Midbrain dopaminergic neurons play an important role in the etiology of neurodevelopmental and neurodegenerative diseases.They also represent a potential source of transplanted cells for therapeutic applications.In vi...Midbrain dopaminergic neurons play an important role in the etiology of neurodevelopmental and neurodegenerative diseases.They also represent a potential source of transplanted cells for therapeutic applications.In vitro differentiation of functional midbrain dopaminergic neurons provides an accessible platform to study midbrain neuronal dysfunction and can be used to examine obstacles to dopaminergic neuronal development.Emerging evidence and impressive advances in human induced pluripotent stem cells,with tuned neural induction and differentiation protocols,makes the production of induced pluripotent stem cell-derived dopaminergic neurons feasible.Using SB431542 and dorsomorphin dual inhibitor in an induced pluripotent stem cell-derived neural induction protocol,we obtained multiple subtypes of neurons,including 20%tyrosine hydroxylase-positive dopaminergic neurons.To obtain more dopaminergic neurons,we next added sonic hedgehog(SHH)and fibroblast growth factor 8(FGF8)on day 8 of induction.This increased the proportion of dopaminergic neurons,up to 75%tyrosine hydroxylase-positive neurons,with 15%tyrosine hydroxylase and forkhead box protein A2(FOXA2)co-expressing neurons.We further optimized the induction protocol by applying the small molecule inhibitor,CHIR99021(CHIR).This helped facilitate the generation of midbrain dopaminergic neurons,and we obtained 31-74%midbrain dopaminergic neurons based on tyrosine hydroxylase and FOXA2 staining.Thus,we have established three induction protocols for dopaminergic neurons.Based on tyrosine hydroxylase and FOXA2 immunostaining analysis,the CHIR,SHH,and FGF8 combined protocol produces a much higher proportion of midbrain dopaminergic neurons,which could be an ideal resource for tackling midbrain-related diseases.展开更多
Numerous studies have shown that cell replacement therapy can replenish lost cells and rebuild neural circuitry in animal models of Parkinson’s disease.Transplantation of midbrain dopaminergic progenitor cells is a p...Numerous studies have shown that cell replacement therapy can replenish lost cells and rebuild neural circuitry in animal models of Parkinson’s disease.Transplantation of midbrain dopaminergic progenitor cells is a promising treatment for Parkinson’s disease.However,transplanted cells can be injured by mechanical damage during handling and by changes in the transplantation niche.Here,we developed a one-step biomanufacturing platform that uses small-aperture gelatin microcarriers to produce beads carrying midbrain dopaminergic progenitor cells.These beads allow midbrain dopaminergic progenitor cell differentiation and cryopreservation without digestion,effectively maintaining axonal integrity in vitro.Importantly,midbrain dopaminergic progenitor cell bead grafts showed increased survival and only mild immunoreactivity in vivo compared with suspended midbrain dopaminergic progenitor cell grafts.Overall,our findings show that these midbrain dopaminergic progenitor cell beads enhance the effectiveness of neuronal cell transplantation.展开更多
Accumulating studies suggest that neuroinflammation characterized by microglial overactivation plays a pivotal role in the pathogenesis of Parkinson’s disease.As such,inhibition of microglial overactivation might be ...Accumulating studies suggest that neuroinflammation characterized by microglial overactivation plays a pivotal role in the pathogenesis of Parkinson’s disease.As such,inhibition of microglial overactivation might be a promising treatment strategy to delay the onset or slow the progression of Parkinson’s disease.Ginsenoside Rbl,the most active ingredient of ginseng,reportedly exerts neuroprotective effects by suppressing inflammation in vitro.The present study aimed to evaluate the neuroprotective and anti-inflammatory effects of ginsenoside Rbl in a lipopolysaccharide-induced rat Parkinson’s disease model.Rats were divided into four groups.In the control group,sham-operated rats were intraperitoneally administered normal saline for 14 consecutive days.In the ginsenoside Rbl group,ginsenoside Rb1(20 mg/kg)was intraperitoneally injected for 14 consecutive days after sham surgery.In the lipopolysaccharide group,a single dose of lipopolysaccharide was unilaterally microinjected into the rat substantial nigra to establish the Parkinson’s disease model.Lipopolysaccharide-injected rats were treated with normal saline for 14 consecutive days.In the ginsenoside Rbl +lipopolysaccharide group,lipopolysaccharide was unilaterally microinjected into the rat substantial nigra.Subsequently,ginsenoside Rbl was intraperitoneally injected for 14 consecutive days.To investigate the therapeutic effects of ginsenoside Rbl,behavioral tests were performed on day 15 after lipopolysaccharide injection.We found that ginsenoside Rbl treatment remarkably reduced apomorphine-induced rotations in lipopolysaccharide-treated rats compared with the lipopolysaccharide group.To investigate the neurotoxicity of lipopolysaccharide and potential protective effect of ginsenoside Rbl,contents of dopamine and its metabolites in the striatum were measured by high-performance liquid chromatography.Compared with the lipopolysaccharide group,ginsenoside Rbl obviously attenuated the lipopolysaccharide-induced depletion of dopamine and its metabolites in the striatum.To further explore the neuroprotective effect of ginsenoside Rbl against lipopolysaccharide-induced neurotoxicity,immunohistochemistry and western blot assay of tyrosine hydroxylase were performed to evaluate dopaminergic neuron degeneration in the substantial nigra par compacta.The results showed that lipopolysaccharide injection caused a large loss of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra and a significant decrease in overall tyrosine hydroxylase expression.However,ginsenoside Rb1 noticeably reversed these changes.To investigate whether the neuroprotective effect of ginsenoside Rbl was associated with inhibition of lipopolysaccharide-induced microglial activation,we examined expression of the microglia marker Iba-1.Our results confirmed that lipopolysaccharide injection induced a significant increase in Iba-1 expression in the substantia nigra;however,ginsenoside Rbl effectively suppressed lipopolysaccharide-induced microglial overactivation.To elucidate the inhibitory mechanism of ginsenoside Rb1,we examined expression levels of inflammatory mediators(tumor necrosis factor-a,interleukin-1β,inducible nitric oxide synthase,and cyclooxygenase 2)and phosphorylation of nuclear factor kappa B signaling-related proteins(IκB,IKK)in the substantia nigra with enzyme-linked immunosorbent and western blot assays.Our results revealed that compared with the control group,phosphorylation and expression of inflammatory mediators IκB and IKK in the substantia nigra of lipopolysaccharide group rats were significantly increased;whereas,ginsenoside Rbl obviously reduced lipopolysaccharide-induced changes on the lesioned side of the substantial nigra par compacta.These findings confirm that ginsenoside Rbl can inhibit inflammation induced by lipopolysaccharide injection into the substantia nigra and protect dopaminergic neurons,which may be related to its inhibition of the nuclear factor kappa B signaling pathway.This study was approved by the Experimental Animal Ethics Committee of Shandong University of China in April 2016(approval No.KYLL-2016-0148).展开更多
Prolonged activation of adenosine A1 receptor likely leads to damage of dopaminergic neurons and subsequent development of neurodegenerative diseases.However,the pathogenesis underlying long-term adenosine A1 receptor...Prolonged activation of adenosine A1 receptor likely leads to damage of dopaminergic neurons and subsequent development of neurodegenerative diseases.However,the pathogenesis underlying long-term adenosine A1 receptor activation-induced neurodegeneration remains unclear.In this study,rats were intraperitoneally injected with 5 mg/kg of the adenosine A1 receptor agonist N6-cyclopentyladenosine(CPA)for five weeks.The mobility of rats was evaluated by forced swimming test,while their cognitive capabilities were evaluated by Y-maze test.Expression of sortilin,α-synuclein,p-JUN,and c-JUN proteins in the substantia nigra were detected by western blot analysis.In addition,immunofluorescence staining of sortilin andα-synuclein was performed to detect expression in the substantia nigra.The results showed that,compared with adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine(5 mg/kg)+CPA co-treated rats,motor and memory abilities were reduced,surface expression of sortin andα-synuclein in dopaminergic neurons was reduced,and total sortilin and totalα-synuclein were increased in CPA-treated rats.MN9D cells were incubated with 500 nM CPA alone or in combination with 10μM SP600125(JNK inhibitor)for 48 hours.Quantitative real-time polymerase chain reaction analysis of sortilin andα-synuclein mRNA levels in MN9D cells revealed upregulated sortilin expression in MN9D cells cultured with CPA alone,but the combination of CPA and SP600125 could inhibit this expression.Predictions made using Jasper,PROMO,and Alibaba online databases identified a highly conserved sequence in the sortilin promoter that was predicted to bind JUN in both humans and rodents.A luciferase reporter assay of sortilin promoter plasmid-transfected HEK293T cells confirmed this prediction.After sortilin expression was inhibited by sh-SORT1,expression of p-JUN and c-JUN was detected by western blot analysis.Long-term adenosine A1 receptor activation levels upregulatedα-synuclein expression at the post-transcriptional level by affecting sortilin expression.The online tool Raptor-X-Binding and Discovery Studio 4.5 prediction software predicted that sortilin can bind toα-synuclein.Co-immunoprecipitation revealed an interaction between sortilin andα-synuclein in MN9D cells.Our findings indicate that suppression of prolonged adenosine A1 receptor activation potently inhibited sortilin expression andα-synuclein accumulation,and dramatically improved host cognition and kineticism.This study was approved by the University Committee of Animal Care and Supply at the University of Saskatchewan(approval No.AUP#20070090)in March 2007 and the Animals Ethics Committee of University of South China(approval No.LL0387-USC)in June 2017.展开更多
