Alleviated Motor Dysfunction Research Articles

L-NRB alleviates amyotrophic lateral sclerosis by regulating P11-Htr4 signaling pathway

IntroductionL-NRB is a compound formed as a ring cleavage product of butylphthalide and borneol in a molar ratio 1:2. This study aimed to explore the therapeutic effect of L-NRB on amyotrophic lateral sclerosis (ALS) and its possible mechanism. MethodsSOD1-G93A mice were used as an ALS model. Behavioral tests, histopathological staining, Nissl staining, immunohistochemistry, enzyme-linked immunosorbent assays, and Western blotting were used to analyze the therapeutic effect. The underlying mechanism of L-NRB in treating ALS was investigated using transcriptomic analyses. ResultsIt was found that L-NRB alleviated motor dysfunction, pathological changes in the gastrocnemius muscle, and motor neuron injuries.The results indicated that L-NRB had a neuroprotective function associated with the inhibition of neuroinflammation. The anti-apoptotic effect of L-NRB was found to be related to the regulation of the P11-Htr4 signaling pathway. ConclusionIn summary, the results demonstrated the therapeutic effect of L-NRB on ALS and suggest a promising new therapeutic candidate for ALS.

Nurr1 overexpression in the primary motor cortex alleviates motor dysfunction induced by intracerebral hemorrhage in the striatum in mice

Hemorrhage-induced injury of the corticospinal tract (CST) in the internal capsule (IC) causes severe neurological dysfunction in both human patients and rodent models of intracerebral hemorrhage (ICH). A nuclear receptor Nurr1 (NR4A2) is known to exert anti-inflammatory and neuroprotective effects in several neurological disorders. Previously we showed that Nurr1 ligands prevented CST injury and alleviated neurological deficits after ICH in mice. To prove direct effect of Nurr1 on CST integrity, we examined the effect of Nurr1 overexpression in neurons of the primary motor cortex on pathological consequences of ICH in mice. ICH was induced by intrastriatal injection of collagenase type VII, where hematoma invaded into IC. Neuron-specific overexpression of Nurr1 was induced by microinjection of synapsin I promoter-driven adeno-associated virus (AAV) vector into the primary motor cortex. Nurr1 overexpression significantly alleviated motor dysfunction but showed only modest effect on sensorimotor dysfunction after ICH. Nurr1 overexpression also preserved axonal structures in IC, while having no effect on hematoma-associated inflammatory events, oxidative stress, and neuronal death in the striatum after ICH. Immunostaining revealed that Nurr1 overexpression increased the expression of Ret tyrosine kinase and phosphorylation of Akt and ERK1/2 in neurons in the motor cortex. Moreover, administration of Nurr1 ligands 1,1-bis(3′-indolyl)-1-(p-chlorophenyl)methane or amodiaquine increased phosphorylation levels of Akt and ERK1/2 as well as expression of glial cell line-derived neurotrophic factor and Ret genes in the cerebral cortex. These results suggest that the therapeutic effect of Nurr1 on striatal ICH is attributable to the preservation of CST by acting on cortical neurons.

Asiaticoside exerts neuroprotection through targeting NLRP3 inflammasome activation

BackgroundParkinson's disease (PD), a neurodegenerative disorder, is characterized by motor symptoms due to the progressive loss of dopaminergic neurons in the substantia nigra (SN) and striatum (STR), alongside neuroinflammation. Asiaticoside (AS), a primary active component with anti-inflammatory and neuroprotective properties, is derived from Centella asiatica. However, the precise mechanisms through which AS influences PD associated with inflammation are not yet fully understood. PurposeThis study aimed to explore the protective mechanism of AS in PD. MethodsTargets associated with AS and PD were identified from the Swiss Target Prediction, Similarity Ensemble Approach, PharmMapper, and GeneCards database. A protein-protein interaction (PPI) network was constructed to identify potential therapeutic targets. Concurrently, GO and KEGG analyses were performed to predict potential signaling pathways. To validate these mechanisms, the effects of AS on 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD in mice were investigated. Furthermore, neuroinflammation and the activation of the NLRP3 inflammasome were assessed to confirm the anti-inflammatory properties of AS. In vitro experiments in BV2 cells were then performed to investigate the mechanisms of AS in PD. Moreover, CETSA, molecular docking, and molecular dynamics simulations (MDs) were performed for further validation. ResultsNetwork pharmacology analysis identified 17 potential targets affected by AS in PD. GO and KEGG analyses suggested the biological roles of these targets, demonstrating that AS interacts with 149 pathways in PD. Notably, the NOD-like receptor signaling pathway was identified as a key pathway mediating AS's effect on PD. In vivo studies demonstrated that AS alleviated motor dysfunction and reduced the loss of dopaminergic neurons in MPTP-induced PD mice. In vitro experiments demonstrated that AS substantially decreased IL-1β release in BV2 cells, attributing this to the modulation of the NLRP3 signaling pathway. CETSA and molecular docking studies indicated that AS forms a stable complex with NLRP3. MDs suggested that ARG578 played an important role in the formation of the complex. ConclusionIn this study, we first predicted that the potential target and pathway of AS's effect on PD could be NLRP3 protein and NOD-like receptor signaling pathway by network pharmacology analysis. Further, we demonstrated that AS could alleviate symptoms of PD induced by MPTP through its interaction with the NLRP3 protein for the first time by in vivo and in vitro experiments. By binding to NLRP3, AS effectively inhibits the assembly and activation of the inflammasome. These findings suggest that AS is a promising inhibitor for PD driven by NLRP3 overactivation.

