Cypermethrin Activates Autophagosome Formation Albeit Inhibits Autophagy Owing to Poor Lysosome Quality: Relevance to Parkinson’s Disease
Abhishek Kumar Mishra1,2 • Saumya Mishra1,2 • Charul Rajput1,2 • Mohd Sami ur Rasheed1,2 • Devendra Kumar Patel2,3 • Mahendra Pratap Singh1,2
Abstract
Parkinson’s disease (PD) is the second most familiar, progressive and movement-related neurodegenerative disorder after Alzheimer disease. This study aimed to decipher the role of autophagy in cypermethrin-induced Parkinsonism, an animal model of PD. Indicators of autophagy [expression of beclin 1, autophagy-related protein 12 (Atg 12), unc-51 like autophagy activating kinase 1 (Ulk 1), p62 and lysosome-associated mem- brane protein 2 (LAMP 2) and conversion of microtubule- associated protein 1A/1B-light chain 3 (LC3) I to II], signalling cascade [phosphorylated (p) 5′ adenosine monophosphate- activated protein kinase (p-AMPK), sirtuin 1 (Sirt 1), phosphorylated-mammalian target of rapamycin (p-mTOR), tu- berous sclerosis complex 2 (TSC 2), p317Ulk 1 and p757Ulk 1 levels] and lysosome morphology were assessed in control and cypermethrin-treated rat model of PD. Autophagy markers were also measured in cypermethrin-treated neuroblastoma cells in the presence of 3-methyl adenine, a phosphatidylinositol-4,5- bisphosphate 3-kinase (PI3K) class III inhibitor; vinblastine, an autophagosome elongation inhibitor; bafilomycin A1, an autophagolysosome and lysosome fusion/abnormal acidification inhibitor or torin 1, a mechanistic target of rapamycin inhibitor. Cypermethrin reduced LAMP 2 and increased p-AMPK and Sirt 1 without causing any change in other signalling proteins. 3- Methyl adenine did not change LC3 conversion; vinblastine and bafilomycin A1 decreased LAMP 2 expression in controls. While cypermethrin increased LC3 conversion in the presence of 3-methyl adenine, LAMP 2 reduction was more pronounced in vinblastine and bafilomycin A1-treated cells. Torin 1 normalized the expression of LAMP 2 without any change in other autoph- agy markers. Results demonstrate that albeit cypermethrin acti- vates autophagosome formation, it reduces LAMP 2 expression and lysosome quality leading to autophagy inhibition.
Keywords Cypermethrin . Autophagy . Pyrethroid . Parkinson’s disease . Parkinsonism
Introduction
Selective degeneration of the nigrostriatal dopaminergic neurons leading to slowness of movement, tremor at rest, muscular stiff- ness and postural instability is associated with Parkinson’s disease (PD). It is recognized as the second most widespread, mystifying and age-related movement disorder (Singh et al. 2012). While aetiology of PD is elusive, pesticides, heavy metals, genetic pre- disposition and old age have been comprehensively implicated (Klodowska-Duda et al. 2005; Dixit et al. 2013; Srivastava et al. 2012; ur Rasheed et al. 2016). Epidemiological and experimental evidences have revealed that exposure to pesticides, such as cypermethrin, is a key risk factor for PD. An individual exposed to a higher level of cypermethrin is more prone to neurodegener- ation and impaired motor or cognitive functions. Almost all ex- perimental rats imitate the progressive and time-dependent neu- rodegeneration after cypermethrin exposure. A firm wrapping up on the occurrence rate of PD owing to cypermethrin exposure is not yet possible since it is rarely used in isolation rather often used in combination with other pesticides. Besides, epidemiological studies employing single pesticide are also not yet available. However, epidemiological investigation involving cases of acute cypermethrin poisoning, if performed, could predict the occur- rence rate as well as establish the genuine contribution of cypermethrin to human PD. Cypermethrin is a synthetic class II pyrethroid pesticide and is known toalter the mitochondrial mem- brane potential, proteome profile and complex Iactivity leading to oxidative stress and aberrant energy metabolism (Singh et al. 2011; Agrawal et al. 2015a; Agrawal et al. 2015b). It is known to induce the mitochondrial apoptosis-dependent selective loss of dopamine-containing neurons in the nigrostriatal dopaminergic pathway (Singh et al. 2012; Agrawal et al. 2015a; Agrawal et al. 2015b; Singh et al. 2011).
