MPP antagonist

The novel protective effects of loganin against 1‐methyl‐4‐ phenylpyridinium‐induced neurotoxicity: Enhancement of neurotrophic signaling, activation of IGF‐1R/GLP‐1R, and inhibition of RhoA/ROCK pathway

Loganin, a major iridoid glycoside obtained from fruits of Cornus officinalis, possesses anti‐inflammatory, antitumor, antidiabetic, and osteoporosis prevention effects. Loganin has been linked to neuroprotection in several models of neurodegeneration, including Parkinson’s disease (PD). However, mechanisms underlying the neuropro- tective effects of loganin are still mostly unknown. Here, we demonstrated the pro- tective effects of loganin against PD mimetic toxin 1‐methyl‐4‐phenylpyridinium (MPP+) and the important roles of insulin‐like growth factor 1 receptor (IGF‐1R) and glucagon‐like peptide 1 receptor (GLP‐1R) in the neuroprotective mechanisms of loganin. In primary mesencephalic neuronal cultures treated with or without MPP+, loganin up‐regulated expressions of neurotrophic signals including IGF‐1R, GLP‐1R, p‐Akt, BDNF, and tyrosine hydroxylase. Loganin protected against MPP+‐induced apoptosis by up‐regulating antiapoptotic protein and down‐regulating proapoptotic protein. Moreover, loganin attenuated MPP+‐induced neurite damage via up‐ regulation of GAP43 and down‐regulation of membrane‐RhoA/ROCK2/p‐LIMK/p‐ cofilin. Loganin also attenuated MPP+‐induced reactive oxygen species (ROS) produc- tion. However, both AG1024, an IGF‐1R antagonist, and exendin 9‐39, a GLP‐1R antagonist, attenuated the protective effects of loganin on MPP+‐induced cytotoxic- ity, apoptosis, neurite length decrease, and ROS production. Our results suggest that loganin attenuates MPP+‐induced apoptotic death, neurite damage, and oxidative stress through enhancement of neurotrophic signaling, activation of IGF‐1R/GLP‐ 1R, and inhibition of RhoA/ROCK pathway, providing the evidence that loganin possesses novel neuroprotective effects.

1| INTRODUCTION
Parkinson’s disease (PD) is the second most common neurodegenera- tive diseases characterized by progressive dopaminergic neuronal loss in substantia nigra pars compacta (Dawson & Dawson, 2003). Growing evidence suggests that dysfunction of insulin‐like growth factor 1 (IGF‐1) or glucagon‐like peptide 1 (GLP‐1) pathway contributes to the progressive loss of PD (Bassil, Fernagut, Bezard, & Meissner, 2014). Recently, the potential protection of many antidiabetic drugs or glucose‐lowering candidates targeting IGF‐1 or GLP‐1 signaling has been revealed in preclinical studies of PD (Bassil et al., 2014; Kim, Moon, & Park, 2009). IGF‐1 is an endogenous peptide secreted by the liver and also synthesized and expressed in the brain, which contributes to the development, differentiation, and survival of neurons in the central nervous system (D’Ercole, Ye, Calikoglu, & Gutierrez‐Ospina, 1996). Activation of the intrinsic tyrosine kinase activity of IGF‐1 receptor (IGF‐1R) by IGF‐1 leads to the activation of phosphorylated 3‐kinase (PI3K)/protein kinase B (Akt) and several downstream effectors includ- ing those related to protein synthesis, neurotrophic effects, neurite outgrowth, or antiapoptotic effects (Cheng et al., 2011; Tseng, Chen, Jong, Chang, & Lo, 2016).