Hsa_circ_0054220 Upregulates HMGA1 by the Competitive RNA Pattern to Promote Neural Impairment in MPTP Model of Parkinson's Disease.

Parkinson's disease (PD) is a common neurodegenerative disease. Circular RNAs (circRNAs) have been confirmed to regulate neurodegenerative diseases. This study was aimed to explore hsa_circ_0054220 functions in PD.MPP-stimulated SH-SY5Y cells were established as the PD cell model. PD mouse model was established by MPTP. Gene expression in cells and tissues was tested by RT-qPCR. Cell viability and apoptosis were evaluated through CCK-8 and TUNEL assays. The interactions of RNAs were determined by RNA pull-down assay, RIP assay, and luciferase reporter assay.Circ_0054220 expressed at a high level in MPP-treated SH-SY5Y cells. Circ_0054220 inhibition promoted viability and suppressed apoptosis in MPP-stimulated cells. Furthermore, we found that circ_0054220 can competitively bind to miR-145 and miR-625 to upregulate high mobility group A1 (HMGA1) expression. HMGA1 was positively regulated by circ_0054220 and overexpressed in MPP-treated cells as well as the striatum (STR), substantia nigra pars compacta (SNpc), and serum of MPTP-induced mouse model of PD. HMGA1 overexpression counteracted the function of circ_0054220 silencing on cell apoptosis. Furthermore, HMGA1 inhibition notably alleviated motor dysfunction and increased the quantity of neurons in mice resembling PD.Circ_0054220 upregulates HMGA1 by the competitive endogenous RNAs (ceRNA) pattern to promote neural impairment in PD.

NRF1 mitigates motor dysfunction and dopamine neuron degeneration in mice with Parkinson's disease by promoting GLRX m6 A methylation through upregulation of METTL3 transcription.

The feature of Parkinson's disease (PD) is the heavy dopaminergic neuron loss of substantia nigra pars compacta (SNpc), while glutaredoxin (GLRX) has been discovered to modulate the death of dopaminergic neurons. In this context, this study was implemented to uncover the impact of GRX1 on motor dysfunction and dopamine neuron degeneration in PD mice and its potential mechanism. A PD mouse model was established via injection with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into mice. After gain- and loss-of-function assays in mice, motor coordination was assessed using rotarod, pole, and open-field tests, and neurodegeneration in mouse SNpc tissues was determined using immunohistochemistry of tyrosine hydroxylase and Nissl staining. NRF1, methyltransferase-like 3 (METTL3), and GLRX expression in SNpc tissues were evaluated using qRT-PCR, Western blot, and immunohistochemistry. The N6-methyladenosine (m6 A) levels of GLRX mRNA were examined using MeRIP. The relationship among NRF1, METTL3, and GLRX was determined by RIP, ChIP, and dual luciferase assays. Low GLRX, METTL3, and NRF1 expression were observed in MPTP-induced mice, accompanied by decreased m6 A modification level of GLRX mRNA. GLRX overexpression alleviated motor dysfunction and dopamine neuron degeneration in MPTP-induced mice. METTL3 promoted m6 A modification and IGF2BP2-dependent stability of GLRX mRNA, and NRF1 increased METTL3 expression by binding to METTL3 promoter. NRF1 overexpression increased m6 A modification of GLRX mRNA and repressed motor dysfunction and dopamine neuron degeneration in MPTP-induced mice, which was counteracted by METTL3 knockdown. Conclusively, NRF1 constrained motor dysfunction and dopamine neuron degeneration in MPTP-induced PD mice by activating the METTL3/GLRX axis.