Autophagy is a key regulator of cell death and is acknowl- edged even as class II apoptosis. It is a self-sacrificing option that eliminates unwanted cellular components from the cytoplasm under the control of lysosome (Mishra et al. 2015; Niso- Santano et al. 2011). Autophagy eliminates abnormal protein aggregates and repairs or removes the impaired organelles. It is an elimination passageway of aggregated and mutated α- synuclein associated with sporadic PD and Parkinsonism (Pan et al. 2008; Giordano et al. 2013; Hou et al. 2015). Accumulation or clearance of α-synuclein has been shown to indicate the rig- orousness of neurodegeneration/neuroprotection (Mishra et al. 2015). While autophagy is implicated in PD, conflicting mecha- nisms are reported in sporadic PD and toxicant-induced Parkinsonism (Mishra et al. 2015). It is still a demigod question whether activation or inhibition is the primitive event and if autophagy is favourable or damaging process for the survival of dopamine-containing neurons. Autophagy is initiated by a signalling cascade depending on the cell type and cellular energy accessibility or demand. Aberrant autophagy is an outcome of unbalanced status of death or survival-associated elimination pro- cess under the erratic conditions (Niso-Santano et al. 2011). Autophagy determines the severity of degeneration and could help in designing an effective ameliorating or delaying strategy. Therefore, it was worthwhile to explore the mechanism of cypermethrin-mediated autophagy in rat model of PD and its connection with neuronal cell death.
Autophagy comprises of a dynamic cascade of events and is directed by the upstream regulators. In summary, it is achieved by the inhibition of the mammalian target of rapamycin (mTOR) that is regulated by 5′ adenosine monophosphate-activated pro- tein kinase (AMPK). Alternatively, non-canonical AMP regula- tion or mTOR-independent autophagy has also been recognized (Sarkar et al. 2009; Hou et al. 2015). It is accompanied by the extrinsic factors that directly act on the conventional down- stream AMPK mediators. Nonetheless, mTOR is the major cel- lular pathway; AMPK-activated sirtuin 1 (Sirt 1) could also regulate autophagy (ur Rasheed et al. 2016). Level of alteration in autophagy depends on the track pro- vided by the upstream regulators. Phosphorylated AMPK (p- AMPK) inhibits tuberous sclerosis complex 2 (TSC 2) that acts as an activator of mTOR. On the other hand, phosphorylated mTOR (p-mTOR) inhibits unc-51 like autophagy activating kinase (Ulk 1) complex that initiates autophagy. Therefore, p- mTOR is recognized as an autophagy inhibitor. Alternatively, p-AMPK directly activates Ulk 1 complex under some specific situations thereby induces autophagy. Besides, Sirt 1, another upstream regulator, is also regulated by AMPK or vice versa and increases the overall autophagy by increasing the expres- sion of a few autophagy-related proteins. Since the morphology of lysosome and expression of p-AMPK, Sirt 1, TSC 2 and mTOR have been shown to be linked with autophagy in PD (Burbulla et al. 2010; Lee et al. 2012; Mishra et al. 2015); therefore, their levels were measured in the study. Major steps of autophagy include initiation or phagophore formation, autophagosome formation and fusion of autophagosome with the lysosome leading to the formation of autophagolysosome. The acidic environment of lysosome fi- nally degrades and eliminates the abnormal proteins or organ- elles that are internalized within the autophagosome. Ulk 1 complex and beclin 1 are the major proteins involved in phagophore formation. Two phosphorylated forms (p317Ulk 1 and p757Ulk 1) of Ulk 1 have been shown to be linked with autophagy in PD. The former one is associated with an increase while the latter one is linked with a decrease in Ulk 1 activity. Microtubule-associated protein 1A/1B-light chain 3 (LC3) is another crucial protein, which is involved in the initiation and elongation of autophagosome. Its cytosolic form (LC3 I) gets converted into lipidated form (LC3 II) and subsequently at- taches with phagophore leading to the formation of autophagosome. Autophagy-related protein 12 (Atg 12) is also involved in the elongation of phagophore to autophagosome.
On the other hand, lysosome-associated membrane protein 2 (LAMP 2) is a major protein responsible for fusion of autophagosome to lysosome leading to the formation of autophagolysosome. Since Ulk 1, beclin 1 and Atg 12 protein expression, LC3 I to II conversion (II/I ratio) and LAMP 2 expression have been found to be associated with autophagy in PD (Burbulla et al. 2010; Lee et al. 2012; Mishra et al. 2015); their expression along with the ratio of lipidated to cytosolic form of LC3 were measured. Moreover, p62 is a known sub- strate of autophagy, its accumulation is a marker of compro- mised clearance and its level was also measured. Lysosome quality (fusion of autophagosome with lysosome and acidifica- tion) has also been found to be a key step regulating autophagy. Shift in any of the above-mentioned steps could change the overall extent of autophagy (Niso-Santano et al. 2011; Mishra et al. 2015). Measuring the entire phenomenon from phagophore formation to lysosome-mediated clearance could indicate the legitimacy of autophagy inhibition or augmentation in the presence of a chemical entity. Thus, an insightful impli- cation of autophagy in cypermethrin-induced nigrostriatal do- paminergic neurodegeneration was investigated. Cypermethrin-treated cells showed an increase in autophagosome formation; clearance or fusion was therefore assessed in the presence of autophagy modulators. For this purpose, stage-specific inhibitors, 3-methyl adenine, vinblas- tine, torin 1 and bafilomycin A1, were employed in the study. Direct administration of an autophagy inhibitor employing a chronic rodent model of Parkinsonism is not only cumber- some but also cost-consuming, labour intensive and ethically wrong. Autophagy in the presence of modulators was there- fore assessed in the neuroblastoma (SH-SY5Y) cells to mimic cypermethrin-mediated neurodegeneration.