In dopaminergic SH‐SY5Y neuronal cultures, IGF‐1 attenuated hyperglycemia‐induced oxidative stress and neuro- nal injury (Gustafsson, Soderdahl, Jonsson, Bratteng, & Forsby, 2004) and attenuated proteasome inhibition‐induced neuronal apoptosis by PI3K/Akt activation (Cheng et al., 2011). GLP‐1 is an insulinotropic hormone secreted by intestinal endocrine L cells that can lower blood glucose level by stimulating insulin secretion (Baggio & Drucker, 2007). Several GLP‐1 receptor (GLP‐1R) agonists have been suggested to possess neurotrophic and neuroprotective activities. In a rat stroke model, GLP‐1 and exentin‐4 (a GLP‐1R agonist) promoted neuronal survival through activating PI3K/Akt pathway in the brain (Yang, Chen, Chen, Kuo, & Chen, 2016). Activation of GLP‐1R has also been suggested to attenuate advanced glycation end products‐induced oxidative stress and neuronal apoptosis in SH‐SY5Y cells (Chen et al., 2016). Moreover, in adult rat dorsal root ganglion neuron cultures, exentin‐4 enhanced neurite outgrowth and neuronal survival through activating PI3K/Akt and suppressing ras homolog gene family, member A (RhoA) pathway caused by the insulin removal (Tsukamoto et al., 2015). On the other hand, RhoA/Rho‐associated protein kinase (ROCK) plays a pivotal role in the negative regulation of neurite outgrowth, leading to growth cone collapse and neurite retraction (Stankiewicz & Linseman, 2014). Pharmacological inhibition of RhoA/ROCK pathway can improve neurite outgrowth and neuronal differentiation of mouse neural stem cells (Gu, Yu, Gutekunst, Gross, & Wei, 2013) and can attenuate 1‐methyl‐4‐phenylpyridinium (MPP+)‐ induced oxidative stress and neuronal death in SH‐SY5Y cells (Chong et al., 2014).

Loganin is a major iridoid glycoside that exhibits hypoglycemic activity of fruits of Cornus officinalis (He et al., 2016). Loganin has been proved to possess glucose‐lowering ability in diabetic mice (He et al., 2016) and is suggested as a valuable supplement for the treatment of diabetes mellitus and diabetes complications (He et al., 2016; Jiang, Zhang, Hou, & Zhu, 2012; Yamabe et al., 2010). Growing evidence from several cellular and animal models of neurodegenera- tive disorders demonstrates the potential neuroprotective effects of loganin in PD (Xu et al., 2017; Yao et al., 2017). However, whether loganin exerts protective effects on PD and the underlying mecha- nisms are still not well defined. Our previous study demonstrated that loganin provides benefits to spinal muscular atrophy‐like mice via improving survival motor neuron protein restoration, muscle strength, and body weight. We also revealed that loganin exerts its protective functions via activation of neuronal IGF‐1 signaling and muscle protein synthesis positive regulator Akt/mTOR in experimental models of spinal muscular atrophy (Tseng et al., 2016). MPP+, the active metabolite of MPTP, is identified as a broadly used neurotoxin to induce experimental PD model in vitro (Fiskum, Starkov, Polster, & Chinopoulos, 2003). We therefore characterized the protective effects and mechanisms of loganin in MPP+‐injured cellular model. In the present study, we demonstrated that loganin, a glucose‐ lowering and antidiabetic botanical candidate, protects against MPP +‐induced neurotoxicity in primary mesencephalic neurons. We also revealed the critical role of activation of IGF‐1R and GLP‐1 in loganin‐mediated neuroprotection.

2| MATERIALS AND METHODS
3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide (MTT), dimethyl sulfoxide, 2′,7′‐dichloro‐dihydrofluorescein diacetate (H2DCF‐DA), bovine serum albumin (BSA), arabinoside, poly‐L‐lysine, exendin 9‐39 (Ex9‐39), AG1024, and MPP+ were obtained from Sigma‐Aldrich (St. Louis, MO, USA). Minimum essential medium (MEM), fetal bovine serum, horse serum, glutamine, B27, nonessential amino acids, sodium pyruvate, penicillin, amphotericin B, streptomy- cin and Alexa Fluor® 488 goat anti‐rabbit IgG (H + L) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Lactate dehydrogenase (LDH) cytotoxicity assay kit was purchased from G‐Biosciences (St. Louis, MO, USA). FITC Annexin V apoptosis detec- tion kit was purchased from (BD Bioscience, San Jose, CA, USA). Plasma membrane extraction kit was obtained from BioVision (Moun- tain View, CA, USA). Antibodies used for immunoblotting or immuno- staining were as follows: β‐actin (Sigma‐Aldrich, St. Louis, MO, USA); Bcl‐2, Bax, BDNF, MAP 2, RhoA, ROCK2, and all horseradish peroxidase‐conjugated secondary antibodies (Santa Cruz, CA, USA); caspase‐3, IGF‐1R, p‐Akt, t‐Akt, p‐LIMK, t‐LIMK, p‐cofilin, t‐cofilin (Cell Signaling, Danvers, MA, USA); GLP‐1R (Abcam, Cambridge, MA, USA); and tyrosine hydroxylase (TH; Millipore, Bedford, MA, USA). Enhanced chemiluminescence reagent and polyvinylidene difluoride membrane were obtained from Millipore (Bedford, MA, USA). All mate- rials for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) were obtained from Bio‐Rad (Hercules, CA, USA).