BACKGROUND: Rifampicin inhibits the formation of α-synuclein multimer and protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyritine (MPTP)-induced PC12 cell apoptosis.OBJECTIVE: To compare the effect of rifampici...BACKGROUND: Rifampicin inhibits the formation of α-synuclein multimer and protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyritine (MPTP)-induced PC12 cell apoptosis.OBJECTIVE: To compare the effect of rifampicin pre-and post-treatment on tyrosine hydroxylase and α-synuclein expression in substantia nigra pars compacta in a rat model of Parkinson’s disease.DESIGN,TIME AND SETTING: A randomized,controlled experiment was performed at the Experimental Animal Center of Sun Yat-sen University North Campus (China) from November 2006 to October 2008.MATERIALS: Rifampicin was purchased from MD,USA;rotenone was purchased from Sigma,USA;mouse anti-rat α-synuclein monoclonal antibody was purchased from B&D,USA;and rabbit anti-rat tyrosine hydroxylase monoclonal antibody was purchased from Chemicon,USA.METHODS: A total of 72 male,Sprague Dawley rats,aged 8 weeks,were randomly assigned to 5 groups: blank control (n = 12),rifampicin (n = 12),rotenone (n = 16),rifampicin pre-treatment (n = 16),and rifampicin post-treatment (n = 16).Parkinson’s disease model rats were established via a subcutaneous injection of rotenone (1.5 mg/kg per day) in the three treatment groups,once a day for 3 successive weeks.Rifampicin (30 mg/kg per day) was intragastrically administered in the rifampicin pre-treatment group 3 days prior to rotenone induction and in the rifampicin post-treatment group 7 days after rotenone induction.Rats were treated with a subcutaneous injection of 1 mL/kg per day sunflower oil in the blank control group and an intragastric injection of 30 mg/kg per day rifampicin in the rifampicin group,once a day for 3 successive weeks in total.MAIN OUTCOME MEASURES: Prior to treatment and in the end of the 3rd week after treatment,the rats were evaluated using the modified neurological severity score.The substantia nigra from the rats was extracted for hematoxylin-eosin staining.Western blot analysis was performed to determine tyrosine hydroxylase and α-synuclein expression.RESULTS: Hematoxylin-eosin staining revealed a significant reduction in the number of substantia nigral neurons in the rotenone group,in addition to neurodegradation,hypopigmentation,and pyknosis.In the rifampicin pre-treatment and post-treatment groups,the number of dopaminergic neurons was significantly increased compared with the rotenone group (P < 0.01),with slight neuronal damage.Compared with the rotenone group,substantia nigral tyrosine hydroxylase expres-sion was significantly increased in the rifampicin pre-treatment and post-treatment groups (P < 0.01),but α-synuclein expression and modified neurological severity scores were significantly decreased (P < 0.01).In addition,the effect of rifampicin in the pre-treatment group was superior to the post-treatment group.There was no significant difference in tyrosine hydroxylase and α-synuclein expression,or in the modified neurological severity scores,between the blank control and rifampicin groups (P > 0.05).CONCLUSION: Rifampicin significantly attenuated neuropathological and behavioral motor deficits induced by rotenone.Moreover,rifampicin enhanced tyrosine hydroxylase expression,but inhibited α-synuclein expression.The effect of rifampicin pre-treatment was superior to rifampicin post-treatment.展开更多
Astrocytes protect neurons by modulating neuronal function and survival.Astrocytes support neurons in several ways.They provide energy through the astrocyte-neuron lactate shuttle,protect neurons from excitotoxicity,a...Astrocytes protect neurons by modulating neuronal function and survival.Astrocytes support neurons in several ways.They provide energy through the astrocyte-neuron lactate shuttle,protect neurons from excitotoxicity,and internalize neuronal lipid droplets to degrade fatty acids for neuronal metabolic and synaptic support,as well as by their high capacity for glutamate uptake and the conversion of glutamate to glutamine.A recent reported astrocyte system for protection of dopamine neurons against the neurotoxic products of dopamine,such as aminochrome and other o-quinones,were generated under neuromelanin synthesis by oxidizing dopamine catechol structure.Astrocytes secrete glutathione transferase M2-2 through exosomes that transport this enzyme into dopaminergic neurons to protect these neurons against aminochrome neurotoxicity.The role of this new astrocyte protective mechanism in Parkinson´s disease is discussed.展开更多
The expression of major histocompatibility complex class I(MHC-I),a key antigen-presenting protein,can be induced in dopaminergic neurons in the substantia nigra,thus indicating its possible involvement in the occurre...The expression of major histocompatibility complex class I(MHC-I),a key antigen-presenting protein,can be induced in dopaminergic neurons in the substantia nigra,thus indicating its possible involvement in the occurrence and development of Parkinson’s disease.However,it remains unclear whether oxidative stress induces Parkinson’s disease through the MHC-I pathway.In the present study,polymerase chain reaction and western blot assays were used to determine the expression of MHC-I in 1-methyl-4-phenylpyridinium(MPP+)-treated SH-SY5Y cells and a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced Parkinson’s disease mouse model.The findings revealed that MHC-I was expressed in both models.To detect whether the expression of MHC-I was able to trigger the infiltration of cytotoxic T cells,immunofluorescence staining was used to detect cytotoxic cluster of differentiation 8(CD8)+T cell infiltration in the substantia nigra of MPTP-treated mice.The results indicated that the presentation of MHC-I in dopaminergic neurons was indeed accompanied by an increase in the number of CD8+T cells.Moreover,in MPTP-induced Parkinson’s disease model mice,the genetic knockdown of endogenous MHC-I,which was caused by injecting specific adenovirus into the substantia nigra,led to a significant reduction in CD8+T cell infiltration and alleviated dopaminergic neuronal death.To further investigate the molecular mechanisms of oxidative stress-induced MHC-I presentation,the expression of PTEN-induced kinase 1(PINK1)was silenced in MPP+-treated SH-SY5Y cells using specific small interfering RNA(siRNA),and there was more presentation of MHC-I in these cells compared with control siRNA-treated cells.Taken together,MPP+-/MPTP-induced oxidative stress can trigger MHC-I presentation and autoimmune activation,thus rendering dopaminergic neurons susceptible to immune cells and degeneration.This may be one of the mechanisms of oxidative stress-induced Parkinson’s disease,and implies the potential neuroprotective role of PINK1 in oxidative stress-induced MHC-I presentation.All animal experiments were approved by the Southern Medical University Ethics Committee(No.81802040,approved on February 25,2018).展开更多
The function of dopaminergic neurons in the substantia nigra is of central importance to the coordination of movement by the brain’s basal ganglia circuitry.This is evidenced by the loss of these neurons,resulting in...The function of dopaminergic neurons in the substantia nigra is of central importance to the coordination of movement by the brain’s basal ganglia circuitry.This is evidenced by the loss of these neurons,resulting in the cardinal motor deficits associated with Parkinson’s disease.In order to fully understand the physiology of these key neurons and develop potential therapies for their loss,it is essential to determine if and how dopaminergic neurons are replenished in the adult brain.Recent work has presented evidence for adult neurogenesis of these neurons by Nestin^+/Sox2~–neural progenitor cells.We sought to further validate this finding and explore a potential atypical origin for these progenitor cells.Since neural progenitor cells have a proximal association with the vasculature of the brain and subsets of endothelial cells are Nestin^+,we hypothesized that dopaminergic neural progenitors might share a common cell lineage.Therefore,we employed a VE-cadherin promoter-driven CRE^(ERT2):TH^(lox)/TH^(lox) transgenic mouse line to ablate the tyrosine hydroxylase gene from endothelial cells in adult animals.After 26 weeks,but not 13 weeks,following the genetic blockade of tyrosine hydroxylase expression in VE-cadherin^+cells,we observed a significant reduction in tyrosine hydroxylase^+neurons in the substantia nigra.The results from this genetic lineage tracing study suggest that dopaminergic neurons are replenished in adult mice by a VE-cadherin^+progenitor cell population potentially arising from an endothelial lineage.展开更多
BACKGROUND: Induced differentiation strategies and cytochemical properties of human embryonic stem cells (hESCs) have been investigated. However, the electrophysiological functions of tyrosine hydroxylase (TH)-positiv...BACKGROUND: Induced differentiation strategies and cytochemical properties of human embryonic stem cells (hESCs) have been investigated. However, the electrophysiological functions of tyrosine hydroxylase (TH)-positive cells derived from hESCs remain unclear. OBJECTIVE: To investigate the differentiation efficiency of TH-positive cells from hESCs in vitro using modified four-step culture methods, including embryoid body formation, and to examine the functional characteristics of the differentiated TH-positive cells using electrophysiological techniques. DESIGN, TIME AND SETTING: Neuroelectrophysiology was performed at the Reproductive Medicine Center and Stem Cell Research Center, Peking University Third Hospital, and the Neuroscience Research Institute and Department of Neurobiology, Peking University, from September 2004 to August 2008. MATERIALS: The hESC line, PKU-1.1, a monoclonal cell line derived from a pre-implantation human blastocyst in the Reproductive Medical Center of Peking University Third Hospital. The patch clamp recording system was provided by the Neuroscience Research Institute and Department of Neurobiology, Peking University. METHODS: The hESC line was induced to differentiate into TH-positive cells in vitro using a modified four-step culture method, including the formation of embryoid body, as well as the presence of sonic hedgehog and fibroblast growth factor 8. The cell karyotype was assessed by G-banding karyotype analysis techniques and specific markers were detected immunocytochemically. Whole-cell configuration was obtained after obtaining a tight seal of over 1 GΩ. Ionic currents were detected by holding the cells at -70 mV and stepping to test voltages between -80 and 40 mV in 10-mV increments in voltage-clamp configuration. MAIN OUTCOME MEASURES: We measured the cell karyotype, specific cell markers, and the electrophysiological properties of the voltage-gated ion channels on the cell membrane of TH-positive dopaminergic cells differentiated from our hESCs line in vitro. RESULTS: The differentiated cells had a consistent appearance, and the majority of cells (> 90%) expressed TH and β-tubulion, as well as the neural progenitor marker, nestin. Cell karyotype analysis demonstrated that all of the hESCs had a stable and normal karyotype (46, XX) after dif-ferentiation. In addition, patch clamp recording showed that the 10 recorded TH-positive cells exhibited a fast inward current when the test voltage depolarized to -30 mV, and a delayed outward current when the test voltage depolarized to -10 mV. The peak of inward current was obtained at voltage between -10 mV and 0 mV, while the peak of outward current was obtained at 40 mV. The average peak of inward current density was (-50.05 ± 15.50) pA/pF, and the average peak of outward current density was (41.98 ± 13.55) pA/pF. CONCLUSION: More than 90% of the differentiated hESC-derived cells induced by the modified four-step culture method exhibit dopaminergic neuronal properties, including general electrophysiological functional properties, such as functional potassium and sodium channels.展开更多