Effects of albiflorin on oxidative stress and inflammatory responses in rats with acute spinal cord injury.

Oxidative stress and inflammatory responses are often the predominant detrimental factors associated with spinal cord injury (SCI). This study investigates the potential therapeutic effects of albiflorin (AF) on alleviating inflammation and oxidative stress in the rat model with SCI. Initially, the behavior of SCI-induced rats is examined by Basso-Beattie-Bresnahan score and the inclined plane examination. Then, the immunohistochemical staining of inflammasome-related protein (for instance, NACHT, LRR, and PYD domains-containing protein 3, NLRP3) is performed in combination with enzyme-linked immunosorbent assay (ELISA) of corresponding proinflammatory factors to assess the immunomodulatory effects of AF. Further, the markers involved in oxidative stress are examined by ELISA and western blot analysis analyses. These findings indicated that AF could alleviate motor dysfunction and the loss of neuron cells in SCI-induced rats. Mechanistically, AF could attenuate the inflammatory responses by reducing oxidative stress and activating nuclear erythroid-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway in SCI rats. Depleting the antioxidant capacity by inhibiting glutathione biosynthesis could counteract the anti-inflammatory activity of AF in SCI rats. Together, our data suggested that AF could serve as a potential therapeutic agent against the aggravation of SCI in rats.

Effects of Repetitive Transcranial Magnetic Stimulation on Pallidum GABAergic Neurons and Motor Function in Rat Models of Kernicterus.

Kernicterus is a serious complication of hyperbilirubinemia, caused by neuronal injury due to excessive unconjugated bilirubin (UCB) in specific brain areas. This injury induced by this accumulation in the globus pallidus can induce severe motor dysfunction. Repetitive transcranial magnetic stimulation (rTMS) has shown neuroprotective effects in various neurological diseases. This study aimed to investigate the effects of rTMS on pallidal nerve damage and motor dysfunction in a rat model of kernicterus. Rats were divided into a sham group (n = 16), a model group (bilirubin with sham rTMS; n = 16) and an rTMS group (bilirubin with rTMS; n = 16). High-frequency rTMS (10 Hz) was applied starting from 24 h postmodeling for 7 days. The rotarod test, western blotting and immunohistochemical staining were performed to measure motor function and protein expression levels. The rTMS mitigated the negative effects of UCB on the general health of kernicterus-model rats and improved their growth and development. Furthermore, the rTMS alleviated UCB-induced motor dysfunction and increased the expression of GABAergic neuronal marker GAD67 in the globus pallidus. Notably, it also inhibited apoptosis-related protein caspase-3 activation. In conclusion, rTMS could alleviate motor dysfunction by inhibiting apoptosis and increasing globus pallidus GAD67 in kernicterus rat models, indicating that it may be a promising treatment for kernicterus.

Acetylated α-tubulin alleviates injury to the dendritic spines after ischemic stroke in mice.

Functional recovery is associated with the preservation of dendritic spines in the penumbra area after stroke. Previous studies found that polymerized microtubules (MTs) serve a crucial role in regulating dendritic spine formation and plasticity. However, the mechanisms that are involved are poorly understood. This study is designed to understand whether the upregulation of acetylated α-tubulin (α-Ac-Tub, a marker for stable, and polymerized MTs) could alleviate injury to the dendritic spines in the penumbra area and motor dysfunction after ischemic stroke. Ischemic stroke was mimicked both in an in vivo and in vitro setup using middle cerebral artery occlusion and oxygen-glucose deprivation models. Thy1-YFP mice were utilized to observe the morphology of the dendritic spines in the penumbra area. MEC17 is the specific acetyltransferase of α-tubulin. Thy1 CreERT2-eYFP and MEC17fl/fl mice were mated to produce mice with decreased expression of α-Ac-Tub in dendritic spines of pyramidal neurons in the cerebral cortex. Moreover, AAV-PHP.B-DIO-MEC17 virus and tubastatin A (TBA) were injected into Thy1 CreERT2-eYFP and Thy1-YFP mice to increase α-Ac-Tub expression. Single-pellet retrieval, irregular ladder walking, rotarod, and cylinder tests were performed to test the motor function after the ischemic stroke. α-Ac-Tub was colocalized with postsynaptic density 95. Although knockout of MEC17 in the pyramidal neurons did not affect the density of the dendritic spines, it significantly aggravated the injury to them in the penumbra area and motor dysfunction after stroke. However, MEC17 upregulation in the pyramidal neurons and TBA treatment could maintain mature dendritic spine density and alleviate motor dysfunction after stroke. Our study demonstrated that α-Ac-Tub plays a crucial role in the maintenance of the structure and functions of mature dendritic spines. Moreover, α-Ac-Tub protected the dendritic spines in the penumbra area and alleviated motor dysfunction after stroke.