Primary mesencephalic neuronal cultures were prepared from ventral mesencephalon dissected from 15‐day‐old embryos (E15) of pregnant Sprague–Dawley rats as previously described (Lo, Shih, Tseng, & Hsu, 2012; Tseng, Chang, & Lo, 2014). Animal handling was conducted in accordance with the National Institutes of Health (NIH) guidelines. Embryos were collected under aseptic condition in Dulbecco’s phos- phate buffered saline (PBS), and brains were released. Mesencephala were dissected and triturated and then cultured in plate wells pre- coated with poly‐L‐lysine at 1.5 × 106 cells/ml in MEM containing 10% fetal bovine serum, 10% horse serum, 100‐U/ml penicillin, 100‐μg/ml streptomycin, and 0.25‐μg/ml amphotericin B at 37°C in a humidified incubator with an atmosphere containing 5% CO2. After 24‐hr incubation, the culture medium is replaced by MEM supple- mented with 2% B27 and 10‐uM cytosine arabinoside for 48hrs. The culture medium is then further replaced by MEM supplemented with 2% B27 (without 10‐uM cytosine arabinoside)for a further 48 hrs. For drug treatments, cells were incubated with loganin at concentra- tions of 0.01, 0.1, 0.5, and 1 μM for 48 hr or pretreated with loganin (0.1, 0.5, and 1 μM) for 1 hr and then exposed to 10‐μM MPP+ for 48 hr. For antagonist experiments, AG1024 (10 μM) or Ex9‐39 (200 nM) were given 1 hr before loganin pretreatment.

Cell viability was determined by MTT and LDH assays (Tseng et al., 2014). MTT assay is an identified method to estimate cell viability based on dehydrogenases in active mitochondria of living cells that can cleave tetrazolium ring of MTT to formazan crystals. In MTT assay, cells were treated with 0.5‐mg/ml MTT for 3 hr in 37°C, and the formazan crystals were solved with dimethyl sulfoxide. Absorbance was read at 560 nm using a microplate reader (Thermo Scientific, Waltham, MA, USA). Besides, LDH assay is an identified method used to evaluate cytotoxicity. LDH is a soluble cytosolic enzyme in cells that can be released to medium when cell membrane was damage during cell death. Therefore, the presence of LDH in the culture medium is identified as a cell death marker (Kumar, Nagarajan, & Uchil, 2018). In LDH assay, culture medium was collected to measure LDH release using a cytotoxicity detection kit. The tetrazolium salts produced in LDH‐induced enzymatic reaction were then reduced to red formazan,thereby allowing a colorimetric detection by a microplate reader at 490 nm (Thermo Scientific, Waltham, MA, USA).Immunocytochemistry was performed to measure neurite morphology (Tseng et al., 2016; Tseng, Jong, Liang, Chang, & Lo, 2017) in primary mesencephalic neuron cultures. Briefly, cells were fixed with 4% paraformaldehyde for 30 min and then permeabilized with 0.2% Triton X‐100 in PBS at room temperature.