OBJECTIVE To research the effect of naringenin(NAR) on LPS-induced dopaminergic neurons damage and its potential mechanism.METHODS Rats were randomly divided into the following six groups(n=10):control(0.9% NaCl),NAR ...OBJECTIVE To research the effect of naringenin(NAR) on LPS-induced dopaminergic neurons damage and its potential mechanism.METHODS Rats were randomly divided into the following six groups(n=10):control(0.9% NaCl),NAR alone(100 mg·kg-1),LPS(5 μg),LPS+NAR(50 mg·kg-1) and LPS+NAR(100 mg·kg-1).Rats were received a single LPS unilateral injection into the SN pars compacts,after seven daily intragastric administration of NAR,rats′ behavior was analyzed by rotarod test.Then,the expression of TH,IBA-1 and NLRP3 inflammasome were analyzed by Western blotting and immunofluorescence.In vitro experiments,BV-2 cel s were treated with different doses of NAR,and 1 h later,LPS(1 g·L^(-1)) was added to the medium for 24 h,then collect the culture medium and protein for later experiments.The production of IL-1β and IL-18 in culture medium were tested by ELISA,and the production of NO was detected by Griess reagent.The expression of IBA-1,NLRP3 and p-caspase 1 were detected by Western blotting.MN9 D cells were co-cultured with BV2 cells to mimic the animal experiments.MTT assay was used to analyzed the viability of MN9 D cells,and the expression of TH was detected by Western blotting.RESULTS NAR(100 mg · kg-1) could significantly improve the time of rats on the rotating(116.73 s vs 185.45 s,P<0.05).The result of the pathological analysis also suggested that NAR could decrease the activation of microglia as well as the expression of NLRP3 Inflammasome.In addition,NAR also could suppress the expression of pro-inflammatory factor levels,such as IL-1β(P<0.05),IL-18(P<0.05),and the protection of NAR could be inhibited by siR NA NLRP3.Moreover,an in vitro co-culture system with BV2 and MN9 D cells wasused to find the protection of NAR must via microglia,while there is no effect of NAR were directly added to MN9 D cells.CONCLUSION NAR protection of LPS-induced dopaminergic neurons damage might be through mediating NLRP3 inflammasome.展开更多
Introduction:Parkinson’s disease(PD)is a chronic,age-related neurodegenerative disorder that affects 1–2%of the population over the age of 65.PD is characterised by the progressive degeneration of nigrostriatal dopa...Introduction:Parkinson’s disease(PD)is a chronic,age-related neurodegenerative disorder that affects 1–2%of the population over the age of 65.PD is characterised by the progressive degeneration of nigrostriatal dopaminergic(DA)neurons.This leads to disabling motor symptoms,due to the striatal DA denervation.Despite decades of research,展开更多
Background:Generation of neurons is essential in cell replacement therapy for neurodegenerative disorders like Parkinson’s disease.Several studies have reported the generation of dopaminergic(DA)neurons from mouse an...Background:Generation of neurons is essential in cell replacement therapy for neurodegenerative disorders like Parkinson’s disease.Several studies have reported the generation of dopaminergic(DA)neurons from mouse and human fibroblasts by ectopic expression of transcription factors,in which genetic manipulation is associated with potential risks.Methods:The small molecules and protein factors were selected based on their function to directly induce human fetal lung IMR-90 fibroblasts into DA neuron-like cells.Microscopical,immunocytochemical,and RT-qPCR analyses were used to characterize the morphology,phenotype,and gene expression features of the induced cells.The wholecell patch-clamp recordings were exploited to measure the electrophysiological properties.Results:Human IMR-90 fibroblasts were rapidly converted into DA neuron-like cells after the chemical induction using small molecules and protein factors,with a yield of approximately 95%positive TUJ1-positive cells.The induced DA neuron-like cells were immunopositive for pan-neuronal markers MAP2,NEUN,and Synapsin 1 and DA markers TH,DDC,DAT,and NURR1.The chemical induction process did not involve a neural progenitor/stem cell intermediate stage.The induced neurons could fire single action potentials,which reflected partially the electrophysiological properties of neurons.Conclusions:We developed a chemical cocktail of small molecules and protein factors to convert human fibroblasts into DA neuron-like cells without passing through a neural progenitor/stem cell intermediate stage.The induced DA neuron-like cells from human fibroblasts might provide a cellular source for cell-based therapy of Parkinson’s disease in the future.展开更多
As a late endosomal/lysosomal transport protein of the P5-type, ATP13A2 is capable of removing the abnormal accumulation of α-synuclein, which maintains the homeostasis of metal ions and polyamines in the central ner...As a late endosomal/lysosomal transport protein of the P5-type, ATP13A2 is capable of removing the abnormal accumulation of α-synuclein, which maintains the homeostasis of metal ions and polyamines in the central nervous system. Furthermore, ATP13A2 regulates the normal function of several organelles such as lysosomes, endoplasmic reticulum (ER) and mitochondria, and maintains the normal physiological activity of neural cells. Especially, ATP13A2 protects dopaminergic (DA) neurons against environmental or genetically induced Parkinson's disease (PD). As we all know, PD is a neurodegenerative disease characterized by the loss of DA neurons in the substantia nigra pars compacta. An increasing number of studies have reported that the loss-of- function of ATP13A2 affects normal physiological processes of various organelles, leading to abnormalities and the death of DA neurons. Previous studies in our laboratory have also shown that ATP13A2 deletion intensifies the neuroinflammatory response induced by astrocytes, thus inducing DA neuronal injury. In addition to elucidating the normal structure and function of ATP13A2, this review summarized the pathological mechanisms of ATP13A2 mutations leading to PD in existing literature studies, deepening the understanding of ATP13A2 in the pathological process of PD and other related neurodegenerative diseases. This review provides inspiration for investigators to explore the essential regulatory role of ATP13A2 in PD in the future.展开更多
AIM:To find a safe source for dopaminergic neurons,we generated neural progenitor cell lines from human embryonic stem cells.METHODS:The human embryonic stem(hES)cell line H9 was used to generate human neural progenit...AIM:To find a safe source for dopaminergic neurons,we generated neural progenitor cell lines from human embryonic stem cells.METHODS:The human embryonic stem(hES)cell line H9 was used to generate human neural progenitor(HNP)cell lines.The resulting HNP cell lines were differentiated into dopaminergic neurons and analyzed by quantitative real-time polymerase chain reaction and immunofluorescence for the expression of neuronal differentiation markers,including beta-III tubulin(TUJ1)and tyrosine hydroxylase(TH).To assess the risk of teratoma or other tumor formation,HNP cell lines and mouse neuronal progenitor(MNP)cell lines were injected subcutaneously into immunodeficient SCID/beige mice.RESULTS:We developed a fairly simple and fast protocol to obtain HNP cell lines from hES cells.These cell lines,which can be stored in liquid nitrogen for several years,have the potential to differentiate in vitro into dopaminergic neurons.Following day 30 of differentiation culture,the majority of the cells analyzed expressed the neuronal marker TUJ1 and a high proportion of these cells were positive for TH,indicating differentiation into dopaminergic neurons.In contrast to H9 ES cells,the HNP cell lines did not form tumors in immunodeficient SCID/beige mice within 6 mo after subcutaneous injection.Similarly,no tumors developed after injection of MNP cells.Notably,mouse ES cells or neuronal cells directly differentiated from mouse ES cells formed teratomas in more than 90%of the recipients.CONCLUSION:Our findings indicate that neural progenitor cell lines can differentiate into dopaminergic neurons and bear no risk of generating teratomas or other tumors in immunodeficient mice.展开更多
OBJECTIVE Dysfunction of the dopaminergic(DA)neurons is implicated in the pathogenesis of bipolar disorder(BPD).Hista⁃mine receptor 2(Hrh2)is highly expressed in DA neurons,and its antagonists have been reported to in...OBJECTIVE Dysfunction of the dopaminergic(DA)neurons is implicated in the pathogenesis of bipolar disorder(BPD).Hista⁃mine receptor 2(Hrh2)is highly expressed in DA neurons,and its antagonists have been reported to induce mania phase of BPD.However,whether Hrh2 on DA neurons contributes to BPD patho⁃genesis is unclear.The present study aims to explore the role of hrh2 on DA neurons in the pathology of BPD.METHODS AAV-FLEX-shHrh2 was injected into a targeted brain area of DAT-Cre mice,leading to a selective brain-regional loss of Hrh2 on DA neurons.A series of behavior tests were used to measure the sponta⁃neous activity,anxiety and depression level of Hrh2-deficient mice.RESULTS①In the open field test and home-cage activity test,Hrh2-defi⁃cient mice displayed increased spontaneous activity.②Hrh2-deficient mice showed reduced depression level in the tail suspension test,forced swimming test and sucrose preference test.③The anxiety level of Hrh2-deficient mice was decreased in the open field test.CONCLU⁃SION Hrh2 on DA neurons is closely related with mania-like behavior.展开更多