Materials and Methods
Materials
Acrylamide, N,N′-methylenebisacrylamide, ammonium persul- fate, glycine, tris (hydroxymethyl) aminomethane, sodium dode- cyl sulphate (SDS), Tween-20, ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid tetrasodium salt (ethylene gly- col), ethylene diaminetetraacetic acid (EDTA) disodium salt dehydrate, 3,3′-diaminobenzidine (DAB), polyvinylidene difluoride (PVDF), dibutylphtalate xylene (DPX), 5-bromo-4- chloro-3-indolyl phosphate/nitro blue tetrazolium chloride (BCIP/NBT), N,N,N′,N′-tetramethylethylenediamine, vinblas- tine, 3-methyl adenine, bafilomycin A1, phenyl methyl sulfonyl fluoride, mannitol, thiazolyl blue tetrazolium bromide (MTT), cypermethrin, LC3 and LAMP 2 primary antibodies and Folin and Ciocalteu’s phenol reagent were procured from Sigma- Aldrich/Merck, (St. Louis, MO/Darmstadt, Germany). The pri- mary antibody for p317Ulk 1 and p757Ulk 1 and torin 1 was purchased from the Cell Signalling Technology, Inc. (Danvers, MA). A few primary [tyrosine hydroxylase (TH), p62, Sirt-1, p- AMPK-α, Ulk 1, p-mTOR, Atg 12] and secondary [goat anti- mouse immunoglobulin G-fluorescein isothiocyanate (IgG- FITC), goat anti-rabbit IgG F (ab′)2-tetramethylrhodamine (TRITC) and goat anti-rabbit IgG-FITC] antibodies were also purchased from Santa Cruz Biotechnology Inc. (Dallas, TX). While Ulk 1, Beclin 1, Atg 12, LC3, p62, LAMP 2, p-AMPK, p757Ulk 1, p317Ulk 1, mTOR, p-mTOR and Sirt 1 primary anti- bodies were anti-rabbit specific, TH, TSC 2 and B-actin primary antibodies were anti-mouse specific and Tim 44 primary anti- body was anti-goat specific. All primary antibodies showed
reactivity in rat. Antibodies used in this study were also used by others in many research publications (Plank et al. 1998; Dworak et al. 2010; Chen et al. 2011; Xie et al. 2011; Basu et al. 2014; Ma et al. 2014; Song et al. 2014; Agrawal et al. 2015b; Ozeki et al. 2015; Cramer et al. 2017; Zhang et al. 2017). Hydrogen peroxide was purchased from Rankem (Gurugram, India); 4-(2- hydroethyl)piperazine-1-ethane sulphonic acid (HEPES), from Spectrochem (Mumbai, India); and cryomatrix and ProLong Gold Antifade Mountant, from Thermo Fischer Scientific (Waltham, MA). Foetal bovine serum (FBS), Dulbecco’s modi- fied Eagle medium (DMEM) and trypsin were purchased from Gibco Life Technologies (California, CA). All other reagents and chemicals were purchased locally either form Sisco Research Laboratories (Mumbai, India) or from any other local manufacturers.
Animal Treatment
Guidelines of the committee for the purpose of control and supervision of experiments on animals, India, were followed for the study. The study was conducted on the Rattus norvegicus (Wistar rats) after getting the approval from the institutional animal ethics committee. Male pups were treated with cypermethrin [1.5 mg/kg, twice a week during the post- natal days 5–19 followed by 15 mg/kg, twice a week for 12 weeks upon adulthood (treatment initiated around 2 and half months after the postnatal exposure)] along with respec- tive corn oil vehicles (controls) (Singh et al. 2012).