Cells were then incubated with blocking buffer (2% BSA in PBS) for 1 hr at room temperature and then incubated overnight at 4°C with rabbit anti‐MAP 2 (1:500). After that, cells were incubated with secondary antibody Alexa Fluor 488 goat anti‐rabbit IgG (1:1,000) for 1 hr at room temperature. Neurite was confirmed by fluorescent image of MAP 2 (green) staining under a fluorescent microscope (Nikon, Japan). Neurite outgrowth was assessed by measuring the length of neurite for all identified positive neurite‐bearing cells, and the mean of neurite length per cell was calculated using ImageJ software (NIH, Bethesda, MD).The production of reactive oxygen species (ROS) was determined by H2DCF‐DA staining, and the fluorescence of the productdichlorofluorescein (DCF) was analyzed by Coulter CyFlow Cytometer (Partec, Germany; Tseng et al., 2014). Briefly, cells were loaded with 10‐μM H2DCF‐DA (Sigma‐Aldrich, St. Louis, MO, USA) at 37°C for 30 min, and then cells were detached from plates. One hundred thou- sand cells were analyzed at an excitation of 495 nm and emission of 520 nm using Coulter CyFlow Cytometer (Partec, Germany). Apoptotic cells were detected by using FITC Annexin V apoptosis detection kit (Shih et al., 2010). Briefly, after cells were detached from plates, cells were suspended in binding buffer (10‐mM Hepes/NaOH, pH 7.4, 140‐mM NaCl, 2.5‐mM CaCl2) at a concentration of 1 × 106 cells/ml. Cells were then incubated with FITC Annexin V and propidium iodide for 15 min (37°C) in the dark. One hundred thousand cells were ana- lyzed, and the numbers of Annexin V‐positive cell (apoptotic cells) were counted by Coulter CyFlow Cytometer (Partec, Germany).

Western blot analysis was used to determine protein expressions as previously described (Mahmood & Yang, 2012). Cytosolic protein extracts were isolated using lysis buffer (Thermo Scientific, Waltham, MA, USA) supplemented with PMSF (protease inhibitor cocktail). Membrane protein extracts were isolated using commercial membrane protein extraction kit (BioVision, Mountain View, CA, USA). Theextracted lysates were centrifuged at 4°C for 30 min at 13,000 rpm by centrifuge (Kubota Corporation, Japan). Protein concentration was determined by using Bio‐Rad protein assay kit (Bio‐Rad, Hercules, CA, USA). Absorbance was read at 595 nm using a microplate reader (Thermo Scientific, Waltham, MA, USA) and estimated protein concen- tration according to BSA standard curve. Equal amounts of protein were boiled at 100°C for 5 min with sample buffer (Bio‐Rad, Hercules, CA, USA) and separated on 7.5%, 10%, or 12% SDS polyacrylamide gels. Then, proteins were transferred to polyvinylidene difluoride membranes by Electrophoretic Transfer Cell (Bio‐Rad, Hercules, CA, USA).

Membranes were incubated with TBST (50‐mM Tris‐HCl, pH 7.6, 150‐mM NaCl, 0.1% Tween 20) containing 5% nonfat milk for 1 hr at room temperature for nonspecific binding and then with one of the following specific primary antibodies overnight at 4°C: rabbit anti‐IGF‐1R (1:1,000), rabbit anti‐GLP‐1R (1:1,000), rabbit anti‐p‐Akt (1:1,000), rabbit anti‐t‐Akt (1:1,000), rabbit anti‐BDNF (1:500), rabbit anti‐caspase‐3 (1:1,000), rabbit anti‐ROCK2 (1:1,000), rabbit anti‐p‐LIMK (1:1,000), rabbit anti‐t‐LIMK (1:1,000), rabbit anti‐ p‐cofilin (1:1,000), rabbit anti‐t‐cofilin (1:1,000), rabbit anti‐GAP43 (1:1,000), rabbit anti‐TH (1:1,000), mouse anti‐Bcl‐2 (1:1000), mouse anti‐Bax (1:1,000), mouse anti‐RhoA (1:1,000), and mouse anti‐β‐actin (1:10,000). Membranes were then incubated with goat anti‐rabbit IgG‐HRP (1:1,000) or goat anti‐mouse IgG HRP (1:1,000) as secondary antibodies: for 1 hr at room temperature. Protein bands werevisualized with the enhanced chemiluminescence reagent (Millipore, MA, USA). ImageJ software (NIH, Bethesda, MD) was used for protein quantification.Data are shown as the mean ± standard error of the mean. All statistical analyses were performed with InStat version 3.0 (GraphPad Software, San Diego, CA, USA). A one‐way analysis of variance followed by Dunnett’s test was used for all pair comparisons. Differences with p < 0.05 were considered statistically significant. 