Dopaminergic neurotoxicity is characterized by damage and death of dopaminergic neurons.Parkinson's disease(PD)is a neurodegenerative disorder that primarily involves the loss of dopaminergic neurons in the substa...Dopaminergic neurotoxicity is characterized by damage and death of dopaminergic neurons.Parkinson's disease(PD)is a neurodegenerative disorder that primarily involves the loss of dopaminergic neurons in the substantia nigra.Therefore,the study of the mechanisms,as well as the search for new targets for the prevention and treatment of neurodegenerative diseases,is an important focus of modern neuroscience.PD is primarily caused by dysfunction of dopaminergic neurons;however,other neurotransmitter systems are also involved.Research reports have indicated that the glutamatergic system is involved in different pathological conditions,including dopaminergic neurotoxicity.Over the last two decades,the important functional interplay between dopaminergic and glutamatergic systems has stimulated interest in the possible role of metabotropic glutamate receptors(mGluRs)in the development of extrapyramidal disorders.However,the specific mechanisms driving these processes are presently unclear.The participation of the universal neuronal messenger nitric oxide(NO)in the mechanisms of dopaminergic neurotoxicity has attracted increased attention.The current paper aims to review the involvement of mGluRs and the contribution of NO to dopaminergic neurotoxicity.More precisely,we focused on studies conducted on the rotenone-induced PD model.This review is also an outline of our own results obtained using the method of electron paramagnetic resonance,which allows quantitation of NO radicals in brain structures.展开更多
Thirst, which provides the motivation to drink, is an important component of the coordinated sequence of physiological responses that maintain the volume and composition of body fluids. Special structures in the centr...Thirst, which provides the motivation to drink, is an important component of the coordinated sequence of physiological responses that maintain the volume and composition of body fluids. Special structures in the central nervous system like periventricular organs detect changes in these parameters continuously. The present study investigated the interaction between dopaminergic and angiotensinergic systems on water intake in adult male rats. Intracerebroventricular (ICV) injections were carried out in all experiments after 24 h deprivation of water intake. After the deprivation interval, the volume of consumed water was measured for 1h. Administration the angiotensinergic (AT1) receptor antagonist Losartan (45 μg/rat), and the dopaminergic antagonist Chlorpromazine (40 μg/rat) significantly decreased water intake when compared to saline-treated controls. In contrast, ICV microinjection of the dopaminergic agonist Bromocriptine (10 μg/rat) significantly increased water intake when compared to saline-treated controls. ICV injection of Bromocriptine 15min after Losartan administration was able to attenuate the inhibitory effect of Losartan on water intake, whereas administration of Chlorpromazine 15 min after Losartan was unable to change the Losartan effect. These results suggest that the dopaminergic system interactions with the angiotensinergic system to regulate water intake through circumventricular organs. Dopaminergic and angiotensinergic neurons can monitor and regulate water intake via the stimulatory and inhibitory effects on each other, respectively.展开更多
A spontaneous mutation in the Albino Swiss (AS) rat has been shown to be a single point mutation (agu) in the gene coding for the gamma isoform of protein kinase C (PKC-γ). The characteristics of the mutant include m...A spontaneous mutation in the Albino Swiss (AS) rat has been shown to be a single point mutation (agu) in the gene coding for the gamma isoform of protein kinase C (PKC-γ). The characteristics of the mutant include movement disorders, a failure to release dopamine in the striatum and elevations of molecules such as parkin and ubiquitin in the substantia nigra pars compacta (SNC). This present study examined SNC cell bodies and dopaminergic synaptic terminals within the caudate-putamen. Cell volume and nuclear volume were reduced in the AS/AGU mutant compared to the AS control, but the volume fractions of mitochondria and rough endoplasmic reticulum were significantly higher. No Lewy bodies were present in the mutant, although microglia were found adjacent to some SNC cells. Dopaminergic terminals were identified in the caudate-putamen by electron microscopy with low-glutaraldehyde fixation and immunohistochemistry for tyrosine hydroxylase using immuno-gold visualisation. AS/AGU mutant rats had less than half of the synaptic vesicles of AS controls;this was not only true of “readily-releasable” zones adjacent to the synaptic cleft but also “storage pool” zones. The findings support the hypothesis that the initial bar to dopamine availability in the striatum is the reduced release, with nigral cell death being a later phenomenon.展开更多
Objective: Levodopa is the gold-standard of therapy in Parkinson’s disease (PD), but it is associated with motor complications that affect 50% of patients after five years of treatment. Development of delirium and ps...Objective: Levodopa is the gold-standard of therapy in Parkinson’s disease (PD), but it is associated with motor complications that affect 50% of patients after five years of treatment. Development of delirium and psychosis is the main limitation of dopaminergic treatment in older persons. These adverse effects may result from pulsatile stimulation of the dopamine receptors. Dopamine agonists with transdermal delivery that continuously stimulate the dopamine receptors may reduce these complications. The objective of this study was to evaluate the frequencies of acute delirium and psychosis in elderly patients treated with rotigotine vs. levodopa in a newly diagnosed drugnaive Parkinson’s disease (PD). Methods: Patients admitted to the Geriatric-Rehabilitation Department of the University-Hospital of Parma were screened for the presence of Parkinsonism. All subjects admitted with diagnosis of PD according to the UK Brain Bank Criteria were randomly treated with Rotigotine or levodopa. All subjects were assessed by Movement Disorder Society (MDS)-Unified Parkinson's Disease Rating Scale (MDS-UPDRS) Part III. Acute delirium was asessed by CAM Diagnostic Algorithm during the first week after admission. After six months, diagnosis of psychosis was performed according to pro posed diagnostic criteria by NINDS and NIMH. Patients with cognitive impairment (MMSE dementia with Lewy bodies (DLB), were excluded. Results: 60 consecutive newly diagnosed drugnaive PD patients were evaluated. No statistical significant difference between the two groups were observed in term of age, gender, MMSE score, severity of disease expressed by H&Y staging. 30 patients were treated with rotigotine (6 mg/daily) and 30 patients were treated with L-Dopa (250 mg/daily). All participants completed the study. UPDRS Part III was statistical significant lower in both groups after treatment from 26.4 to 18.3 (rotigotine group) and from 26.3 to 17.3 (levodopa group), but comparable within groups (p = 0.83). After 6-month follow-up, acute delirium and/ or psychosis were observed in two cases (6.6%) of patients treated with rotigotine and in three cases (10%) of those treated with levodopa (p = 0.54). Conclusions: Transdermal rotigotine seems comparable to levodopa in regard to motor skill efficacy and neuropsychiatric safety, because provides a more continuous delivery of drug. Dopamine agonists may represent a valid therapeutic option in newly diagnosed older PD patients.展开更多
Dopaminergic therapy can improve motor functioning in Parkinson’s disease (PD), but its effect on spoken discourse is poorly understood. This study addressed whether discourse produced during a one-minute monologue w...Dopaminergic therapy can improve motor functioning in Parkinson’s disease (PD), but its effect on spoken discourse is poorly understood. This study addressed whether discourse produced during a one-minute monologue would be affected by medication withdrawal. We performed a detailed analysis of semistructured speech samples in 38 patients with PD, including measures of efficiency, syntactic complexity, cohesion (i.e., lexical and grammatical ties between sentences), and coherence (i.e., semantic relationship between the utterances and the topic, or between utterances). We found that patients produced more efficient and coherent monlogues when on versus off medication. As discourse relies heavily on cognitive processes such as working memory, these findings are consistent with research showing improved working memory and executive processes with dopaminegic medication.展开更多
基金supported by the National Natural Science Foundation of China,No.81771222(to LS)Guangzhou Key Research Program on Brain Science,Nos.202007030011,202206060001(to LS)the Program of Introducing Talents of Discipline to Universities of China,No.B14036(to KFS)。
文摘Midbrain dopaminergic neurons play an important role in the etiology of neurodevelopmental and neurodegenerative diseases.They also represent a potential source of transplanted cells for therapeutic applications.In vitro differentiation of functional midbrain dopaminergic neurons provides an accessible platform to study midbrain neuronal dysfunction and can be used to examine obstacles to dopaminergic neuronal development.Emerging evidence and impressive advances in human induced pluripotent stem cells,with tuned neural induction and differentiation protocols,makes the production of induced pluripotent stem cell-derived dopaminergic neurons feasible.Using SB431542 and dorsomorphin dual inhibitor in an induced pluripotent stem cell-derived neural induction protocol,we obtained multiple subtypes of neurons,including 20%tyrosine hydroxylase-positive dopaminergic neurons.To obtain more dopaminergic neurons,we next added sonic hedgehog(SHH)and fibroblast growth factor 8(FGF8)on day 8 of induction.This increased the proportion of dopaminergic neurons,up to 75%tyrosine hydroxylase-positive neurons,with 15%tyrosine hydroxylase and forkhead box protein A2(FOXA2)co-expressing neurons.We further optimized the induction protocol by applying the small molecule inhibitor,CHIR99021(CHIR).This helped facilitate the generation of midbrain dopaminergic neurons,and we obtained 31-74%midbrain dopaminergic neurons based on tyrosine hydroxylase and FOXA2 staining.Thus,we have established three induction protocols for dopaminergic neurons.Based on tyrosine hydroxylase and FOXA2 immunostaining analysis,the CHIR,SHH,and FGF8 combined protocol produces a much higher proportion of midbrain dopaminergic neurons,which could be an ideal resource for tackling midbrain-related diseases.