Cryosectioning, TH-Immunoreactivity and Dopamine Content
Cryosectioning and TH-immunoreactivity were performed as described elsewhere (Singh et al. 2012). In summary, the brain was dissected out from the skull of ether-anaesthetized rats once perfused transcardially with saline. The brain was kept overnight in paraformaldehyde and incubated in sucrose gra- dient. Cryosectioning (20 μm) was done across the substantia nigra. Sections were incubated in blocking buffer, hydrogen peroxide and monoclonal anti-TH antibody. Sections were washed and incubated with anti-mouse biotin-conjugated sec- ondary antibody, horseradish peroxidase-conjugated streptavidin complex followed by DAB, dehydrated in graded ethanol and mounted with DPX. The images were captured, and counting of TH-positive neurons was done employing an image analysis system (QWin Pro, Leica, Germany). Striatal dopamine was measured and calculated as described previous- ly (Singh et al. 2012; Agrawal et al. 2015a)
Cell Culture and Lysates
Human neuroblastoma (SH-SY5Y) cells were obtained from the National Centre for Cell Science, Pune, India. Cells were grown in DMEM supplemented with FBS (10%), penicillin (100 U/mL) and streptomycin (100 μg/mL). Cells were main- tained at 37 °C in 5% carbon dioxide. Six-well culture dish was used to grow the cells for 48 h followed by the addition of the variable doses of cypermethrin (0.1–30 μm). The mixed content was incubated for 24 h. On the basis of the cell via- bility assay (mentioned in the subsequent section), specific cypermethrin concentration (15 μm) and incubation time (24 h) were selected for further study. Selected concentration and time used in the study were also in accordance with a previous study (Kakko et al. 2004). In a few sets, cells were also treated with 3-methyl adenine (10 mM), vinblastine (10 μM), bafilomycin A1 (100 nM) and torin 1 (1 μM). Autophagy inhibitor was administered 6 h prior to cypermethrin treatment. Dish containing the cultured cells was placed on ice and medium was aspirated to terminate the reaction. A total of 400 μL of cold cell lysis buffer containing protease and phosphatase inhibitor cocktail was added and left on ice for 30 min. Mixed content was centrifuged (14,000×g for 10 min at 4 °C) and protein concentration was determined in the supernatant employing the Lowry method (Lowry et al. 1951).
MTT Assay
Cells were detached by vigorous washing, centrifuged at 200×g for 5 min and cultured in a 96-well dish at a density of 3000 cells/well in 2% FBS. Cypermethrin (0.1, 1, 5, 10, 15, 20, 25 and 30 μm) was added to the culture medium for 24 h on the second day. After 24 h, the cells were incubated with 2-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2-tetrazolium bro- mide (MTT, 0.5 mg/mL) for 2 h at 37 °C. Formazan product appeared after reduction of MTT was dissolved in DMSO (150 μL) and quantified by taking the absorbance (OD) at 570 nm employing an automatic microplate reader (spectro- photometer-TECAN, infinite, M200PRO). immunofluorescence assay at ×40 magnification and the images were captured under a fluorescent microscope. Values were calculated as mean ± SEM (n = 4). Significant changes are expressed as
***p ≤ 0.001 in comparison with those of controls
Immunofluorescence
Coronal sections of the brain across the substantia nigra (20 μm thick)/SH-SY5Y cells were processed for dual immunofluorostaining as described elsewhere (Mak et al. 2010). In brief, the sections/cells were incubated in blocking buffer followed by mouse anti-TH (1:600) along with rabbit anti-LAMP-2A (1:200)/rabbit anti-LC3 (1:200)/rabbit anti- p62 (1:200) and rinsed with phosphate-buffered saline. Sections/cells were further incubated with goat anti-mouse immunoglobulin G conjugated with tetramethylrhodamine (1:200) and goat anti-rabbit secondary antibody conjugated with fluorescein isothiocyanate (1:200). Lysotracker dye (50–75 nM) was added to the sections/cells, washed and coat- ed with cover slips. Images were captured at higher magnifi- cation (×40) using a fluorescence microscope (Leica, Germany).
Protein Extraction and Estimation
Protein was extracted, as described previously (Agrawal et al. 2015a). In summary, the tissue was homogenized and sonicated in lysis buffer containing protease inhibitor cocktail, centrifuged at 4 °C and supernatant was collected. Protein content was measured in the supernatant employing the Lowry method (Lowry et al. 1951).
Western Blot Analysis
Proteins were separated on SDS-polyacrylamide gel electro- phoresis and electroblotted onto PVDF membrane. Non- specific sites were blocked either by 5% non-fat dry milk or by 2% BSA in tris-buffered saline (135 mM NaCl, 2.5 mM KCl, 50 mM Tris and 0.1% Tween 20, pH 7.4). PVDF mem- brane was incubated in the primary antibody [anti-beclin 1 (1:5000), anti-Ulk 1 (1:5000), anti-Atg 12 (1:5000), anti-
LAMP 2(1:5000), anti-LC3 (1:5000), anti-p62 (1:5000), anti-mTOR (1:3000), anti-p-mTOR (1:3000), anti-p-Ulk 1/p317(1:5000), anti-p-Ulk 1/p757 (1:5000), anti-TSC2 (1:3000), anti-p-AMPK (1:5000) or anti-Sirt 1 (1:3000)] for 4 h followed by an incubation with anti-mouse/anti-rabbit alkaline phosphate-conjugated secondary antibody (1:15,000) for 2 h. Bands were developed using BCIP/NBT as the substrates. Band density was calculated employing computerized densitometry system in relation to respective β-actin.
