam University before the study (ap- proval number: 2016-004). All animal experiments complied with the Behavioral tests were carried out on Day 0 and 5 to evaluate the ARRIVE guidelines and were carried out in accordance with the motor deficits in the MPTP and rotenone-intoxicated animals.