3| RESULTS First, we investigated the effects of loganin on the expressions of IGF‐1R and GLP‐1R in primary mesencephalic neurons treated with or without MPP+. Results indicated that up‐regulated expressions of IGF‐1R (Figure 1a) and GLP‐1R (Figure 1c) were observed in loganin‐ treated neurons. In MPP+‐treated neurons, the expressions of IGF‐1R (Figure 1b) and GLP‐1R (Figure 1d) in the cells were significantly reduced. However, loganin attenuated the MPP+‐induced down‐ regulation of IGF‐1R (Figure 1b) and GLP‐1R (Figure 1d). We also examined the effects of loganin on neurotrophic factor p‐Akt and BDNF, which are closely associated with neurite outgrowth. The results indicated that loganin along increased Akt phosphorylation (Figure 2a) and BDNF expression (Figure 2c). Loganin also attenuated MPP+‐induced down‐regulation of pAkt (Figure 2b) and BDNF (Figure 2d) in neurons. Moreover, loganin attenuated MPP+‐induced decease of TH protein expression (Figure 3), which is the identified marker of dopaminergic neurons.To evaluate the role of IGF‐1R and GLP‐1R in the protective effects of loganin on MPP+‐induced cytotoxicity, AG1024 (IGF‐1R tyrosine kinase inhibitor) and Ex9‐39 (GLP‐1R antagonist) were used, respectively. Results from MTT and LDH assays indicated that loganin attenuated MPP+‐induced decrease of MTT reduction (Figure 4a) and MPP+‐induced increase of LDH release (Figure 4b) in primary mesen- cephalic neurons. These results suggested that loganin could increase cell viability and attenuate cytotoxicity induced by MPP+. However, both IGF‐1R tyrosine kinase inhibitor AG1024 and GLP‐1R antagonist Ex9‐39 attenuated the protection of loganin against MPP+‐induced cytotoxicity (Figure 4c and 4d).To determine whether the antiapoptotic pathway involves the neuroprotective mechanism of loganin, the effects of loganin onantiapoptotic protein (Bcl‐2) and proapoptotic proteins (Bax and caspase‐3) were examined. MPP+‐induced down‐regulation of Bcl‐2 (Figure 5a), up‐regulation of Bax (Figure 5b), and cleavage of caspase‐3 (Figure 5c) in neurons were inhibited by loganin pretreatment. Moreover, detecting apoptotic cells by FITC Annexin V/propidium iodide double staining, loganin attenuated MPP+‐induced increase of Annexin V‐positive cell numbers (apoptotic neuronal numbers); however, the antiapoptotic effect of loganin on MPP+ was attenuated by AG1024 and Ex9‐39 (Figure 5d).As the results shown in Figure 6a, MPP+ induced reduction of neurite length; however, loganin pretreatment attenuated the neurite retraction caused by MPP+. We next examined the effect of loganin on RhoA and its downstream effectors, in which pathway involves in the inhibition of axon regeneration. Results indicated that loganin attenuated MPP+‐induced translocation of RhoA from cytosol tomembrane (Figure 6b). Moreover, the downstream effectors of RhoA pathway, including ROCK2 (Figure 6c), p‐LIMK (Figure 6d), and p‐cofilin (Figure 6e), caused by MPP+ were also attenuated byloganin. In addition, loganin increased protein level of growth associated protein 43 (GAP43) in MPP+‐treated primary mesence- phalic neurons (Figure 6f).To determine the protective effects of loganin on MPP+‐induced oxidative stress, the effects of loganin on ROS production were measured by H2DCF‐DA fluorescence staining and the fluorescence of the product DCF was analyzed by a flow cytometer. Results indi- cated that MPP+ induced ROS overproduction, which effect was attenuated by loganin (Figure 7a). However, the inhibition of loganin on MPP+‐induced ROS production was attenuated by AG1024 and Ex9‐39 (Figure 7a). Similarly, loganin attenuated MPP+‐induced neurite damage; however, AG1024 and Ex9‐39 attenuated the protective effect of loganin against MPP+‐induced reduction of neurite length (Figure 7b). 