MPTP Injection Research Articles

Gastric Enteric Glial Cells: A New Contributor to the Synucleinopathies in the MPTP-Induced Parkinsonism Mouse.

Accumulating evidence has shown that Parkinson's disease (PD) is a systemic disease other than a mere central nervous system (CNS) disorder. One of the most important peripheral symptoms is gastrointestinal dysfunction. The enteric nervous system (ENS) is regarded as an essential gateway to the environment. The discovery of the prion-like behavior of α-synuclein makes it possible for the neurodegenerative process to start in the ENS and spread via the gut-brain axis to the CNS. We first confirmed that synucleinopathies existed in the stomachs of chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/probenecid (MPTP/p)-induced PD mice, as indicated by the significant increase in abnormal aggregated and nitrated α-synuclein in the TH-positive neurons and enteric glial cells (EGCs) of the gastric myenteric plexus. Next, we attempted to clarify the mechanisms in single MPTP-injected mice. The stomach naturally possesses high monoamine oxidase-B (MAO-B) activity and low superoxide dismutase (SOD) activity, making the stomach susceptible to MPTP-induced oxidative stress, as indicated by the significant increase in reactive oxygen species (ROS) in the stomach and elevated 4-hydroxynonenal (4-HNE) in the EGCs after MPTP exposure for 3 h. Additionally, stomach synucleinopathies appear before those of the nigrostriatal system, as determined by Western blotting 12 h after MPTP injection. Notably, nitrated α-synuclein was considerably increased in the EGCs after 3 h and 12 h of MPTP exposure. Taken together, our work demonstrated that the EGCs could be new contributors to synucleinopathies in the stomach. The early-initiated synucleinopathies might further influence neighboring neurons in the myenteric plexus and the CNS. Our results offer a new experimental clue for interpreting the etiology of PD.

Dexmedetomidine alleviates pain in MPTP-treated mice by activating the AMPK/mTOR/NF-κB pathways in astrocytes

Pain is a major non-motor symptom that contributes to impaired quality of life in Parkinson's disease (PD). However, the mechanisms and treatment of pain in PD have not been well studied. Dexmedetomidine (Dex) is used for analgesia and sedation during deep brain stimulation (DBS) and may reverse the progression of PD. Here, we explored the effect of Dex on Parkinson’s pain and the underlying mechanism. C57BL/6 mice were intraperitoneally injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 30 mg/kg) to establish a PD model. Then, the mice were treated with Dex (50 µg/kg) or Compound C (CC, 10 mg/kg, AMPK inhibitor). A motor behavioral test was used to validate the PD model, and a plantar test was conducted to assess mechanical and thermal stimulation thresholds. Immunofluorescence and western blotting were used to analyze the level of tyrosine hydroxylase (TH) in the substantia nigra (SN) and the expression of c-Fos, GFAP, p-AMPK, mTOR, NF-κB, TNFα, and IL-6 in the dorsal horn of the spinal cord (DHSC). We found that mice exhibited motor dysfunction and mechanical allodynia and thermal hyperalgesia after MPTP injection, and these changes were partially reversed by Dex. Dex also reduced MPTP-induced astrocyte activation and TNFα and IL-6 expression, increased p-AMPK and reduced mTOR and NF-κB expression in DHSC. Moreover, the effects of Dex were partially reversed by the AMPK inhibitor Compound C. Conclusions: These findings reveal that Dex protects dopaminergic neurons in PD and alleviates pain by reducing the activation of DHSC astrocytes through the AMPK/mTOR/NF-κB pathway. Therefore, Dex may be a potential drug for treating Parkinson’s pain.

Caloric Vestibular Stimulation Alleviates the Motor Dysfunctions in a Mouse Model of Parkinson’s Disease

Background: Parkinson’s disease (PD) is one of the most common degenerative disorders of the ageing brain. Currently, there is no cure for PD and most of the available treatments only aim to reverse the dopamine deficiency and relieve its symptoms. Caloric vestibular stimulation (CVS) is believed to help in relieving motor symptoms of PD. Hence, the present study is planned to evaluate the effect of CVS on behavioral changes in PD Mice. Methods: Twenty-four healthy male Swiss albino mice divided into four groups (n=6) were used for the study. PD was induced by giving an intraperitoneal injection of MPTP for 5 consecutive days. Bilateral CVS was given with hot water (temperature 40°C) for 15 days. Changes in behaviour (locomotor activity, grip strength, motor coordination, immobilization time) were measured on day 1 and day 15 and the results were statistically analysed. Results: The PD group showed a significant decrease in locomotor activity, muscular strength (fall on time), grip strength and immobilization time when compared to the control group, whereas CVS prevented the symptoms of PD when compared with the PD group. Conclusion: Caloric vestibular stimulation was effective in alleviating behavioral alterations in Parkinson-induced mice.