Statistical Analysis
For comparison among the groups, Student t test or one-way analysis of variance (ANOVA) with Newman-Keuls test was used. Data were expressed as mean ± standard error of the mean (SEM). Differences were considered statistically signif- icant if ‘p’ was less than 0.05.
Results
Striatal Dopamine Content and TH-Positive Neurons Cypermethrin reduced the striatal dopamine content (59.86 ± 0.73%) and number of the nigral TH-positive neu- rons (56.63 ± 0.51%) in comparison with controls showing that cypermethrin degenerated dopaminergic neurons.
Autophagy Proteins
Cypermethrin increased Ulk 1, beclin 1 and Atg 12 con- tents, p62 accumulation and LC3 I to LC3 II conversion (Fig. 1) indicating a significant increase in autophagosome formation. Expression of p62 and LC3 proteins was augmented in TH-positive neurons of cypermethrin- exposed group (Fig. 2). Expression of LAMP 2 was reduced, which showed that the quality of lysosome was compromised in cypermethrin-exposed rats in comparison with that of con- trols. Compromised quality (as assessed by reduced intensity of lysotracker dye positivity) and decreased LAMP 2 expres- sion indicated that there was some problem with fusion or clearance of autophagosome with lysosome despite an in- creased autophagosome formation (Fig. 3). Moreover, no sig- nificant change in mTOR, p-mTOR, TSC 2 and p757Ulk 1 levels was noticed in either group, cypermethrin induced p- AMPK and Sirt 1, a downstream protein of activated AMPK along with p317Ulk 1 (Fig. 4).
MTT Assay
MTT was performed to select an appropriate dose of cypermethrin, and therefore a dose-response study was per- formed (0.1 to 30 μm) (Fig. 5). Cells exposed to cypermethrin for 24 h at 15μm concentration were used for further study. Autophagy Proteins in the Presence/Absence of Autophagy Modulators Cypermethrin increased p62, LC3 II/I ratio and reduced LAMP 2 expression (Fig. 5). LC3-positive sub-cellular bodieswere unable to fuse with lysotracker or LAMP 2-positive ly- sosome in cypermethrin-treated cells (Fig. 6). 3-Methyl ade- nine did not alter LC3 conversion; vinblastine and bafilomycin A1 decreased LAMP 2 expression in controls. While cypermethrin increased LC3 conversion in the presence of 3-methyl adenine, LAMP 2 reduction was more pro- nounced in vinblastine- and bafilomycin A1-treated cells. Torin 1 normalizes the expression of LAMP 2 without any change in other autophagy markers in cypermethrin-treated cells (Fig. 7).
Discussion
Cypermethrin induced the degeneration of the nigral TH-positive neurons and reduced the striatal dopamine content and TH-ex- pression. Such indexes were measured just to assess if cypermethrin induced Parkinsonism in the current experimental paradigm. Degeneration and reduction in dopamine content were in accordance with the previous investigations, which have shown the onset of Parkinsonism in rats exposed to cypermethrin (Singh et al. 2012; Agrawal et al. 2015a; Agrawal et al. 2015b; Tripathi et al. 2017). Increased Beclin 1, Atg 12 and Ulk1 levels and LC3 I to II conversion in cypermethrin-treated rats suggested that vesicle/autophagosome was formed and autophagy could be involved. Increased accumulation of p62 after cypermethrin exposure indicated that in spite of autophagosome formation, probably autophagy was impaired. It could be due to improper fusion with lysosome, aberrant degradation and defective elimi- nation of damaged proteins and organelles. Status of lysosome was therefore assessed in the nigrostriatal tissue of control and cypermethrin-treated rats employing lysotracker dye and LAMP 2 expression. An increase in the level of upstream proteins (Ulk1, beclin1 and Atg12) could also be contributed by an inhibition of downstream mediators, such as LAMP 2. However, reduced colour intensity of lysotracker binding and reduced expression of LAMP 2 have been known to demonstrate poor lysosome quality (Bove et al. 2014; Harlan et al. 2016). Expression of LAMP 2 and binding of lysotracker dye with lysosome were also reduced in cypermethrin-treated rats, which showed that quality of lysosome was severely compromised in cypermethrin- exposed rats.
Results indicated that reduced lysosome quality/acidification/fusion with autophagosome could be compromised after cypermethrin exposure. Co-localization of TH with p62, LC3 or LAMP 2 was assessed to investi- gate the specificity of neurodegeneration in TH-positive neurons. Increased co-localization in the first two and re- duced co-localization in the last one with TH indicated the specificity of autophagy in TH-positive neurons. Autophagy is known to occur under the control of a few signalling molecules, which decides the fate of autophagy. Cypermethrin increased the levels of p-AMPK, Sirt 1 and p317Ulk 1 without any change in mTOR, p-mTOR, TSC 2 and p757Ulk 1 levels suggesting that induction of autophagosome formation could be AMPK dependent but independent to mTOR. AMPK is an energy sensor protein that helps to estimate the ratio of ATP and AMP (Richter and Ruderman 2009; Viollet et al. 2010). ATP/AMP ratio could possibly be depleted owing to reduced mitochondrial function (Agrawal et al. 2015b) sug- gesting that depleted ATP/AMP ratio could be partially/fully responsible for cypermethrin-induced aberrant autophagy.