基金supported by the National Key Research and Development Program of China,Nos.2017YFE0122900(to BH),2019YFA0110800(to WL),2019YFA0903802(to YW),2021YFA1101604(to LW),2018YFA0108502(to LF),and 2020YFA0804003(to JW)the National Natural Science Foundation of China,Nos.31621004(to WL,BH)and 31970821(to YW)+1 种基金CAS Project for Young Scientists in Basic Research,No.YSBR-041(to YW)Joint Funds of the National Natural Science Foundation of China,No.U21A20396(to BH)。
文摘Numerous studies have shown that cell replacement therapy can replenish lost cells and rebuild neural circuitry in animal models of Parkinson’s disease.Transplantation of midbrain dopaminergic progenitor cells is a promising treatment for Parkinson’s disease.However,transplanted cells can be injured by mechanical damage during handling and by changes in the transplantation niche.Here,we developed a one-step biomanufacturing platform that uses small-aperture gelatin microcarriers to produce beads carrying midbrain dopaminergic progenitor cells.These beads allow midbrain dopaminergic progenitor cell differentiation and cryopreservation without digestion,effectively maintaining axonal integrity in vitro.Importantly,midbrain dopaminergic progenitor cell bead grafts showed increased survival and only mild immunoreactivity in vivo compared with suspended midbrain dopaminergic progenitor cell grafts.Overall,our findings show that these midbrain dopaminergic progenitor cell beads enhance the effectiveness of neuronal cell transplantation.
基金supported by the Medical and Health Technology Development Plan of Shandong Province of China,No.2011HD009(to AHW)the Chinese Medicine Science and Technology Development Plan Project of Shandong Province of China,No.2017-163(to AHW)+1 种基金the Natural Science Foundation of Shandong Province of China,No.ZR2016HP23(to AHW)the Science and Technology Development Plan Project of Taian City of China,No.2017NS0151(to XCS)
文摘Accumulating studies suggest that neuroinflammation characterized by microglial overactivation plays a pivotal role in the pathogenesis of Parkinson’s disease.As such,inhibition of microglial overactivation might be a promising treatment strategy to delay the onset or slow the progression of Parkinson’s disease.Ginsenoside Rbl,the most active ingredient of ginseng,reportedly exerts neuroprotective effects by suppressing inflammation in vitro.The present study aimed to evaluate the neuroprotective and anti-inflammatory effects of ginsenoside Rbl in a lipopolysaccharide-induced rat Parkinson’s disease model.Rats were divided into four groups.In the control group,sham-operated rats were intraperitoneally administered normal saline for 14 consecutive days.In the ginsenoside Rbl group,ginsenoside Rb1(20 mg/kg)was intraperitoneally injected for 14 consecutive days after sham surgery.In the lipopolysaccharide group,a single dose of lipopolysaccharide was unilaterally microinjected into the rat substantial nigra to establish the Parkinson’s disease model.Lipopolysaccharide-injected rats were treated with normal saline for 14 consecutive days.In the ginsenoside Rbl +lipopolysaccharide group,lipopolysaccharide was unilaterally microinjected into the rat substantial nigra.Subsequently,ginsenoside Rbl was intraperitoneally injected for 14 consecutive days.To investigate the therapeutic effects of ginsenoside Rbl,behavioral tests were performed on day 15 after lipopolysaccharide injection.We found that ginsenoside Rbl treatment remarkably reduced apomorphine-induced rotations in lipopolysaccharide-treated rats compared with the lipopolysaccharide group.To investigate the neurotoxicity of lipopolysaccharide and potential protective effect of ginsenoside Rbl,contents of dopamine and its metabolites in the striatum were measured by high-performance liquid chromatography.Compared with the lipopolysaccharide group,ginsenoside Rbl obviously attenuated the lipopolysaccharide-induced depletion of dopamine and its metabolites in the striatum.To further explore the neuroprotective effect of ginsenoside Rbl against lipopolysaccharide-induced neurotoxicity,immunohistochemistry and western blot assay of tyrosine hydroxylase were performed to evaluate dopaminergic neuron degeneration in the substantial nigra par compacta.The results showed that lipopolysaccharide injection caused a large loss of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra and a significant decrease in overall tyrosine hydroxylase expression.However,ginsenoside Rb1 noticeably reversed these changes.To investigate whether the neuroprotective effect of ginsenoside Rbl was associated with inhibition of lipopolysaccharide-induced microglial activation,we examined expression of the microglia marker Iba-1.Our results confirmed that lipopolysaccharide injection induced a significant increase in Iba-1 expression in the substantia nigra;however,ginsenoside Rbl effectively suppressed lipopolysaccharide-induced microglial overactivation.To elucidate the inhibitory mechanism of ginsenoside Rb1,we examined expression levels of inflammatory mediators(tumor necrosis factor-a,interleukin-1β,inducible nitric oxide synthase,and cyclooxygenase 2)and phosphorylation of nuclear factor kappa B signaling-related proteins(IκB,IKK)in the substantia nigra with enzyme-linked immunosorbent and western blot assays.Our results revealed that compared with the control group,phosphorylation and expression of inflammatory mediators IκB and IKK in the substantia nigra of lipopolysaccharide group rats were significantly increased;whereas,ginsenoside Rbl obviously reduced lipopolysaccharide-induced changes on the lesioned side of the substantial nigra par compacta.These findings confirm that ginsenoside Rbl can inhibit inflammation induced by lipopolysaccharide injection into the substantia nigra and protect dopaminergic neurons,which may be related to its inhibition of the nuclear factor kappa B signaling pathway.This study was approved by the Experimental Animal Ethics Committee of Shandong University of China in April 2016(approval No.KYLL-2016-0148).
基金supported by the National Natural Sciences Foundation of China,No.81770460(to YCL)the Postdoctoral Research Fellowship of the Saskatchewan Health Research Foundation,No.SHRF,4144(to YCL)+2 种基金the third level of the Chuanshan Talent project of the University of South China,No.2017CST20(to YCL)the Aid Program,No.2017KJ268 and the Key Lab for Clinical Anatomy&Reproductive Medicine,No.2017KJ182 from the Science and Technology Bureau of Hengyang City,China(to YCL and XC)the Postgraduate Student Research Innovation Projects of Hunan Province,China,No.CX2018B62(to ABG)
文摘Prolonged activation of adenosine A1 receptor likely leads to damage of dopaminergic neurons and subsequent development of neurodegenerative diseases.However,the pathogenesis underlying long-term adenosine A1 receptor activation-induced neurodegeneration remains unclear.In this study,rats were intraperitoneally injected with 5 mg/kg of the adenosine A1 receptor agonist N6-cyclopentyladenosine(CPA)for five weeks.The mobility of rats was evaluated by forced swimming test,while their cognitive capabilities were evaluated by Y-maze test.Expression of sortilin,α-synuclein,p-JUN,and c-JUN proteins in the substantia nigra were detected by western blot analysis.In addition,immunofluorescence staining of sortilin andα-synuclein was performed to detect expression in the substantia nigra.The results showed that,compared with adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine(5 mg/kg)+CPA co-treated rats,motor and memory abilities were reduced,surface expression of sortin andα-synuclein in dopaminergic neurons was reduced,and total sortilin and totalα-synuclein were increased in CPA-treated rats.MN9D cells were incubated with 500 nM CPA alone or in combination with 10μM SP600125(JNK inhibitor)for 48 hours.Quantitative real-time polymerase chain reaction analysis of sortilin andα-synuclein mRNA levels in MN9D cells revealed upregulated sortilin expression in MN9D cells cultured with CPA alone,but the combination of CPA and SP600125 could inhibit this expression.Predictions made using Jasper,PROMO,and Alibaba online databases identified a highly conserved sequence in the sortilin promoter that was predicted to bind JUN in both humans and rodents.A luciferase reporter assay of sortilin promoter plasmid-transfected HEK293T cells confirmed this prediction.After sortilin expression was inhibited by sh-SORT1,expression of p-JUN and c-JUN was detected by western blot analysis.Long-term adenosine A1 receptor activation levels upregulatedα-synuclein expression at the post-transcriptional level by affecting sortilin expression.The online tool Raptor-X-Binding and Discovery Studio 4.5 prediction software predicted that sortilin can bind toα-synuclein.Co-immunoprecipitation revealed an interaction between sortilin andα-synuclein in MN9D cells.Our findings indicate that suppression of prolonged adenosine A1 receptor activation potently inhibited sortilin expression andα-synuclein accumulation,and dramatically improved host cognition and kineticism.This study was approved by the University Committee of Animal Care and Supply at the University of Saskatchewan(approval No.AUP#20070090)in March 2007 and the Animals Ethics Committee of University of South China(approval No.LL0387-USC)in June 2017.