Targeting the differentiation of astrocytes by Bilobalide in the treatment of Parkinson’s disease model

Background: The etiology of Parkinson’s disease (PD), a chronic and progressive neurodegenerative disease, is multifactorial but not fully unknown. Until now, no drug has been proven to have neuroprotective or neuroregenerative effects in patients with PD. Objectives: To observe the therapeutic potential of Bilobalide (BB), a constituent of ginkgo biloba, in MPTP-induced PD model, and explore its possible mechanisms of action. Material and Methods: Mice were randomly divided into three groups: healthy group, MPTP group and MPTP + BB group. PD-related phenotypes were induced by intraperitoneal injection of MPTP into male C57BL/6 mice, and BB (40 mg/kg/day) was intraperitoneally given for 7 consecutive days at the end of modeling. The injection of saline was set up as the control in a similar manner. Results: BB induced M2 polarization of microglia, accompanied by inhibition of neuroinflammation in the brain. Simultaneously, BB promoted the expression of BDNF in astrocytes and neurons, and expression of GDNF in neurons. Most interestingly, BB enhanced the formation of GFAP+ astrocytes expressing nestin, Brn2 and Ki67, as well as the transformation of GFAP+ astrocytes expressing tyrosine hydroxylase around subventricular zone, providing experimental evidence that BB could promote the conversion of astrocytes into TH+ dopamine neurons in vivo and in vitro. Conclusions: These results suggest the natural product BB may utilize multiple pathways to modify degenerative process of TH+ neurons, revealing an exciting opportunity for novel neuroprotective therapeutics. However, its multi-target and important mechanisms need to be further explored.

SUN11602, a bFGF mimetic, modulated neuroinflammation, apoptosis and calcium-binding proteins in an in vivo model of MPTP-induced nigrostriatal degeneration

BackgroundParkinson’s disease (PD) is the second most frequent neurodegenerative disease. PD etiopathogenesis is multifactorial and not yet fully known, however, the scientific world advised the establishment of neuroinflammation among the possible risk factors. In this field, basic fibroblast growth factor/fibroblast growth factor receptor-1 (bFGF/FGFR1) could be a promising way to treat CNS-mediated inflammation; unfortunately, the use of bFGF as therapeutic agent is limited by its side effects. The novel synthetic compound SUN11602 exhibited neuroprotective activities like bFGF. With this perspective, this study aimed to evaluate the effect of SUN11602 administration in a murine model of MPTP-induced dopaminergic degeneration.MethodsSpecifically, nigrostriatal degeneration was induced by intraperitoneal injection of MPTP (80 mg/kg). SUN11602 (1 mg/kg, 2.5 mg/kg, and 5 mg/kg) was administered daily by oral gavage starting from 24 h after the first administration of MPTP. Mice were killed 7 days after MPTP induction.ResultsThe results obtained showed that SUN11602 administration significantly reduced the alteration of PD hallmarks, attenuating the neuroinflammatory state via modulation of glial activation, NF-κB pathway, and cytokine overexpression. Furthermore, we demonstrated that SUN11602 treatment rebalanced Ca2+ overload in neurons by regulating Ca2+-binding proteins while inhibiting the apoptotic cascade.ConclusionTherefore, in the light of these findings, SUN11602 could be considered a valuable pharmacological strategy for PD.

Toxic Exposure to Endocrine Disruptors Worsens Parkinson's Disease Progression through NRF2/HO-1 Alteration.

Human exposure to endocrine disruptors (EDs) has attracted considerable attention in recent years. Different studies showed that ED exposure may exacerbate the deterioration of the nervous system’s dopaminergic capacity and cerebral inflammation, suggesting a promotion of neurodegeneration. In that regard, the aim of this research was to investigate the impact of ED exposure on the neuroinflammation and oxidative stress in an experimental model of Parkinson’s disease (PD). PD was induced by intraperitoneally injections of MPTP for a total dose of 80 mg/kg for each mouse. Mice were orally exposed to EDs, starting 24 h after the first MPTP administration and continuing through seven additional days. Our results showed that ED exposure raised the loss of TH and DAT induced by the administration of MPTP, as well as increased aggregation of α-synuclein, a key marker of PD. Additionally, oral exposure to EDs induced astrocytes and microglia activation that, in turn, exacerbates oxidative stress, perturbs the Nrf2 signaling pathway and activates the cascade of MAPKs. Finally, we performed behavioral tests to demonstrate that the alterations in the dopaminergic system also reflected behavioral and cognitive alterations. Importantly, these changes are more significant after exposure to atrazine compared to other EDs. The results from our study provide evidence that exposure to EDs may play a role in the development of PD; therefore, exposure to EDs should be limited.