Cyclophilin D-induced mitochondrial impairment confers axonal injury after intracerebral hemorrhage in mice.

The mitochondrial permeability transition pore is a nonspecific transmembrane channel. Inhibition of mitochondrial permeability transition pore opening has been shown to alleviate mitochondrial swelling, calcium overload, and axonal degeneration. Cyclophilin D is an important component of the mitochondrial permeability transition pore. Whether cyclophilin D participates in mitochondrial impairment and axonal injury after intracerebral hemorrhage is not clear. In this study, we established mouse models of intracerebral hemorrhage in vivo by injection of autologous blood and oxyhemoglobin into the striatum in Thy1-YFP mice, in which pyramidal neurons and axons express yellow fluorescent protein. We also simulated intracerebral hemorrhage in vitro in PC12 cells using oxyhemoglobin. We found that axonal degeneration in the early stage of intracerebral hemorrhage depended on mitochondrial swelling induced by cyclophilin D activation and mitochondrial permeability transition pore opening. We further investigated the mechanism underlying the role of cyclophilin D in mouse models and PC12 cell models of intracerebral hemorrhage. We found that both cyclosporin A inhibition and short hairpin RNA interference of cyclophilin D reduced mitochondrial permeability transition pore opening and mitochondrial injury. In addition, inhibition of cyclophilin D and mitochondrial permeability transition pore opening protected corticospinal tract integrity and alleviated motor dysfunction caused by intracerebral hemorrhage. Our findings suggest that cyclophilin D is used as a key mediator of axonal degeneration after intracerebral hemorrhage; inhibition of cyclophilin D expression can protect mitochondrial structure and function and further alleviate corticospinal tract injury and motor dysfunction after intracerebral hemorrhage. Our findings provide a therapeutic target for preventing axonal degeneration of white matter injury and subsequent functional impairment in central nervous diseases.

Effect of Electroacupuncture on Short-Chain Fatty Acids in Peripheral Blood after Middle Cerebral Artery Occlusion/Reperfusion in Rats Based on Gas Chromatography-Mass Spectrometry.

Previous fundamental and clinical research has shown that electroacupuncture (EA) at the acupoints of Quchi (LI11) and Zusanli (ST36) can successfully alleviate motor dysfunction following stroke. Additionally, it has been discovered that gut microbiota and their metabolites play an essential role in stroke. However, the relationship between the metabolites of gut microbiota and the efficacy of EA is still unclear. Therefore, the aim of this study was to evaluate the mechanism of EA at LI11 and ST36 in the treatment of motor dysfunction after middle cerebral artery occlusion/reperfusion (MCAO/R) in model rats by comparing the differences and correlation between different short-chain fatty acids (SCFAs) and the recovery of motor function. The results indicated that EA at LI11 and ST36 acupoints enhanced the neurological function, motor function, and infarct volume of MCAO/R rats. The levels of acetic acid, propionic acid, and total SCFAs were considerably lower in the MCAO/R group than in the sham group (P < 0.05). Acetic acid, propionic acid, and total SCFA concentrations were substantially higher in the MCAO/R + EA group than in the MCAO/R group (P < 0.05). Finally, Pearson correlation analysis revealed that the propionic acid concentration was substantially favorably connected with the duration on the rotarod (r = 0.633 and P < 0.05) and highly negatively correlated with the modified neurological severity score (mNSS) (r = −0.698 and P < 0.05) and the percentage of cerebral infarct volume (r = −0.729 and P < 0.05). Taken together, these findings indicate that the increase in propionic acid may be one of the mechanisms and targets of EA at LI11 and ST36 acupoints to improve poststroke motor dysfunction in MCAO/R rats.

Ononin Alleviates Sublesional Bone Loss and Inflammation Following Spinal Cord Injury in Rats by Regulating Adenosine 5′-Monophosphate-Activated Protein Kinase Signaling Pathway

Inhibition of bone loss is one of the ways to alleviate motor dysfunction caused by spinal cord injury, a lifelong and lifechanging injury. Isoflavone from legumes that include ononin, Calycosin-7-O-β-Glucoside, and others have been shown to have potential for the treatment of orthopedic diseases. While there are potentials for ononin to improve the motor function of spinal cord injury, studies on the subject are lacking. To this end, we examined the ameliorative role of ononin in an animal model of spinal cord injury. Our results show the ononin reduced inflammatory response, decreased femoral bone loss, and promoted osteoblast formation in rats with spinal cord injury through promotion of adenosine 5′′-monophosphate-activated protein kinase signaling pathway.