基金the Natural Science Foundation of Guangdong Province,No.04009355Science and Technology Planning Project of Guandong Province,China,05B33801003
文摘BACKGROUND: Rifampicin inhibits the formation of α-synuclein multimer and protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyritine (MPTP)-induced PC12 cell apoptosis.OBJECTIVE: To compare the effect of rifampicin pre-and post-treatment on tyrosine hydroxylase and α-synuclein expression in substantia nigra pars compacta in a rat model of Parkinson’s disease.DESIGN,TIME AND SETTING: A randomized,controlled experiment was performed at the Experimental Animal Center of Sun Yat-sen University North Campus (China) from November 2006 to October 2008.MATERIALS: Rifampicin was purchased from MD,USA;rotenone was purchased from Sigma,USA;mouse anti-rat α-synuclein monoclonal antibody was purchased from B&D,USA;and rabbit anti-rat tyrosine hydroxylase monoclonal antibody was purchased from Chemicon,USA.METHODS: A total of 72 male,Sprague Dawley rats,aged 8 weeks,were randomly assigned to 5 groups: blank control (n = 12),rifampicin (n = 12),rotenone (n = 16),rifampicin pre-treatment (n = 16),and rifampicin post-treatment (n = 16).Parkinson’s disease model rats were established via a subcutaneous injection of rotenone (1.5 mg/kg per day) in the three treatment groups,once a day for 3 successive weeks.Rifampicin (30 mg/kg per day) was intragastrically administered in the rifampicin pre-treatment group 3 days prior to rotenone induction and in the rifampicin post-treatment group 7 days after rotenone induction.Rats were treated with a subcutaneous injection of 1 mL/kg per day sunflower oil in the blank control group and an intragastric injection of 30 mg/kg per day rifampicin in the rifampicin group,once a day for 3 successive weeks in total.MAIN OUTCOME MEASURES: Prior to treatment and in the end of the 3rd week after treatment,the rats were evaluated using the modified neurological severity score.The substantia nigra from the rats was extracted for hematoxylin-eosin staining.Western blot analysis was performed to determine tyrosine hydroxylase and α-synuclein expression.RESULTS: Hematoxylin-eosin staining revealed a significant reduction in the number of substantia nigral neurons in the rotenone group,in addition to neurodegradation,hypopigmentation,and pyknosis.In the rifampicin pre-treatment and post-treatment groups,the number of dopaminergic neurons was significantly increased compared with the rotenone group (P < 0.01),with slight neuronal damage.Compared with the rotenone group,substantia nigral tyrosine hydroxylase expres-sion was significantly increased in the rifampicin pre-treatment and post-treatment groups (P < 0.01),but α-synuclein expression and modified neurological severity scores were significantly decreased (P < 0.01).In addition,the effect of rifampicin in the pre-treatment group was superior to the post-treatment group.There was no significant difference in tyrosine hydroxylase and α-synuclein expression,or in the modified neurological severity scores,between the blank control and rifampicin groups (P > 0.05).CONCLUSION: Rifampicin significantly attenuated neuropathological and behavioral motor deficits induced by rotenone.Moreover,rifampicin enhanced tyrosine hydroxylase expression,but inhibited α-synuclein expression.The effect of rifampicin pre-treatment was superior to rifampicin post-treatment.
基金supported by ANID-FONDECYT 1170033(to JSA)ANID-STINT-CONICYT CS2018-7940(to JSA,IN,JI,MV)Swedish Research Council grant 2015-04222 to BM.
文摘Astrocytes protect neurons by modulating neuronal function and survival.Astrocytes support neurons in several ways.They provide energy through the astrocyte-neuron lactate shuttle,protect neurons from excitotoxicity,and internalize neuronal lipid droplets to degrade fatty acids for neuronal metabolic and synaptic support,as well as by their high capacity for glutamate uptake and the conversion of glutamate to glutamine.A recent reported astrocyte system for protection of dopamine neurons against the neurotoxic products of dopamine,such as aminochrome and other o-quinones,were generated under neuromelanin synthesis by oxidizing dopamine catechol structure.Astrocytes secrete glutathione transferase M2-2 through exosomes that transport this enzyme into dopaminergic neurons to protect these neurons against aminochrome neurotoxicity.The role of this new astrocyte protective mechanism in Parkinson´s disease is discussed.
基金This work was supported by the National Natural Science Foundation of China,Nos.81671240(to SZZ),81560220(to GHL)the Youth Science Foundation of Jiangxi Province of China,No.20151BAB215014(to GHL)Health and Family Planning Commission of Jiangxi Province of China,No.20195109(to GHL)。
文摘The expression of major histocompatibility complex class I(MHC-I),a key antigen-presenting protein,can be induced in dopaminergic neurons in the substantia nigra,thus indicating its possible involvement in the occurrence and development of Parkinson’s disease.However,it remains unclear whether oxidative stress induces Parkinson’s disease through the MHC-I pathway.In the present study,polymerase chain reaction and western blot assays were used to determine the expression of MHC-I in 1-methyl-4-phenylpyridinium(MPP+)-treated SH-SY5Y cells and a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced Parkinson’s disease mouse model.The findings revealed that MHC-I was expressed in both models.To detect whether the expression of MHC-I was able to trigger the infiltration of cytotoxic T cells,immunofluorescence staining was used to detect cytotoxic cluster of differentiation 8(CD8)+T cell infiltration in the substantia nigra of MPTP-treated mice.The results indicated that the presentation of MHC-I in dopaminergic neurons was indeed accompanied by an increase in the number of CD8+T cells.Moreover,in MPTP-induced Parkinson’s disease model mice,the genetic knockdown of endogenous MHC-I,which was caused by injecting specific adenovirus into the substantia nigra,led to a significant reduction in CD8+T cell infiltration and alleviated dopaminergic neuronal death.To further investigate the molecular mechanisms of oxidative stress-induced MHC-I presentation,the expression of PTEN-induced kinase 1(PINK1)was silenced in MPP+-treated SH-SY5Y cells using specific small interfering RNA(siRNA),and there was more presentation of MHC-I in these cells compared with control siRNA-treated cells.Taken together,MPP+-/MPTP-induced oxidative stress can trigger MHC-I presentation and autoimmune activation,thus rendering dopaminergic neurons susceptible to immune cells and degeneration.This may be one of the mechanisms of oxidative stress-induced Parkinson’s disease,and implies the potential neuroprotective role of PINK1 in oxidative stress-induced MHC-I presentation.All animal experiments were approved by the Southern Medical University Ethics Committee(No.81802040,approved on February 25,2018).
基金provided by the Institutional Development Awards(IDe A)from the National Institute of General Medical Sciences of the National Institutes of Health under Grants No.P20GM103408 and P20GM109095provided by The Biomolecular Research Center at Boise State University
文摘The function of dopaminergic neurons in the substantia nigra is of central importance to the coordination of movement by the brain’s basal ganglia circuitry.This is evidenced by the loss of these neurons,resulting in the cardinal motor deficits associated with Parkinson’s disease.In order to fully understand the physiology of these key neurons and develop potential therapies for their loss,it is essential to determine if and how dopaminergic neurons are replenished in the adult brain.Recent work has presented evidence for adult neurogenesis of these neurons by Nestin^+/Sox2~–neural progenitor cells.We sought to further validate this finding and explore a potential atypical origin for these progenitor cells.Since neural progenitor cells have a proximal association with the vasculature of the brain and subsets of endothelial cells are Nestin^+,we hypothesized that dopaminergic neural progenitors might share a common cell lineage.Therefore,we employed a VE-cadherin promoter-driven CRE^(ERT2):TH^(lox)/TH^(lox) transgenic mouse line to ablate the tyrosine hydroxylase gene from endothelial cells in adult animals.After 26 weeks,but not 13 weeks,following the genetic blockade of tyrosine hydroxylase expression in VE-cadherin^+cells,we observed a significant reduction in tyrosine hydroxylase^+neurons in the substantia nigra.The results from this genetic lineage tracing study suggest that dopaminergic neurons are replenished in adult mice by a VE-cadherin^+progenitor cell population potentially arising from an endothelial lineage.