SUN11602, a novel bFGF mimetic, exerts neuroprotective effects in Parkinson's disease

Parkinson's disease (PD) is the second most frequent neurodegenerative disease, with a large prevalence in industrialized countries. The etiopathogenesis of PD is multifactorial and not yet fully known, however, in the last few decades, scientific world advised the establishment of a neuroinflammatory state among the possible risk factors. In this field, basic fibroblast growth factor/fibroblast growth factor receptor 1 (bFGF/FGFR1) could be a promising way to treat CNS‐mediated inflammation; however, the use of bFGF as a therapeutic agent is limited by its side effects.The novel synthetic compound SUN11602 exhibited neuroprotective activities like bFGF, but demonstrating greater safety, thus constituting a potential candidate for the treatment of neurodegenerative diseases including PD. In this perspective, this study aimed to evaluate the effect of SUN11602 administration in a murine model of MPTP‐induced PD. PD was induced by intraperitoneal injection of MPTP (80 mg/kg). SUN11602 (1, 2.5, and 5 mg/kg) was administered daily by oral gavage starting from 24 hours after the first administration of MPTP, mice were sacrificed 7 days after MPTP induction. The results obtained showed that SUN11602 administration significantly reduced the alteration of PD hallmarks, attenuating the neuroinflammatory state via modulation of glial activation, NF‐kB pathway and cytokine overexpression. Furthermore, we demonstrated that SUN11602 treatment rebalanced Ca2+ overload in neurons by regulating Ca2+‐binding proteins, inhibiting the apoptotic cascade. Therefore, considering these findings, SUN11602 could be considered a valuable pharmacological strategy for PD.

The protective effect of docosahexaenoic acid on the stomach in Parkinson’s disease induced by MPTP in male C57BL/6 mice

The purpose of this study was to detect gastric changes in Parkinson’s disease induced by 1-methyl-4-phenyl-1.2.3.6.-tetrahydropyridine (MPTP), and to investigate the protective effect of docosahexaenoic acid (DHA) against these changes in mice. The mice were divided into 4 groups (n=10 in per group) as control, DHA, Parkinson and DHA+Parkinson groups. DHA was administered by gavage for 30 days. On the 23rd day of gavage treatment, the animals of the Parkinson and DHA+Parkinson groups were intraperitoneally injected with MPTP. Seven days after the injection of MPTP, their locomotor activity, bradykinesia and rotarod performance were measured. Tyrosine hydroxylase expression in substantia nigra and the apoptotic index, the concentrations of tumor necrosis factor-α and histamine, and the number of mast cells in the stomach were evaluated. Administration of DHA significantly prevented the reduction in motor functions (P<0.001) and nigral TH neurons (P<0.05), and apoptosis (P<0.05), and an increase in TNF-α concentration (P<0.01) in the stomach. An increase in the number of mast cells in the stomach wall was observed in PD (P<0.001). DHA prevented the increase in the number of mast cells (P<0.05) and the histamine level (P<0.01) due to PD. As a result, MPTP administration in mice caused changes in the stomach as well as impairment in motor functions, and DHA was observed to reduce these changes.

Dexmedetomidine Can Enhance PINK1/Parkin-Mediated Mitophagy in MPTP-Induced PD Mice Model by Activating AMPK.