Dexmedetomidine attenuates motor deficits via restoring the function of neurons in the nigrostriatal circuit in Parkinson's disease model mice

Parkinson's disease (PD) is a neurodegenerative disorder characterized by a progressive degeneration in nigrostriatal dopamine pathway that is essential to control motor functions. Dexmedetomidine (DEX), a sedative and analgesic drug, is often used in patients with PD undergoing surgery. Although DEX seems to have promising future applications in neuroprotection, whether and how DEX alter the function of nigrostriatal circuit and its roles on motor deficits in PD remain unclear. Here we report that DEX attenuated motor deficits in a dose-dependent manner and protected the degeneration of dopaminergic neurons in MPTP-induced PD model mice. The DEX acted on the neurons in the nigrostriatal circuits, including activation of dopaminergic neurons and the reduction of the excitabilities of striatal neurons via dopamine D2 receptors. We further found that DEX prevented the increase in glutamatergic transmission of cholinergic interneurons (CINs) to alleviate motor dysfunction. It also decreased the intrinsic excitability and glutamatergic transmission of striatal D2 medium spiny neurons (D2-MSNs). Finally, D2 receptor antagonists prevented the restoration of DEX on motor deficits. These results demonstrate that DEX, a neuroprotective drug, restores the function of nigrostriatal neurons and improves the motor deficits, providing a potential neural mechanism of the effects of anesthetic drugs on PD progression.

HOTTIP downregulation reduces neuronal damage and microglial activation in Parkinson's disease cell and mouse models.

HOXA transcript at the distal tip (HOTTIP), a newly identified long noncoding RNA, has been shown to exhibit anti-inflammatory effects and inhibit oxygen-glucose deprivation-induced neuronal apoptosis. However, its role in Parkinson's disease (PD) remains unclear. 1-Methyl-4-phenylpyridium (MPP+) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were used to establish PD models in SH-SY5Y and BV2 cells and in C57BL/6 male mice, respectively. In vitro, after HOTTIP knockdown by sh-HOTTIP transfection, HOTTIP and FOXO1 overexpression promoted SH-SY5Y apoptosis, BV2 microglial activation, proinflammatory cytokine expression, and nuclear factor kappa-B and NACHT, LRR and PYD domains-containing protein 3 inflammasome activation. Overexpression of miR-615-3p inhibited MPP+-induced neuronal apoptosis and microglial inflammation and ameliorated HOTTIP- and FOXO1-mediated nerve injury and inflammation. In vivo, HOTTIP knockdown alleviated motor dysfunction in PD mice and reduced neuronal apoptosis and microglial activation in the substantia nigra. These findings suggest that inhibition of HOTTIP mitigates neuronal apoptosis and microglial activation in PD models by modulating miR-615-3p/FOXO1. This study was approved by the Ethics Review Committee of the Affiliated Hospital of Qingdao University, China (approval No. UDX-2018-042) in June 2018.

Treadmill exercise alleviates neuronal damage by suppressing NLRP3 inflammasome and microglial activation in the MPTP mouse model of Parkinson’s disease

Treadmill exercise has been recognized as an effectively therapeutic strategy for Parkinson's disease (PD). However, its exact molecular mechanism of promoting PD remain unclear. Recently, the NLRP3 inflammasome is considered to play a critical role in the pathogenesis of PD. In this study, we investigated whether NLRP3 inflammasome was involved in treadmill exercise-induced neuroprotection and anti-inflammation effect in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD. 8-week-old male mice (C57BL/6 strain) were divided into four groups: Control, MPTP, MPTP + EX and EX. MPTP was intraperitoneally injected into mice to establish chronic PD model. The open-field test and pole test were used to assess motor function. The results showed that treadmill exercise significantly alleviated motor dysfunction and dopaminergic neuron degeneration induced by MPTP. In addition, we also found that treadmill exercise suppressed MPTP-triggered microglia activation and the co-localization of NLRP3+/Iba-1+ cells in the substantia nigra. These effects were associated with suppression NLRP3 inflammasome via down-regulation of TLR4/MyD88/NF-κB pathway. Overall, our study demonstrated that treadmill exercise could effectively alleviates neuronal damage via inhibition of NLRP3 inflammasome and microglial activation in MPTP‐induced PD mouse model.