4| DISCUSSION PD is a multifactorial disease triggered by multiple pathogenic factors. So far, there is no effective drug treatment on PD, but a considerable number of studies have suggested the potential targets for therapeutic interventions. Growing evidence demonstrated that drugs targeting on activations of IGF‐1R or GLP‐1R pathways possess their therapeutic potential on neurodegenerative disease, including PD (Bassil et al., 2014; Kim et al., 2009). Loganin is a glucose‐lowering botanic compound (He et al., 2016), and we previously reported its neuroprotection‐mediated activation of IGF‐1R signaling in motor neurons (Tseng et al., 2016). Recently, the potential protection of loganin has been revealed in different experimental models of PD (Xu et al., 2017; Yao et al., 2017). In this study, we demonstrated that loganin protects neuron against MPP+‐induced apoptotic death, neurite damage, and oxidative stress, and activations of IGF‐1R and GLP‐1R play the important roles in the neuroprotective effects of loganin (Figure 8).The IGF‐1 signaling pathway is clearly essential for normal devel- opment of the central nervous system. It possesses neurotrophic effects including neuronal survival and neurite outgrowth. Activation of IGF‐1 signaling has been reported to involve in dopaminergic neu- rons' protection of estrogen in PD rats (Quesada & Micevych, 2004). In a progressive PD model caused by 6‐hydroxydopamine, IGF‐1 attenuated nigrostriatal dopaminergic neuronal loss via activation of survival signaling cascades PI3K/Akt (Ayadi, Zigmond, & Smith, 2016). Recently, we have demonstrated that loganin attenuated sur- vival motor neuron deficiency‐induced neurotoxicity, and IGF‐1R pathway plays an important role in loganin‐mediated protection (Tseng et al., 2016). In this study, loganin up‐regulated protein expres- sion of IGF‐1R in neuronal cultures treated with or without MPP+. AG1024, an IGF‐1R antagonist, attenuated the protection of loganin on MPP+‐induced neurotoxicity. Therefore, we reconfirmed that acti- vation of IGF‐1R pathway contributes to loganin neuroprotection. Moreover, GLP‐1 is known to possess similar functions and properties as IGF‐1, and dysfunction of either pathway leads to neurodegenera- tion (Bassil et al., 2014). The potential therapeutic application of GLP‐ 1R agonists has been suggested for the treatment of neurodegenera- tive disorders in experimental models of PD. For example, exendin‐4, a GLP‐1 agonist, attenuated dopaminergic neuronal loss in MPTP‐ induced mouse model of PD by attenuating inflammatory and oxida- tive insults (Kim et al., 2009). Another GLP‐1 agonist, liraglutide, a drug that is on the market as a treatment for type 2 diabetes, reversed nigrostriatal degeneration through anti‐inflammatory, antiapoptotic, and neurotrophic mechanistic activities in PD‐like rats induced by rotenone (Badawi, Abd El Fattah, Zaki, & El Sayed, 2017). Moreover, intrathecal injection of loganin in rats has been reported to attenuate formalin‐induced tonic pain in an Ex9‐39‐reverse manner, suggesting the activation of spinal GLP‐1R involved in the antinociceptive effects of loganin (Gong, Fan, Ma, Xiao, & Wang, 2014). In this study, loganin significantly enhanced GLP‐1R expression in primary mesencephalic neuronal cultures. In MPP+‐treated neurons, loganin attenuated MPP +‐induced down‐regulation of GLP‐1R. Ex9‐39, a GLP‐1R antagonist,also attenuated the protection of loganin on MPP+‐induced cytotoxic- ity. Thus, activation of IGF‐1R and GLP‐1R signaling pathways should be one of the important mechanisms for the neuroprotective effects of loganin.Akt and BDNF have been reported to be associated with activa- tion of IGF‐1R and GLP‐1R, in which signaling implicated in numerous drug‐mediated neuroprotection, including neuronal survival, neurite outgrowth, and other neurotrophic effects (Hoppe et al., 2013). Increasing evidence reported that drugs targeting on the activation of PI3K/Akt pathway might be the candidates against MPP+/MPTP‐induced neurotoxicity (Cao et al., 2017). Results from this study demonstrated that MPP+ decreased Akt phosphorylation and BDNF expression in primary mesencephalic neurons, in which effects could be attenuated by loganin, suggesting the beneficial protective effects of loganin via enhancing Akt‐ and BDNF‐related neurotrophic path- ways. Moreover, TH is the rate‐limiting enzyme that catalyzes the hydroxylation of tyrosine to L‐DOPA and is identified as the important marker of dopaminergic neurons. The improvement of loganin on TH expression in MPP+‐treated primary mesencephalic neurons