基金the National Natural Science Foundation of China, No. 30672239
文摘BACKGROUND: Induced differentiation strategies and cytochemical properties of human embryonic stem cells (hESCs) have been investigated. However, the electrophysiological functions of tyrosine hydroxylase (TH)-positive cells derived from hESCs remain unclear. OBJECTIVE: To investigate the differentiation efficiency of TH-positive cells from hESCs in vitro using modified four-step culture methods, including embryoid body formation, and to examine the functional characteristics of the differentiated TH-positive cells using electrophysiological techniques. DESIGN, TIME AND SETTING: Neuroelectrophysiology was performed at the Reproductive Medicine Center and Stem Cell Research Center, Peking University Third Hospital, and the Neuroscience Research Institute and Department of Neurobiology, Peking University, from September 2004 to August 2008. MATERIALS: The hESC line, PKU-1.1, a monoclonal cell line derived from a pre-implantation human blastocyst in the Reproductive Medical Center of Peking University Third Hospital. The patch clamp recording system was provided by the Neuroscience Research Institute and Department of Neurobiology, Peking University. METHODS: The hESC line was induced to differentiate into TH-positive cells in vitro using a modified four-step culture method, including the formation of embryoid body, as well as the presence of sonic hedgehog and fibroblast growth factor 8. The cell karyotype was assessed by G-banding karyotype analysis techniques and specific markers were detected immunocytochemically. Whole-cell configuration was obtained after obtaining a tight seal of over 1 GΩ. Ionic currents were detected by holding the cells at -70 mV and stepping to test voltages between -80 and 40 mV in 10-mV increments in voltage-clamp configuration. MAIN OUTCOME MEASURES: We measured the cell karyotype, specific cell markers, and the electrophysiological properties of the voltage-gated ion channels on the cell membrane of TH-positive dopaminergic cells differentiated from our hESCs line in vitro. RESULTS: The differentiated cells had a consistent appearance, and the majority of cells (> 90%) expressed TH and β-tubulion, as well as the neural progenitor marker, nestin. Cell karyotype analysis demonstrated that all of the hESCs had a stable and normal karyotype (46, XX) after dif-ferentiation. In addition, patch clamp recording showed that the 10 recorded TH-positive cells exhibited a fast inward current when the test voltage depolarized to -30 mV, and a delayed outward current when the test voltage depolarized to -10 mV. The peak of inward current was obtained at voltage between -10 mV and 0 mV, while the peak of outward current was obtained at 40 mV. The average peak of inward current density was (-50.05 ± 15.50) pA/pF, and the average peak of outward current density was (41.98 ± 13.55) pA/pF. CONCLUSION: More than 90% of the differentiated hESC-derived cells induced by the modified four-step culture method exhibit dopaminergic neuronal properties, including general electrophysiological functional properties, such as functional potassium and sodium channels.
基金National Natural Science Foundation of China(8146055681760658).
文摘OBJECTIVE To research the effect of naringenin(NAR) on LPS-induced dopaminergic neurons damage and its potential mechanism.METHODS Rats were randomly divided into the following six groups(n=10):control(0.9% NaCl),NAR alone(100 mg·kg-1),LPS(5 μg),LPS+NAR(50 mg·kg-1) and LPS+NAR(100 mg·kg-1).Rats were received a single LPS unilateral injection into the SN pars compacts,after seven daily intragastric administration of NAR,rats′ behavior was analyzed by rotarod test.Then,the expression of TH,IBA-1 and NLRP3 inflammasome were analyzed by Western blotting and immunofluorescence.In vitro experiments,BV-2 cel s were treated with different doses of NAR,and 1 h later,LPS(1 g·L^(-1)) was added to the medium for 24 h,then collect the culture medium and protein for later experiments.The production of IL-1β and IL-18 in culture medium were tested by ELISA,and the production of NO was detected by Griess reagent.The expression of IBA-1,NLRP3 and p-caspase 1 were detected by Western blotting.MN9 D cells were co-cultured with BV2 cells to mimic the animal experiments.MTT assay was used to analyzed the viability of MN9 D cells,and the expression of TH was detected by Western blotting.RESULTS NAR(100 mg · kg-1) could significantly improve the time of rats on the rotating(116.73 s vs 185.45 s,P<0.05).The result of the pathological analysis also suggested that NAR could decrease the activation of microglia as well as the expression of NLRP3 Inflammasome.In addition,NAR also could suppress the expression of pro-inflammatory factor levels,such as IL-1β(P<0.05),IL-18(P<0.05),and the protection of NAR could be inhibited by siR NA NLRP3.Moreover,an in vitro co-culture system with BV2 and MN9 D cells wasused to find the protection of NAR must via microglia,while there is no effect of NAR were directly added to MN9 D cells.CONCLUSION NAR protection of LPS-induced dopaminergic neurons damage might be through mediating NLRP3 inflammasome.
基金supported by grants from the Irish Research Council(R15897SVH/AMS/GWO’K)+4 种基金the National University of Ireland(R16189SVH/AMS/GWO’K)Royal Irish Academy(SVH/AMS/GWO’K)Science Foundation Ireland(15/CDA/3498GWO’K)
文摘Introduction:Parkinson’s disease(PD)is a chronic,age-related neurodegenerative disorder that affects 1–2%of the population over the age of 65.PD is characterised by the progressive degeneration of nigrostriatal dopaminergic(DA)neurons.This leads to disabling motor symptoms,due to the striatal DA denervation.Despite decades of research,
基金supported in part by the National Nature Science Foundation of China(81830064,81721092)the National Key Research and Development Plan(2017YFC1103304)+1 种基金the CAMS Innovation Fund for Medical Sciences(CIFMS,2019-I2M-5-059)the Military Medical Research and Development Projects(AWS17J005,2019–126)。
文摘Background:Generation of neurons is essential in cell replacement therapy for neurodegenerative disorders like Parkinson’s disease.Several studies have reported the generation of dopaminergic(DA)neurons from mouse and human fibroblasts by ectopic expression of transcription factors,in which genetic manipulation is associated with potential risks.Methods:The small molecules and protein factors were selected based on their function to directly induce human fetal lung IMR-90 fibroblasts into DA neuron-like cells.Microscopical,immunocytochemical,and RT-qPCR analyses were used to characterize the morphology,phenotype,and gene expression features of the induced cells.The wholecell patch-clamp recordings were exploited to measure the electrophysiological properties.Results:Human IMR-90 fibroblasts were rapidly converted into DA neuron-like cells after the chemical induction using small molecules and protein factors,with a yield of approximately 95%positive TUJ1-positive cells.The induced DA neuron-like cells were immunopositive for pan-neuronal markers MAP2,NEUN,and Synapsin 1 and DA markers TH,DDC,DAT,and NURR1.The chemical induction process did not involve a neural progenitor/stem cell intermediate stage.The induced neurons could fire single action potentials,which reflected partially the electrophysiological properties of neurons.Conclusions:We developed a chemical cocktail of small molecules and protein factors to convert human fibroblasts into DA neuron-like cells without passing through a neural progenitor/stem cell intermediate stage.The induced DA neuron-like cells from human fibroblasts might provide a cellular source for cell-based therapy of Parkinson’s disease in the future.
基金The work reported herein was supported by the grants from the National Natural Science Foundation of China(Grant No.81803505)Jiangsu Research Hospital Association for Precision Medication(Grant No.JY202134).
文摘As a late endosomal/lysosomal transport protein of the P5-type, ATP13A2 is capable of removing the abnormal accumulation of α-synuclein, which maintains the homeostasis of metal ions and polyamines in the central nervous system. Furthermore, ATP13A2 regulates the normal function of several organelles such as lysosomes, endoplasmic reticulum (ER) and mitochondria, and maintains the normal physiological activity of neural cells. Especially, ATP13A2 protects dopaminergic (DA) neurons against environmental or genetically induced Parkinson's disease (PD). As we all know, PD is a neurodegenerative disease characterized by the loss of DA neurons in the substantia nigra pars compacta. An increasing number of studies have reported that the loss-of- function of ATP13A2 affects normal physiological processes of various organelles, leading to abnormalities and the death of DA neurons. Previous studies in our laboratory have also shown that ATP13A2 deletion intensifies the neuroinflammatory response induced by astrocytes, thus inducing DA neuronal injury. In addition to elucidating the normal structure and function of ATP13A2, this review summarized the pathological mechanisms of ATP13A2 mutations leading to PD in existing literature studies, deepening the understanding of ATP13A2 in the pathological process of PD and other related neurodegenerative diseases. This review provides inspiration for investigators to explore the essential regulatory role of ATP13A2 in PD in the future.