Melatonin Attenuates Neuroinflammation by Down-Regulating NLRP3 Inflammasome via a SIRT1-Dependent Pathway in MPTP-Induced Models of Parkinson’s Disease

BackgroundInflammasome-induced neuroinflammation is a key contributor to the pathology of Parkinson’s disease (PD). NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation has been implicated in PD in postmortem human PD brains, indicating it as a potential target for PD treatment. Melatonin, a multitasking molecule, has been found to have anti-inflammatory activities, mediated by silence information regulator 1 (SIRT1). However, whether and how melatonin is involved in inflammasome-induced neuroinflammation in PD pathogenesis remains unclear.MethodsWe investigated the potential anti-inflammatory effects of melatonin in vitro and in vivo, using 1-methyl-4-phenylpyridinium (MPP+)-simulated BV2 and primary microglia cell models, and a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced murine PD model, with or without melatonin treatment. Rotarod, grip strength, and open-field tests were performed to measure the effects of melatonin on MPTP-induced motor disorders. Degeneration of dopaminergic neurons was evaluated by immunofluorescence. Changes in microglia were examined by immunofluorescence and Western blotting, and the expression levels of the involved signaling molecules were assessed by Western blotting and enzyme-linked immunosorbent assay (ELISA). Intracellular reactive oxygen species (ROS) was detected using fluorescent probes via flow cytometry.ResultsWe found that melatonin significantly alleviated motor dysfunction and prevented MPTP-induced neurotoxicity in dopaminergic neurons. Additionally, melatonin reduced MPTP-induced microglial activation and suppressed NLRP3 inflammasome activity, and also inhibited IL-1β secretion. Moreover, in MPP+-primed BV2 cells, melatonin markedly restored the downregulation of SIRT1 and attenuated the activation of the NLRP3 inflammasome. This was reversed by SIRT1 inhibitor treatment.ConclusionIn conclusion, our data demonstrated that melatonin attenuates neuroinflammation by negatively regulating NLRP3 inflammasome activation via a SIRT1-dependent pathway in MPTP-induced PD models. These findings provide novel insights into the mechanism underlying the anti-inflammatory effects of melatonin in PD.

Glucagon-like peptide-1/glucose-dependent insulinotropic polypeptide dual receptor agonist DA-CH5 is superior to exendin-4 in protecting neurons in the 6-hydroxydopamine rat Parkinson model.

Patients with Parkinson's disease (PD) have impaired insulin signaling in the brain. Incretin hormones, including glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), can re-sensitize insulin signaling. In a recent phase II clinical trial, the first GLP-1 mimic, exendin-4, has shown reliable curative effect in patients with PD. DA-CH5 is a novel GLP-1/GIP receptor unimolecular co-agonist with a novel peptide sequence added to cross the blood-brain barrier. Here we showed that both exendin-4 and DA-CH5 protected against 6-hydroxydopamine (6-OHDA) cytotoxicity, inhibited apoptosis, improved mitogenesis and induced autophagy flux in SH-SY5Y cells via activation of the insulin receptor substrate-1 (IRS-1)/alpha serine/threonine-protein kinase (Akt)/cAMP response element-binding protein (CREB) pathway. We also found that DA-CH5 (10 nmol/kg) daily intraperitoneal administration for 30 days post-lesion alleviated motor dysfunction in rats and prevented stereotactic unilateral administration of 6-OHDA induced dopaminergic neurons loss in the substantia nigra pars compacta. However, DA-CH5 showed curative effects in reducing the levels of α-synuclein and the levels of pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-1β). It was also more effective than exendin-4 in inhibiting apoptotic process and protecting mitochondrial functions. In addition, insulin resistance was largely alleviated and the expression of autophagy-related proteins was up-regulated in PD model rats after DA-CH5 treatment. These results in this study indicate DA-CH5 plays a therapeutic role in the 6-OHDA-unilaterally lesioned PD rat model and is superior to GLP-1 analogue exendin-4. The study was approved by the Animal Ethics Committee of Shanxi Medical University of China.

Effective inhibition of miR-330/SHIP1/NF-κB signaling pathway via miR-330 sponge repolarizes microglia differentiation.