Parkinson's disease (PD) is a common neurodegenerative disease characterized by the degeneration of dopaminergic (DA) neurons in the substantia nigra (SN). Our previous study has shown that dexmedetomidine (Dex) can protect mitochondrial function and reduce apoptosis in MPP+-induced SH-SY5Y cells. Evidences have shown that mitophagy is related to the development of PD. In this study, we investigated whether Dex can enhance mitophagy in MPTP-induced mice to play a neuroprotective effect. In our experiment, mice were injected with MPTP 30 mg/kg intraperitoneally for 5 consecutive days to establish a PD subacute model. Dex (30, 50, and 100 μg/kg) was injected intraperitoneally 30 minutes before each injection of MPTP, respectively. Our results showed that Dex (50 μg/kg) most significantly attenuated MPTP-induced motor dysfunction and restored TH-positive neurons in the SN, increased the expression of the antiapoptotic protein Bcl-2, and decreased the expression of apoptotic proteins cleaved casepase3, cleaved casepase9, and Bax. Moreover, Dex increased the activity of mitochondrial Complexes I-IV and decreased the level of oxidative stress, manifesting as decreased MDA levels and increased SOD and GSH-PX levels. Besides, under transmission electron microscopy, Dex increased the mitophagosome which is an autophagosome with a mitochondrion-like structure inside under the electron microscope. In addition, Dex could also increase the expression of mitophagy-related proteins p-AMPK, LC3II/I, PINK1, and Parkin and decrease P62. However, after using Compound C (CC, 10 mg/kg, AMPK inhibitor), the effects of Dex on increasing PINK1/Parkin-induced mitophagy and neuroprotection were attenuated. In conclusion, Dex may improve mitochondrial function by activating AMPK to enhance PINK1/Parkin-induced mitophagy, thereby protecting dopaminergic neurons.

P 12 Expansion of regulatory T cells by early CD28 Superagonist treatment attenuates neurodegeneration in MPTP mice

Introduction: The neuropathology induced by injection of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a commonly used and well characterized animal model of Parkinson's disease (PD). It shows pathophysiological hallmarks of PD such as the loss of dopaminergic neurons from the substantia nigra (SN) as well as neuroinflammation. Expansion of CD4 + Foxp3 + regulatory T cells (Tregs) by administration of a single dose of a CD28 Superagonist monoclonal antibody (CD28-SA) in the MPTP mouse model was evaluated as a potential anti-inflammatory therapy for PD. Materials and Methods: An acute PD-like disease was induced in mice by a regimen of four intraperitoneal injections at 2 h intervals of MPTP-HCl (20 mg MPTP/kg bodyweight in NaCl). The same injection intervals were used for saline injected control animals (ctrl). The CD28-SA (clone D665, in PBS) or vehicle (PBS) was subcutaneously injected at an early (three days before MPTP intoxication) or late time point (30 minutes after the last MPTP injection). The total number of Nissl + neurons and dopaminergic neurons (TH + ) of the SN were analysed by unbiased stereology seven days after MPTP intoxication. Two groups (MPTP + vehicle; MPTP + CD28-SA) were compared for the early treatment time point and four groups (ctrl + vehicle; ctrl + CD28-SA; MPTP + vehicle; MPTP + CD28-SA) for the late treatment time point. Results: At the late treatment time point significantly decreased total cell numbers of TH + and Nissl + neurons were observed in MPTP mice treated with vehicle compared to ctrl + vehicle and ctrl + CD28-SA mice (TH + neurons: ctrl + vehicle: 7840 ± 405; ctrl + CD28-SA: 8422 ± 286; MPTP + vehicle: 5289 ± 230 / Nissl + neurons: ctrl + vehicle: 18949 ± 309; ctrl + CD28-SA: 19490 ± 212; MPTP + vehicle: 14921 ± 783). CD28-SA treatment of MPTP mice did not rescue TH + and Nissl + neurons within the SN (TH + neurons: MPTP + D665: 5933 ± 462 / Nissl + neurons: MPTP + CD28-SA: 15523 ± 754). In contrast, early treatment of MPTP mice with the CD28-SA led to higher numbers of both TH + and Nissl + neurons compared to vehicle injected mice (TH + neurons: MPTP + CD28-SA: 5645 ± 313; MPTP + vehicle: 4912 ± 171 / Nissl + neurons: MPTP + CD28-SA: 13171 ± 574; MPTP + vehicle: 11062 ± 497). Discussion/Conclusion: Our delayed-start design study demonstrates a neuroprotective effect of early CD28-SA-induced CD4 + Foxp3 + Treg expansion within the SN in the MPTP mouse model of PD.