MTT assay of human neuroblastoma cells was performed to examine the effective concentration of cypermethrin and 15μm cypermethrin was found to be optimum since maxi- mum effect on TH expression (data not shown) and minimum cell death were observed at this concentration. In order to assess the fate of the cells, defect in maturation of autophagosome, status of autophagy and role of lysosome, LC3 conversion, p62 accumulation and LAMP 2 expression were checked in control and cypermethrin-treated cells. Expression of p62 and LC3 conversion were significantly increased while expression of LAMP 2 proteins was reduced akin to the nigrostriatal tissue. Co-localization study was per- formed in neuroblastoma cells to check the status of LC3 positivity and lysosome quality employing fluorescent lysotracker dye and fluorescent-labelled LC3 antibody (Lan et al. 2012). Results showed that LC3-positive sub-cellular bodies were unable to fuse with lysosome since two fluores- cent signals did not merge together to yield an expected colour (Lan et al. 2012). Results indicated that autophagy plays a crucial role in the nigrostriatal dopaminergic neurodegenera- tion in cypermethrin-induced Parkinsonism. Loss of quality control in dopaminergic neurons exposed to cypermethrin could be a consequence of lysosomal impairment. Impairment of lysosome could be a central event that led to defective elimination of aberrant proteins and defective organ- elles (Moors et al. 2016). Mitochondrial dysfunction, α-synuclein aggregation and oxidative stress are found to be key events in cypermethrin- induced PD (Agrawal et al. 2015a; Agrawal et al. 2015b). Degradation and removal of α-synuclein is known to occur through lysosome-mediated autophagy (Webb et al. 2003; Cuervo et al. 2004; Ebrahimi-Fakhari et al. 2012; Xilouri et al. 2013).
In order to investigate whether quality of lysosome is responsible for aberrant autophagy, LC3 positivity was mea- sured in the presence or absence of the selected autophagy modulators. 3-Methyl adenine, a specific inhibitor of phos- phatidylinositol-4,5-bisphosphate 3-kinase, vinblastine and bafilomycin A1, specific inhibitors of autophagosome elongation/fusion of autophagolysosome with lysosome/ abnormal acidification and torin 1, a mTOR inhibitor were used in the study in the presence or absence of cypermethrin expo- sure (Wu et al. 2010; Sarkar 2013; Musiwaro et al. 2013). 3- Methyl adenine did not alter LC3 conversion, p62 accumula- tion and expression of LAMP 2 and TH. Increased LC3 I to II conversion and p62 level and decreased expression of LAMP 2 and TH were observed in the cells treated with cypermethrin in combination with bafilomycin A1. Vinblastine reduced only LAMP 2 level in cypermethrin-treated cells. Torin 1 ameliorat- ed autophagosome formation and rescued from decreased TH content in cypermethrin-treated group. Results confirmed that poor/reduced quality of lysosome, defective fusion or compro- mised acidification could be blamed for cypermethrin-induced aberrant autophagy.
Acknowledgements The authors sincerely acknowledge the Council of Scientific and Industrial Research (CSIR), University Grants Commission and Department of Science and Technology India, respec- tively, for extending fellowship to Abhishek Kumar Mishra and Charul Rajput; Saumya Mishra and Mohd Sami ur Rasheed. The Science and Engineering Research Board (SERB), India (Project Reference No.: EMR/2016/005041), is gratefully appreciated for approving the study for financial support. CSIR-IITR communication number of this article is 3446.
Compliance with Ethical Standards Animal study was performed as per the guidelines of CPCSEA, India.
Conflict of Interest The authors declare that they have no conflict of interest.