Neuroinflammation mediated by microglia has been identified as vital pathogenesis in Parkinson's disease (PD). This study aimed to investigate the role and potential regulatory mechanism of microRNA-330 in the lipopolysaccharide (LPS)-induced chronic neuroinflammatory model. Primary microglia chronic inflammation model and PD animal model were established by LPS treatment. Bulged microRNA-330 sponges containing six microRNA binding sites were constructed and delivered by plasmid or recombinant adeno-associated virus (rAAV2)/5-green fluorescent protein (GFP) vector. The expression levels of microRNA-330 were assessed by a quantitative real-time polymerase chain reaction. Primary microglia polarization was determined by flow cytometry; meanwhile,dopamine and pro-(anti-)inflammatory cytokines were measured by enzyme-linked immunosorbent assay. Expression levels of GFAP, lba1, inducible nitric oxide synthase (iNOS), Arg1, SHIP1, cytoplasmic, and nuclear factor-κB (NF-κB) were analyzed by Western blot. The behavioral deficit was determined by the rotarod test. The expression of microRNA-330 increased in the first 4 days and reached a plateau subsequently after LPS treatment. The sponges-mediated repression effect on M1 polarization was gradually enhanced with time. Treatment of miR-330 sponges increased the SHIP1 and Arg1 expression, and decreased the translocation of NF-κB and iNOS expression, suggesting the repression of inflammation. In the LPS-induced PD mice, administration of rAAV-sponge-GFP suppressed activation of microglia, downregulated proinflammatory cytokines, resumed the secretion of dopamine, rescued the dopaminergic neurons, and alleviated motor dysfunction. Our results demonstrated that microRNA-330 sponges could sustainably suppress LPS-induced polarization of microglia both in vivo and in vitro probably by negatively regulating NF-κB activity via target SHIP1 in microglia, which might be a promising neuroprotective strategy in neurological diseases, such as PD.

Inhibition of copper transporter 1 prevents α-synuclein pathology and alleviates nigrostriatal degeneration in AAV-based mouse model of Parkinson's disease

The formation of α-synuclein aggregates is a major pathological hallmark of Parkinson's disease. Copper promotes α-synuclein aggregation and toxicity in vitro. The level of copper and copper transporter 1, which is the only known high-affinity copper importer in the brain, decreases in the substantia nigra of Parkinson's disease patients. However, the relationship between copper, copper transporter 1 and α-synuclein pathology remains elusive. Here, we aim to decipher the molecular mechanisms of copper and copper transporter 1 underlying Parkinson's disease pathology. We employed yeast and mammalian cell models expressing human α-synuclein, where exogenous copper accelerated intracellular α-synuclein inclusions and silencing copper transporter 1 reduced α-synuclein aggregates in vitro, suggesting that copper transporter 1 might inhibit α-synuclein pathology. To study our hypothesis in vivo, we generated a new transgenic mouse model with copper transporter 1 conditional knocked-out specifically in dopaminergic neuron. Meanwhile, we unilaterally injected adeno-associated viral human-α-synuclein into the substantia nigra of these mice. Importantly, we found that copper transporter 1 deficiency significantly reduced S129-phosphorylation of α-synuclein, prevented dopaminergic neuronal loss, and alleviated motor dysfunction caused by α-synuclein overexpression in vivo. Overall, our data indicated that inhibition of copper transporter 1 alleviated α-synuclein mediated pathologies and provided a novel therapeutic strategy for Parkinson's disease and other synucleinopathies.

Silencing RNF13 Alleviates Parkinson's Disease - Like Problems in Mouse Models by Regulating the Endoplasmic Reticulum Stress-Mediated IRE1α-TRAF2-ASK1-JNK Pathway.

The objective of this study was to understand if RNF13 can affect Parkinson's disease (PD) model mice by modulating the endoplasmic reticulum stress (ERS)-mediated IRE1α-TRAF2-ASK1-JNK pathway. C57BL/6 mice injected with MPTP to establish PD mice models were divided into Control, MPTP, MPTP + sh-RNF13, and MPTP + sh-NC groups. Rotarod, balance beam, and open-field tests were used to assess the behavioral changes of experimental mice. Immunofluorescence assay was used to determine TH-positive expression in substantia nigra, TUNEL staining to detect apoptosis, and Western blotting to measure the expression of IRE1α-TRAF2-ASK1-JNK pathway. Besides, SH-SY5Y cells treated with MPP+ were assigned into Control, MPP+, MPP+ + sh-RNF13, and MPP+ + sh-NC groups in vitro to detect cell viability, apoptosis and Ca2+ level. When compared with those Control mice, MPTP mice showed decreased retention time spent on rotarod performance and prolonged time on balance beam test, as well as evident reductions in floor plane (FP) movements, moving time, moving distance, and mean velocity in open-field test, which had an obvious increase of TUNEL-positive cells, significant decrease of TH-positive cells, and remarkable up-regulations of RNF13, p-IRE1α/IRE1α, TRAF2, ASK1, and p-JNK/JNK. Meanwhile, MPTP mice treated with sh-RNF13 were improved in all above indexes. In vitro, MPP+ treated SH-SY5Y cells had decreased cell viability and increased cell apoptosis, as well as the upregulated IRE1α-TRAF2-ASK1-JNK pathway proteins and Ca2+ level. RNF13 knockdown improved all above indexes in SH-SY5Y cells treated with MPP+. Silencing RNF13 can alleviate motor dysfunction and dopamine neuronal damage in PD mice by inhibiting ERS-mediated IRE1α-TRAF2-ASK1-JNK pathway.