Cerebral metabolic pattern associated with progressive parkinsonism in non-human primates reveals early cortical hypometabolism

Dopaminergic denervation in patients with Parkinson's disease is associated with changes in brain metabolism. Cerebral in-vivo mapping of glucose metabolism has been studied in severe stable parkinsonian monkeys, but data on brain metabolic changes in early stages of dopaminergic depletion of this model is lacking. Here, we report cerebral metabolic changes associated with progressive nigrostriatal lesion in the pre-symptomatic and symptomatic stages of the progressive 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkey model of Parkinson's Disease. Monkeys (Macaca fascicularis) received MPTP injections biweekly to induce progressive grades of dopamine depletion. Monkeys were sorted according to motor scale assessments in control, asymptomatic, recovered, mild, and severe parkinsonian groups. Dopaminergic depletion in the striatum and cerebral metabolic patterns across groups were studied in vivo by positron emission tomography (PET) using monoaminergic ([11C]-dihydrotetrabenazine; 11C-DTBZ) and metabolic (2-[18F]-fluoro-2-deoxy-d-glucose; 18F-FDG) radiotracers. 11C-DTBZ-PET analysis showed progressive decrease of binding potential values in the striatum of monkeys throughout MPTP administration and the development of parkinsonian signs. 18F-FDG analysis in asymptomatic and recovered animals showed significant hypometabolism in temporal and parietal areas of the cerebral cortex in association with moderate dopaminergic nigrostriatal depletion. Cortical hypometabolism extended to involve a larger area in mild parkinsonian monkeys, which also exhibited hypermetabolism in the globus pallidum pars interna and cerebellum. In severe parkinsonian monkeys, cortical hypometabolism extended further to lateral-frontal cortices and hypermetabolism also ensued in the thalamus and cerebellum. Unbiased histological quantification of neurons in Brodmann's area 7 in the parietal cortex did not reveal neuron loss in parkinsonian monkeys versus controls. Early dopaminergic nigrostriatal depletion is associated with cortical, mainly temporo-parietal hypometabolism unrelated to neuron loss. These findings, together with recent evidence from Parkinson's Disease patients, suggest that early cortical hypometabolism may be associated and driven by subcortical changes that need to be evaluated appropriately. Altogether, these findings could be relevant when potential disease modifying therapies become available.

Idebenone improves motor dysfunction, learning and memory by regulating mitophagy in MPTP-treated mice

The progression of Parkinson’s disease (PD) is often accompanied by the loss of substantia nigra dopaminergic neurons, mitophagy damage, learning, and memory impairment. Idebenone is a therapeutic drug that targets the mitochondria of neurodegenerative diseases, but its role in Parkinson’s disease and its pathological mechanism are still unclear. The purpose of this study was to investigate whether idebenone could improve behavioral disorders, especially motor, learning, and memory disorders, in mouse PD models and to explore its molecular mechanism. In the present study, C57BL-6 mice underwent intraperitoneal injection of MPTP (30 mg/kg) once a day for five consecutive days. Then, a 200 mg/kg dose was given as a single daily gavage of idebenone dissolved in water for 21 days after the successful establishment of the subacute MPTP model. Motor, learning, and memory were measured by a water maze and a rotarod test. Our results showed that idebenone could reduce MPTP-induced dopaminergic neuron damage and improve movement disorders, memory, and learning ability, which may be associated with upregulating mitochondrial autophagy-related outer membrane proteins VDAC1 and BNIP3 and activating the Parkin/PINK1 mitochondrial autophagy pathway. To confirm whether idebenone promotes the smooth progression of autophagy, we used eGFP-mCherry-LC3 mice to construct a subacute model of Parkinson’s disease and found that idebenone can increase autophagy in dopaminergic neurons in Parkinson’s disease. In summary, our results confirm that idebenone can regulate the expression of the mitochondrial outer membrane proteins VDAC1 and BNIP3, activate Parkin/PINK1 mitophagy, promote the degradation of damaged mitochondria, reduce dopaminergic neuron damage, and improve behavioral disorders in Parkinson’s disease mice.

Selenium reduces nociceptive response in acute 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced neurotoxicity.

The potential of Se to alleviate pain associated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity was investigated.Swiss mice were intraperitoneally injected with MPTP (20 mg/kg) 4 times with an interval of 2 h in 1 day. Seven days after MPTP injection, the mice (n = 5 mice per group) were randomly assigned to groups: MPTP-, DOPA (50 mg/kg)-, Se4 (0.4 mg/kg)-, Se6 (0.6 mg/kg)-, DOPA+Se4-, and DOPA+Se6-treated groups were compared with controls. MPTP mice were treated for seven days; thereafter, motor-coordination and nociceptive-motor reactions were assessed. Pro-inflammatory cytokines (IL-1β, IL-6 and TNFα), and selected pain biomarkers (substance P (SP), glutamate and β-endorphin) were assessed in the serum and the substantial nigra pars compacta (SNpc).Motor activity was increased slightly by Se (0.6 or 0.4 mg/kg) vs. MPTP (10.48 ± 2.71 or 11.81 ± 1.28 s vs. 3.53 ± 0.06 s respectively) but considerably increased by DOPA (50 mg/kg) vs. MPTP (50.47 ± 3.06 s vs. 3.53 ± 0.06 s respectively). Se and DOPA increased nociceptive threshold but Se alone reduced both serum and SN pro-inflammatory cytokines. Se modulates SP while DOPA modulates SP and glutamate in the SNpc of mice treated with MPTP.Se suppressed pro-inflammatory cytokines and restored the basal pain biomarkers in the SNpc of mice treated with MPTP. Se requires further study as analgesic adjuvant.