References
Agrawal S, Dixit A, Singh A, Tripathi P, Singh D, Patel DK, Singh MP (2015a) Cyclosporine A and MnTMPyP alleviate alpha-synuclein expression and aggregation in cypermethrin-induced Parkinsonism. Mol Neurobiol 52:1619–1628
Agrawal S, Singh A, Tripathi P, Mishra M, Singh PK, Singh MP (2015b) Cypermethrin-induced nigrostriatal dopaminergic neurodegenera- tion alters the mitochondrial function: a proteomics study. Mol Neurobiol 51:448–465
Basu S, Rajakaruna S, Reyes B, Van Bockstaele E, Menko AS (2014) Suppression of MAPK/JNK-MTORC1 signaling leads to premature loss of organelles and nuclei by autophagy during terminal differen- tiation of lens fiber cells. Autophagy 10(7):1193–1211
Bove J, Martinez-Vicente M, Dehay B, Perier C, Recasens A, Bombrun A, Antonsson B, Vila M (2014) BAX channel activity mediates lysosomal disruption linked to Parkinson disease. Autophagy 10: 889–900
Burbulla LF, Krebiehl G, Kruger R (2010) Balance is the challenge-the impact of mitochondrial dynamics in Parkinson’s disease. Eur J Clin Investig 40:1048–1060
Chen L, Xu B, Liu L, Luo Y, Zhou H, Chen W, Shen T, Han X, Kontos CD, Huang S (2011) Cadmium induction of reactive oxygen species activates the mTOR pathway, leading to neuronal cell death. Free Radic Biol Med 50(5):624–632
Cramer SL, Saha A, Liu J, Tadi S, Tiziani S, Yan W, Triplett K, Lamb C, Alters SE, Rowlinson S, Zhang YJ, Keating MJ, Huang P, DiGiovanni J, Georgiou G, Stone E (2017) Systemic depletion of L-cyst(e)ine with cyst(e)inase increases reactive oxygen species and suppresses tumor growth. Nat Med 23(1):120–127
Cuervo AM, Stefanis L, Fredenburg R, Lansbury PT, Sulzer D (2004) Impaired degradation of mutant alpha-synuclein by chaperone- mediated autophagy. Science 305:1292–1295
Dixit A, Srivastava G, Verma D, Mishra M, Singh PK, Prakash O, Singh MP (2013) Minocycline, levodopa and MnTMPyP induced changes in the mitochondrial proteome profile of MPTP and maneb and paraquat mice models of Parkinson’s disease. Biochim Biophys Acta 1832:1227–1240
Dworak M, McCarley RW, Kim T, Kalinchuk AV, Basheer R (2010) Sleep and brain energy levels: ATP changes during sleep. J Neurosci 30(26):9007–9016
Ebrahimi-Fakhari D, McLean PJ, Unni VK (2012) Alpha-synuclein’s degradation in vivo: opening a new (cranial) window on the roles of Giordano S, Darley-Usmar V, Zhang J (2013) Autophagy as an essential cellular antioxidant pathway in neurodegenerative disease. Redox Biol 2:82–90
Harlan FK, Lusk JS, Mohr BM, Guzikowski AP, Batchelor RH, Jiang Y, Naleway JJ (2016) Fluorogenic substrates for visualizing acidic or- ganelle enzyme activities. PLoS One 11:e0156312
Hou YS, Guan JJ, Xu HD, Wu F, Sheng R, Qin ZH (2015) Sestrin2 protects dopaminergic cells against rotenone toxicity through AMPK-dependent autophagy activation. Mol Cell Biol 35:2740– 2751
Kakko I, Toimela T, Tahti H (2004) The toxicity of pyrethroid com- pounds in neural cell cultures studied with total ATP, mitochondrial enzyme activity and microscopic photographing. Environ Toxicol Pharmacol 15:95–102
Klodowska-Duda G, Jasinska-Myga B, Safranow K, Boczarska-Jedynak M, Opala G (2005) The role of environmental factors in Parkinson’s disease may depend on disease onset age. Neurol Neurochir Pol 39: 445–450
Lan DM, Liu FT, Zhao J, Chen Y, Wu JJ, Ding ZT, Yue ZY, Ren HM, Jiang YP, Wang J (2012) Effect of trehalose on PC12 cells overex- pressing wild-type or A53T mutant alpha-synuclein. Neurochem Res 37:2025–2032
Lee J, Giordano S, Zhang J (2012) Autophagy, mitochondria and oxida- tive stress: cross-talk and redox signalling. Biochem J 441:523–540 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein mea- surement with the Folin phenol reagent. J Biol Chem 193:265–275
Ma B, Cao W, Li W, Gao C, Qi Z, Zhao Y, Du J, Xue H, Peng J, Wen J, Chen H, Ning Y, Huang L, Zhang H, Gao X, Yu L, Chen YG (2014) Dapper1 promotes autophagy by enhancing the Beclin1-Vps34- Atg14L complex formation. Cell Res 24(8):912–924
Mak SK, McCormack AL, Manning-Bog AB, Cuervo AM, Di Monte DA (2010) Lysosomal degradation of alpha-synuclein in vivo. J Biol Chem 285:13621–13629
Mishra AK, ur Rasheed MS, Shukla S, Tripathi MK, Dixit A, Singh MP (2015) Aberrant autophagy and Parkinsonism: does correction res- cue from disease progression? Mol Neurobiol 51:893–908