in this study also suggests the dopaminergic neurons' protective potential of loganin.MPP+ induces oxidative stress and mitochondrial dysfunction and further induces cell death through apoptotic mechanism in dopaminer- gic neurons (Fiskum et al., 2003). MPP+ induces apoptotic death by down‐regulation of Bcl‐2 and up‐regulation of Bax, leading to activate downstream effector caspase. The present results indicated that both AG1024 and Ex9‐39 attenuated loganin protection against MPP+‐ induced neuronal death. Moreover, loganin attenuated MPP+‐induced ROS production and neuronal apoptosis accompanied with up‐ regulation of Bcl‐2 and down‐regulation of Bax and cleaved caspase‐3. Furthermore, loganin attenuated MPP+‐induced neurite damage. However, both AG1024 and Ex9‐39 attenuated the protective effects of loganin on MPP+‐induced ROS production, apoptosis, and neurite damage. It is known that IGF‐1 and GLP‐1 share many similar downstream signaling such as PI3K/Akt signaling (Bassil et al., 2014). IGF‐1R‐mediated PI3K/Akt signaling has been reported to induce cell growth and differentiation of SH‐SY5Y cells (Kurihara, Hakuno, & Takahashi, 2000). Activation of GLP‐1R has been reported to activate PI3K/Akt signaling and protect cortical neurons against menadione‐ induced oxidative DNA damage (Yang et al., 2016). Besides, brain GLP‐1 and IGF‐1 signaling have also been reported to mediate the antiapoptotic effect of exendin‐4 in type 2 diabetic rats (Candeias et al., 2018). These present results demonstrated that IGF‐1R and GLP‐1R signaling play the important roles in loganin‐induced antioxi- dative, antiapoptotic, and neurite protective abilities. However, there is no significant difference between the inhibitory effects of IGF‐1R tyrosine kinase inhibitor (AG1024) and GLP‐1R antagonist (Ex9‐39) on loganin‐mediated neuroprotection. More investigation will be needed to identify which receptor is the first activating target of loganin. Recently, RhoA/ROCK inhibition is suggested as a new neuropro-tective strategy for PD. Inhibition of RhoA/ROCK signaling has been reported to activate neuroprotective survival cascades in dopaminer- gic neurons against Parkinson's toxin (Labandeira‐Garcia et al., 2015). RhoA signaling activation induces actin cytoskeleton rearrangement leading axon regeneration inhibition and neuron apoptosis (Koh, 2006; Stankiewicz & Linseman, 2014). Once RhoA has been activated, it will switch from inactive GDPbound form to active GTPbound form and translocate from cytosol to membrane. Our results demonstrated that loganin attenuated MPP+‐induced membrane‐RhoA expression as well as ROCK2 expression. Moreover, activation of RhoA/ROCK leads to phosphorylate LIMK and inactivate cofilin, which further causes neurite retraction (Lingor et al., 2007). The present results also demon- strated that loganin attenuated protein expressions of p‐LIMK and p‐cofilin induced by MPP+. Thus, RhoA/ROCK inhibition involves inthe mechanisms of loganin‐mediated neuroprotection. The regulation of IGF‐1‐ or GLP‐1‐mediaied signaling on RhoA/ROCK inhibition has been mentioned. IGF‐1 is reported to promote neurite initiation via inactivation of RhoA/ROCK pathway in SH‐SY5Y neurons (Shiraishi, Tanabe, Saito, & Sasaki, 2006). Activation of GLP‐1R by exendin‐4 is able to restore the reduced neurite outgrowth and viability of rat dorsal root ganglion neurons caused by the insulin removal via activation of PI3K pathway and inhibition of RhoA activity (Tsukamoto et al., 2015). Therefore, loganin attenuated MPP+‐induced neurotoxic- ity via activating IGF‐1R/GLP‐1R and inhibiting RhoA/ROCK pathway. In addition, GAP43 is a protein expressed in neurite growth cone, in which induction is a determinant of neurite growth (Hocquemiller et al., 2010). The present study also showed that loganin increased protein level of GAP43 in MPP+‐treated primary mesencephalic neurons. In conclusion, our results demonstrated that loganin attenuated MPP+‐induced apoptotic MPP antagonist death, neurite damage, and oxidative stress via activation of IGF‐1R and GLP‐1R and inhibition of RhoA/ROCK pathway, revealing the novel neuroprotective mechanisms of loganin.