Withdrawn: MiR-132-3p promotes neuroinflammation and dopaminergic neurodegeneration by suppressing glutaredoxin expression.

This study aimed to investigate the effect of miR-132-3p/GLRX on neuroinflammation and dopaminergic neurodegeneration in Parkinson's disease (PD). In clinical experiments, the levels of miR-132-3p and glutaredoxin (GLRX) in the midbrain tissuesfrom 5 patients with PD were analyzed by qRT-PCR and Western blotting. In cell experiments, inflammatory cell models were established by lipopolysaccharide (LPS) stimulation. miR-132-3p loss- and gain-of-function assays were carried out to investigate the function of miR-132-3p in the BV-2 cell inflammatory response and neuronal injury. RIP experiments and dual-luciferase reporter assays were used to verify the binding of miR-132-3p to GLRX. Gene cotransfection in rescue assays was used to confirm the role of miR-132-3p/GLRX in microglial cell activation and neuronal injury. In animal experiments, mouse models of PD were established by MPTP injection. The effect of miR-132-3p on neuroinflammation and dopaminergic neurodegeneration in mice with PD was explored with fluorescence in situ hybridization (FISH), immunofluorescence, and behavioral tests. The results showed increased miR-132-3p expression and decreased GLRX expression in the midbrain tissues of PD patients. Overexpression of miR-132-3p increased the expression of TNF-α, IL-1β and IL-6 in LPS-treated BV-2 cells, and after coculture with conditioned medium of BV-2 cells, SH-SY5Y cells exhibited increased apoptosis and decreased viability in response to miR-132-3p overexpression, while the opposite results were obtained in miR-132-3p-knockdown cells, indicating that miR-132-3p can promote microglial cell activation and enhance neuronal injury. GLRX was a target gene of miR-132-3p, and GLRX could abolish miR-132-3p-induced neuronal injury and microglial activation. Depletion of miR-132-3p ameliorated dopaminergic neuron degeneration and neuroinflammation in mice with PD. Conclusively, miR-132-3p promoted the activation of microglial cells and further facilitated the loss of dopaminergic neurons by negatively regulating GLRX expression.

Silibinin ameliorates depression/anxiety-like behaviors of Parkinson's disease mouse model and is associated with attenuated STING-IRF3-IFN-β pathway activation and neuroinflammation

Depression and anxiety are common neuropsychiatric symptom of Parkinson's disease (PD), reflecting reduced quality of life in patients with PD. Silibinin (silybin), a flavonoid extracted and isolated from the fruit of Silybum marianum (L.) Gaertn, is widely used for the treatment of hepatic diseases. We report here that silibinin shows anti-depressant and anti-anxiety effects on 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced model mice with PD. All the results of open field test, elevated plus maze test, tail suspension test and forced swimming test demonstrated that silibinin administration significantly attenuated MPTP-induced depression/anxiety. Hematoxylin-eosin (HE) staining and Nissl staining results showed that MPTP injection caused the damage of hippocampal neurons, but this was ameliorated by oral administration of silibinin. Silibinin significantly restored hippocampal levels of 5-hydroxyptramine (5-HT) and noradrenaline (NA), two important neurotransmitters for regulating mood, which decreased in MPTP-injected mice. Neuroinflammation, as reflected by the increased expressions of IL-1β, TNFα and IFN-β, was marked in the hippocampus of MPTP-treated mice, accompanying increased stimulator of interferon genes (STING) and interferon regulatory factor-3 (IRF3). Silibinin administration, however, down-regulated the levels of IL-1β, TNFα and IFN-β, as well as STING and IRF3, protecting MPTP-induced PD model mice. These findings indicate that silibinin has a potential of being further developed as a therapeutic for depression and anxiety in PD.