Moors T, Paciotti S, Chiasserini D, Calabresi P, Parnetti L, Beccari T, van de Berg WD (2016) Lysosomal dysfunction and alpha-synuclein aggregation in Parkinson’s disease: diagnostic links. Mov Disord 31:791–801
Musiwaro P, Smith M, Manifava M, Walker SA, Ktistakis NT (2013) Characteristics and requirements of basal autophagy in HEK 293 cells. Autophagy 9:1407–1417
Niso-Santano M, Bravo-San Pedro JM, Gomez-Sanchez R, Climent V, Soler G, Fuentes JM, Gonzalez-Polo RA (2011) ASK1 overexpres- sion accelerates paraquat-induced autophagy via endoplasmic retic- ulum stress. Toxicol Sci 119:156–168
Ozeki N, Hase N, Hiyama T, Yamaguchi H, Kawai R, Kondo A, Matsumoto T, Nakata K, Mogi M (2015) Interleukin-1β-induced autophagy-related gene 5 regulates proliferation of embryonic stem cell-derived odontoblastic cells. PLoS One 10(4):e0124542
Pan T, Kondo S, Le W, Jankovic J (2008) The role of autophagy- lysosome pathway in neurodegeneration associated with Parkinson’s disease. Brain 131:1969–1978
Plank TL, Yeung RS, Henske EP (1998) Hamartin, the product of the tuberous sclerosis 1 (TSC1) gene, interacts with tuberin and appears to be localized to cytoplasmic vesicles. Cancer Res 58(21):4766–4770
Richter EA, Ruderman NB (2009) AMPK and the biochemistry of exer- cise: implications for human health and disease. Biochem J 418: 261–275
Sarkar S (2013) Regulation of autophagy by mTOR-dependent and mTOR-independent pathways: autophagy dysfunction in neurode- generative diseases and therapeutic application of autophagy en- hancers. Biochem Soc Trans 41:1103–1130
Sarkar S, Ravikumar B, Floto RA, Rubinsztein DC (2009) Rapamycin and mTOR-independent autophagy inducers ameliorate toxicity of polyglutamine-expanded huntingtin and related proteinopathies. Cell Death Differ 16:46–56
Singh AK, Tiwari MN, Dixit A, Upadhyay G, Patel DK, Singh D, Prakash O, Singh MP (2011) Nigrostriatal proteomics of cypermethrin-induced dopaminergic neurodegeneration: microglial activation-dependent and -independent regulations. Toxicol Sci 122: 526–538
Singh AK, Tiwari MN, Upadhyay G, Patel DK, Singh D, Prakash O, Singh MP (2012) Long term exposure to cypermethrin induces nigrostriatal dopaminergic neurodegeneration in adult rats: postnatal exposure enhances the susceptibility during adulthood. Neurobiol Aging 33:404–415
Song L, Chen L, Zhang X, Li J, Le W (2014) Resveratrol ameliorates motor neuron degeneration and improves survival in SOD1(G93A) mouse model of amyotrophic lateral sclerosis. Biomed Res 2014: 483501
Srivastava G, Dixit A, Yadav S, Patel DK, Prakash O, Singh MP (2012) Resveratrol potentiates cytochrome P450 2 d22-mediated neuropro- tection in maneb and paraquat-induced Parkinsonism in the mouse. Free Radic Biol Med 52:1294–1306
Tripathi P, Singh A, Singh MP (2017) Ibuprofen protects from cypermethrin-induced changes in the striatal dendritic length and spine density. Mol Neurobiol. https://doi.org/10.1007/s12035-017- 0491-9
Ur Rasheed MS, Tripathi MK, Mishra AK, Shukla S, Singh MP (2016) Resveratrol protects from toxin-induced Parkinsonism: plethora of proofs hitherto petty translational value. Mol Neurobiol 53:2751– 2760
Viollet B, Horman S, Leclerc J, Lantier L, Foretz M, Billaud M, Giri S, Andreelli F (2010) AMPK inhibition in health and disease. Crit Rev Biochem Mol Biol 45:276–295
Webb JL, Ravikumar B, Atkins J, Skepper JN, Rubinsztein DC (2003) Alpha-Synuclein is degraded by both autophagy and the protea- some. J Biol Chem 278:25009–25013
Wu YT, Tan HL, Shui G, Bauvy C, Huang Q, Wenk MR, Ong CN, Codogno P, Shen HM (2010) Dual role of 3-methyladenine in mod- ulation of autophagy via different temporal patterns of inhibition on class I and III phosphoinositide 3-kinase. J Biol Chem 285:10850– 10861
Xie X, Zhang D, Zhao B, Lu MK, You M, Condorelli G, Wang CY, Guan KL (2011) IkappaB kinase epsilon and TANK-binding kinase 1 activate AKT by direct phosphorylation. Proc Natl Acad Sci 108(16):6474–6479
Xilouri M, Brekk OR, Stefanis L (2013) α-Synuclein and protein degrada- tion systems: a reciprocal relationship. Mol Neurobiol 47:537–551
Zhang L, Fu L, Zhang S, Zhang J, Zhao Y, Zheng Y, He G, Yang S, Ouyang L, Liu B (2017) Discovery of a small molecule targeting ULK1-modulated cell death of triple negative breast cancer in Torin 1 vitro and in vivo. Chem Sci 8(4):2687–2701