Study on Effect of Striatal mGluR2/3 in Alleviating Motor Dysfunction in Rat PD Model Treated by Exercise Therapy.

Background: Exercise therapy has been widely applied in clinical rehabilitation as an important practical and side effect—free adjuvant therapy, with a significant effect in alleviating motor dysfunction of patients with Parkinson’s disease (PD) or animal PD models. This study focuses on the effect of exercise therapy in reducing the concentration of extracellular glutamate (Glu) in the striatum in a rat PD model by upregulating the expression of group II metabotropic Glu receptor (mGluR2/3), so as to alleviate motor dysfunction in the rat PD model.Methods: Neurotoxin 6-hydroxydopamine (6-OHDA) was injected into the right medial forebrain bundle (MFB) of the rats to establish the semi-lateral cerebral damage PD model. The sham-operated group was given an equal amount of normal saline at the same site and taken as the control group. The apomorphine (APO)-induced rotational behavior test combined with immunohistochemical staining with tyrosine hydroxylase (TH) in the substantia nigra (SNc) and striatum was performed to assess the reliability of the model. The exercise group was given treadmill exercise intervention for 4 weeks (11 m/min, 30 min/day, 5 days/week) 1 week after the operation. The open field test (OFT) was performed to assess the locomotor activity of the rats; the Western blot technique was used to detect SNc TH and striatal mGluR2/3 protein expressions; real-time polymerase chain reaction (RT-PCR) was applied to detect striatal mGluR2 and mGluR3 mRNA expressions; the microdialysis—high-performance liquid chromatography (HPLC) method was adopted to detect the concentration of extracellular Glu in striatal neurons.Results: Compared with the control group, the number of rotations of each model group at the first week was significantly increased (P < 0.01); compared with the PD group, the number of rotations of the PD + exercise group at the third week and the fifth week was significantly decreased (P < 0.05, P < 0.01). Compared with the control group, the total movement distance, the total movement time, and the mean velocity of each model group at the first week were significantly reduced (P < 0.05); compared with the PD group, the total movement distance, the total movement time, and the mean velocity of the PD + exercise group at the third week and the fifth week were significantly increased (P < 0.01). Compared with the control group, the count of immunopositive cells and protein expression of SNc TH, and the content of immunopositive fiber terminals in the striatal TH of each model group significantly declined (P < 0.01). Compared with the PD group, the striatal mGluR2/3 protein expression of the PD + exercise group significantly rose (P < 0.01). Compared with the control group, the concentration of extracellular Glu in striatal neurons of each model group at the first week significantly grew (P < 0.05); compared with the PD group, the concentration of extracellular Glu in striatal neurons of the PD + exercise group at the third week and the fifth week was significantly decreased (P < 0.01); compared with the PD + exercise group, the concentration of extracellular Glu in striatal neurons of the group injected with mGluR2/3 antagonist (RS)-1-amino-5-phosphonoindan-1-carboxylic acid (APICA) into the striatum at the third week and the fifth week was significantly increased (P < 0.05, P < 0.01). Compared with the control group, the striatal mGluR2/3 protein expression of the PD group was significantly downregulated (P < 0.01); compared with the PD group, the striatal mGluR2/3 protein expression of the PD + exercise group was significantly upregulated (P < 0.05); compared with the control group, the striatal mGluR3 mRNA expression of the PD group was significantly downregulated (P < 0.01); compared with the PD group, the striatal mGluR3 mRNA expression of the PD + exercise group was significantly upregulated (P < 0.01); 6-OHDA damage and exercise intervention had no significant effect on the striatal mGluR2 mRNA expression (P > 0.05). Compared with the PD + exercise group, the total movement distance, the total movement time, and the mean velocity of the PD + exercise + APICA group were significantly decreased (P < 0.05); compared with the PD group, the PD + exercise + APICA group had no significant change in the total movement distance, the total movement time, and the mean velocity (P > 0.05).Conclusion: These data collectively demonstrate that the mGluR2/3-mediated glutamatergic transmission in the striatum is sensitive to dopamine (DA) depletion and may serve as a target of exercise intervention for mediating the therapeutic effect of exercise intervention in a rat model of PD.