Effect of the glycine transporter 1 inhibitor ALX-5407 on dyskinesia, psychosis-like behaviours and parkinsonism in the MPTP-lesioned marmoset

Dyskinesia and psychosis are complications encountered in advanced Parkinson's disease (PD) following long-term therapy with L-3,4-dihydroxyphenylalanine (L-DOPA). Disturbances in the glutamatergic system have been associated with both dyskinesia and psychosis, making glutamatergic modulation a potential therapeutic approach for these. Treatments thus far have sought to dampen glutamatergic transmission, for example through blockade of N-methyl-D-aspartate (NMDA) receptors or modulation of metabotropic glutamate receptors 5. In contrast, activation of the glycine-binding site on NMDA receptors is required for their physiological response. Here, we investigated whether indirectly enhancing glutamatergic transmission through inhibition of glycine re-uptake would be efficacious in diminishing both dyskinesia and psychosis-like behaviours (PLBs) in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned common marmoset.Six marmosets were rendered parkinsonian by MPTP injection. Following repeated administration of L-DOPA to induce dyskinesia and PLBs, they underwent acute challenges of the glycine transporter 1 (GlyT1) inhibitor ALX-5407 (0.01, 0.1 and 1 mg/kg) or vehicle, in combination with L-DOPA, after which the severity of dyskinesia, PLBs and parkinsonian disability was evaluated.In combination with L-DOPA, ALX-5407 0.1 and 1 mg/kg significantly reduced the severity of dyskinesia, by 51% and 41% (both P < 0.001), when compared to vehicle. ALX-5407 0.01, 0.1 and 1 mg/kg also decreased the severity of global PLBs, by 25%, 51% and 38% (all P < 0.001), when compared to vehicle. The benefits on dyskinesia and PLBs were achieved without compromising the therapeutic effect of L-DOPA on parkinsonism.Our results suggest that GlyT1 inhibition may be a novel strategy to attenuate dyskinesia and PLBs in PD, without interfering with L-DOPA anti-parkinsonian action.

Failure of Diphtheria Toxin Model to Induce Parkinson-Like Behavior in Mice.

Rodent models of Parkinson’s disease are based on transgenic expression of mutant synuclein, deletion of PD genes, injections of MPTP or rotenone, or seeding of synuclein fibrils. The models show histopathologic features of PD such as Lewi bodies but mostly only subtle in vivo manifestations or systemic toxicity. The models only partly mimic a predominant loss of dopaminergic neurons in the substantia nigra. We therefore generated mice that express the transgenic diphtheria toxin receptor (DTR) specifically in DA neurons by crossing DAT-Cre mice with Rosa26 loxP-STOP-loxP DTR mice. After defining a well-tolerated DTx dose, DAT-DTR and DTR-flfl controls were subjected to non-toxic DTx treatment (5 × 100 pg/g) and subsequent histology and behavioral tests. DAT protein levels were reduced in the midbrain, and tyrosine hydroxylase-positive neurons were reduced in the substantia nigra, whereas the pan-neuronal marker NeuN was not affected. Despite the promising histologic results, there was no difference in motor function tests or open field behavior. These are tests in which double mutant Pink1−/−SNCAA53T Parkinson mice show behavioral abnormalities. Higher doses of DTx were toxic in both groups. The data suggest that DTx treatment in mice with Cre/loxP-driven DAT-DTR expression leads to partial ablation of DA-neurons but without PD-reminiscent behavioral correlates.

Fecal Microbiota Transplantation Exerts a Protective Role in MPTP-Induced Parkinson’s Disease via the TLR4/PI3K/AKT/NF-κB Pathway Stimulated by α-Synuclein

Gut microbiota is closely related to the Parkinson's disease (PD) pathogenesis. Additionally, aggregation of α-synuclein (α-syn) is central to PD pathogenesis. Here we identified the further mechanisms of gut microbiota in PD. A mouse model with PD was established via injection of MPTP. Normal or MPTP-induced PD like animals were treated with FMT from healthy normal mice. Pole test and traction test were performed to examine the effectsof FMT on motor function of PD mice. Fecal SCFAs were assessed by gas chromatography-mass spectrometry. The α-syn level in the substantia nigra pars compacta (SN) of mice was measured using western blot. Dopaminergic neurons and microglial activation in the SN were analyzed by immunohistochemistry (IHC) and immunofluorescence (IF) staining. FMT alleviated physical impairment, decreased fecal SCFAs in a mouse model of PD. Additionally, FMT decreased the expression of α-syn, as well as inhibited the activation of microglia in the SN, and blocked the TLR4/PI3K/AKT/NF-κB signaling in the SN and striatum. FMT could protect mice against PD via suppressing α-syn expression and inactivating the TLR4/PI3K/AKT/NF-κB signaling.