基金Supported by The German Federal Ministry for Education and Research(BMBF),No.01GN0818 and No.01GN0819the Max-Planck Society,and initially by the Dr.Helmut Storz Stiftung
文摘AIM:To find a safe source for dopaminergic neurons,we generated neural progenitor cell lines from human embryonic stem cells.METHODS:The human embryonic stem(hES)cell line H9 was used to generate human neural progenitor(HNP)cell lines.The resulting HNP cell lines were differentiated into dopaminergic neurons and analyzed by quantitative real-time polymerase chain reaction and immunofluorescence for the expression of neuronal differentiation markers,including beta-III tubulin(TUJ1)and tyrosine hydroxylase(TH).To assess the risk of teratoma or other tumor formation,HNP cell lines and mouse neuronal progenitor(MNP)cell lines were injected subcutaneously into immunodeficient SCID/beige mice.RESULTS:We developed a fairly simple and fast protocol to obtain HNP cell lines from hES cells.These cell lines,which can be stored in liquid nitrogen for several years,have the potential to differentiate in vitro into dopaminergic neurons.Following day 30 of differentiation culture,the majority of the cells analyzed expressed the neuronal marker TUJ1 and a high proportion of these cells were positive for TH,indicating differentiation into dopaminergic neurons.In contrast to H9 ES cells,the HNP cell lines did not form tumors in immunodeficient SCID/beige mice within 6 mo after subcutaneous injection.Similarly,no tumors developed after injection of MNP cells.Notably,mouse ES cells or neuronal cells directly differentiated from mouse ES cells formed teratomas in more than 90%of the recipients.CONCLUSION:Our findings indicate that neural progenitor cell lines can differentiate into dopaminergic neurons and bear no risk of generating teratomas or other tumors in immunodeficient mice.
文摘OBJECTIVE Dysfunction of the dopaminergic(DA)neurons is implicated in the pathogenesis of bipolar disorder(BPD).Hista⁃mine receptor 2(Hrh2)is highly expressed in DA neurons,and its antagonists have been reported to induce mania phase of BPD.However,whether Hrh2 on DA neurons contributes to BPD patho⁃genesis is unclear.The present study aims to explore the role of hrh2 on DA neurons in the pathology of BPD.METHODS AAV-FLEX-shHrh2 was injected into a targeted brain area of DAT-Cre mice,leading to a selective brain-regional loss of Hrh2 on DA neurons.A series of behavior tests were used to measure the sponta⁃neous activity,anxiety and depression level of Hrh2-deficient mice.RESULTS①In the open field test and home-cage activity test,Hrh2-defi⁃cient mice displayed increased spontaneous activity.②Hrh2-deficient mice showed reduced depression level in the tail suspension test,forced swimming test and sucrose preference test.③The anxiety level of Hrh2-deficient mice was decreased in the open field test.CONCLU⁃SION Hrh2 on DA neurons is closely related with mania-like behavior.
文摘Dopaminergic neurotoxicity is characterized by damage and death of dopaminergic neurons.Parkinson's disease(PD)is a neurodegenerative disorder that primarily involves the loss of dopaminergic neurons in the substantia nigra.Therefore,the study of the mechanisms,as well as the search for new targets for the prevention and treatment of neurodegenerative diseases,is an important focus of modern neuroscience.PD is primarily caused by dysfunction of dopaminergic neurons;however,other neurotransmitter systems are also involved.Research reports have indicated that the glutamatergic system is involved in different pathological conditions,including dopaminergic neurotoxicity.Over the last two decades,the important functional interplay between dopaminergic and glutamatergic systems has stimulated interest in the possible role of metabotropic glutamate receptors(mGluRs)in the development of extrapyramidal disorders.However,the specific mechanisms driving these processes are presently unclear.The participation of the universal neuronal messenger nitric oxide(NO)in the mechanisms of dopaminergic neurotoxicity has attracted increased attention.The current paper aims to review the involvement of mGluRs and the contribution of NO to dopaminergic neurotoxicity.More precisely,we focused on studies conducted on the rotenone-induced PD model.This review is also an outline of our own results obtained using the method of electron paramagnetic resonance,which allows quantitation of NO radicals in brain structures.
文摘Thirst, which provides the motivation to drink, is an important component of the coordinated sequence of physiological responses that maintain the volume and composition of body fluids. Special structures in the central nervous system like periventricular organs detect changes in these parameters continuously. The present study investigated the interaction between dopaminergic and angiotensinergic systems on water intake in adult male rats. Intracerebroventricular (ICV) injections were carried out in all experiments after 24 h deprivation of water intake. After the deprivation interval, the volume of consumed water was measured for 1h. Administration the angiotensinergic (AT1) receptor antagonist Losartan (45 μg/rat), and the dopaminergic antagonist Chlorpromazine (40 μg/rat) significantly decreased water intake when compared to saline-treated controls. In contrast, ICV microinjection of the dopaminergic agonist Bromocriptine (10 μg/rat) significantly increased water intake when compared to saline-treated controls. ICV injection of Bromocriptine 15min after Losartan administration was able to attenuate the inhibitory effect of Losartan on water intake, whereas administration of Chlorpromazine 15 min after Losartan was unable to change the Losartan effect. These results suggest that the dopaminergic system interactions with the angiotensinergic system to regulate water intake through circumventricular organs. Dopaminergic and angiotensinergic neurons can monitor and regulate water intake via the stimulatory and inhibitory effects on each other, respectively.
文摘A spontaneous mutation in the Albino Swiss (AS) rat has been shown to be a single point mutation (agu) in the gene coding for the gamma isoform of protein kinase C (PKC-γ). The characteristics of the mutant include movement disorders, a failure to release dopamine in the striatum and elevations of molecules such as parkin and ubiquitin in the substantia nigra pars compacta (SNC). This present study examined SNC cell bodies and dopaminergic synaptic terminals within the caudate-putamen. Cell volume and nuclear volume were reduced in the AS/AGU mutant compared to the AS control, but the volume fractions of mitochondria and rough endoplasmic reticulum were significantly higher. No Lewy bodies were present in the mutant, although microglia were found adjacent to some SNC cells. Dopaminergic terminals were identified in the caudate-putamen by electron microscopy with low-glutaraldehyde fixation and immunohistochemistry for tyrosine hydroxylase using immuno-gold visualisation. AS/AGU mutant rats had less than half of the synaptic vesicles of AS controls;this was not only true of “readily-releasable” zones adjacent to the synaptic cleft but also “storage pool” zones. The findings support the hypothesis that the initial bar to dopamine availability in the striatum is the reduced release, with nigral cell death being a later phenomenon.
文摘Objective: Levodopa is the gold-standard of therapy in Parkinson’s disease (PD), but it is associated with motor complications that affect 50% of patients after five years of treatment. Development of delirium and psychosis is the main limitation of dopaminergic treatment in older persons. These adverse effects may result from pulsatile stimulation of the dopamine receptors. Dopamine agonists with transdermal delivery that continuously stimulate the dopamine receptors may reduce these complications. The objective of this study was to evaluate the frequencies of acute delirium and psychosis in elderly patients treated with rotigotine vs. levodopa in a newly diagnosed drugnaive Parkinson’s disease (PD). Methods: Patients admitted to the Geriatric-Rehabilitation Department of the University-Hospital of Parma were screened for the presence of Parkinsonism. All subjects admitted with diagnosis of PD according to the UK Brain Bank Criteria were randomly treated with Rotigotine or levodopa. All subjects were assessed by Movement Disorder Society (MDS)-Unified Parkinson's Disease Rating Scale (MDS-UPDRS) Part III. Acute delirium was asessed by CAM Diagnostic Algorithm during the first week after admission. After six months, diagnosis of psychosis was performed according to pro posed diagnostic criteria by NINDS and NIMH. Patients with cognitive impairment (MMSE dementia with Lewy bodies (DLB), were excluded. Results: 60 consecutive newly diagnosed drugnaive PD patients were evaluated. No statistical significant difference between the two groups were observed in term of age, gender, MMSE score, severity of disease expressed by H&Y staging. 30 patients were treated with rotigotine (6 mg/daily) and 30 patients were treated with L-Dopa (250 mg/daily). All participants completed the study. UPDRS Part III was statistical significant lower in both groups after treatment from 26.4 to 18.3 (rotigotine group) and from 26.3 to 17.3 (levodopa group), but comparable within groups (p = 0.83). After 6-month follow-up, acute delirium and/ or psychosis were observed in two cases (6.6%) of patients treated with rotigotine and in three cases (10%) of those treated with levodopa (p = 0.54). Conclusions: Transdermal rotigotine seems comparable to levodopa in regard to motor skill efficacy and neuropsychiatric safety, because provides a more continuous delivery of drug. Dopamine agonists may represent a valid therapeutic option in newly diagnosed older PD patients.
文摘Dopaminergic therapy can improve motor functioning in Parkinson’s disease (PD), but its effect on spoken discourse is poorly understood. This study addressed whether discourse produced during a one-minute monologue would be affected by medication withdrawal. We performed a detailed analysis of semistructured speech samples in 38 patients with PD, including measures of efficiency, syntactic complexity, cohesion (i.e., lexical and grammatical ties between sentences), and coherence (i.e., semantic relationship between the utterances and the topic, or between utterances). We found that patients produced more efficient and coherent monlogues when on versus off medication. As discourse relies heavily on cognitive processes such as working memory, these findings are consistent with research showing improved working memory and executive processes with dopaminegic medication.