MiR-29c-3p regulates TET2 expression and inhibits autophagy process in Parkinson's disease models.

Autophagy in dopamine (DA) neurons is concerned to be associated with Parkinson's disease (PD), but the detailed mechanism remains unknown. Herein, we aimed to investigate the function of microRNA (miR)-29c-3p in autophagy in PD models. Intraperitoneal injection of MPTP (20mg/kg) was given to C57BL/6 mice to establish PD mouse model. SH-SY5Y cells were treated with MPP+ (1mmol/L) to establish in vitro PD model. The results indicated that in the substantia nigra pars compacta (SNpc) DA neurons of PD mice, autophagy was activated accompanied by down-regulated miR-29c-3p and up-regulated ten-eleven translocation 2 (TET2) expression. Up-regulation of miR-29c-3p inhibited TET2 expression and SNpc (including DA neurons) autophagy in PD mice. In vitro PD model confirmed that MPP+ treatment markedly down-regulated miR-29c-3p expression and up-regulated TET2 expression in SH-SY5Y cells in a dose/time-dependent manner. Moreover, miR-29c-3p up-regulation also inhibited autophagy and TET2 expression in vitro. Additionally, TET2 was proved to be targeted and down-regulated by miR-29c-3p. TET2 knockdown inhibited MPP+ -induced autophagy, whereas TET2 over-expression reversed the effects of miR-29c-3p over-expression on SH-SY5Y cell autophagy. Overall, miR-29c-3p over-expression inhibits autophagy in PD models, which may be mediated by TET2. Our finding may provide new insights for regulating autophagy to improve PD progression.

Macrophage migration inhibitory factor meditates MPP+/MPTP-induced NLRP3 inflammasome activation in microglia cells

To explore the mechanisms of macrophage migration inhibitory factor (MIF)/nucleus factor-κB (NF-κB) in mediating 1-methyl-4-phenylpyridinium iodide (MPP +)/1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced activation of Nod-like receptor protein 3 (NLRP3) inflammasome in microglials and the its effects on neurons. Murine microglial cell line Bv-2 was infected with a lentivirus carrying MIF shRNA for MIF knockdown and then treated with MPP+. The total protein levels of NLRP3, caspase-1, p65 and p65 in the cell nuclei and cytoplasm were detected. ELISA was used to detect the levels of IL-1β and IL-18 in the culture supernatant, which served as the conditioned culture medium for MN9D cells, whose TH expression level was detected using Western blotting. The effect of stereotactic injection of an adeno-associated virus (AAV) carrying MIF shRNA on behaviors was assessed in a C57BL/6 mouse model of Parkinson disease (PD) induced by intraperitoneal MPTP injection. TH and Iba-1 immunohistochemistry was used to evaluate the number of substantia nigra neurons and the activation of microglia cells, and the protein expressions of MIF, NLRP3 and TH in the substantia nigra were detected with Western blotting. MPP+ significantly increased NLRP3 and MIF expressions in Bv-2 cells (P < 0.05). MIF knockdown in Bv-2 cells significantly lowered NLRP3 and caspase-1 protein expressions and IL-1β and IL-18 levels in the culture supernatant (P < 0.05) without affecting total protein level of p65. Bv-2 cells with MIF knockdown showed significantly lowered p65 protein expression in the nuclei but increased p65 expression in the cytoplasm (P < 0.05). The conditioned medium derived from Bv-2 cells with MIF knockdown, as compared with that from than MPP +-treated Bv-2 cells, significantly increased TH expression in MN9D cells (P=0.01). Compared with those in MPTP group, the mice receiving injections of AAV-MIF-shRNA had higher scores in pole test and open field test with lower scores in traction test, and showed increased TH-positive neurons, decreased Iba-1 microglia cell activation, reduced expressions of MIF and NLRP3, and increased expression of TH in he substantia nigra (all P < 0.05). Inhibition of MIF can reduce the expression of NLRP3 inflammasomes and inflammatory factor caused by MPP+ in microglia cells to relieve the damage of dopaminergic neurons and alleviate microglia cell activation, thus offering protection against neuroinflammation in Parkinson's disease.