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
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
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
OBJECTIVE:To explore the possible mechanism of Tongdu Tiaoshen acupuncture combined with Xiaoxuming decoction(小续命汤,XXMD)in the treatment of Parkinson’s disease(PD).METHODS:C57BL/6 mice were randomly divided into ...OBJECTIVE:To explore the possible mechanism of Tongdu Tiaoshen acupuncture combined with Xiaoxuming decoction(小续命汤,XXMD)in the treatment of Parkinson’s disease(PD).METHODS:C57BL/6 mice were randomly divided into eight groups(n=12),including blank group,model group,medication group,acupuncture group,high-dose XXMD group(XXMD-H),low-dose XXMD group(XXMD-L),acupuncture combined with high-dose XXMD group(A+H),and acupuncture combined with low-dose XXMD group(A+L).After treatment for 6 weeks,dopamine(DA)neurons and the pathological changes of tyrosine hydroxylase(TH)positive cells were observed.The enzyme-linked immunosorbent assay(ELISA)was used to measure the content of DA and the level of interleukin-1β(IL-1β),interleukin-6(IL-6),interleukin-10(IL-10)and tumor necrosis factor alpha(TNF-α).The m RNA level of PINK1 and Parkin and the protein expression of Nix,PINK1 and Parkin in the substantia nigra were also detected.RESULTS:Combination treatment effectively ameliorated the symptoms of PD.Compared with model group,combined treatment significantly up-regulated the protein expression of Nix,Parkin and PINK1 and the m RNA levels of PINK1 and Parkin in the substantia nigra(P<0.0001,P<0.001,P<0.01 or P<0.05).Furthermore,the levels of pro-inflammation cytokines were obviously decreased after combination therapy,while IL-10 content was increased remarkably(P<0.01).CONCLUSION:Compared with each treatment alone,combination therapy improved the pathological damage of DA neurons of PD mice more effectively.The possible mechanism may be attributed to the up-regulated level of mitochondrial autophagy and improved mitochondrial function.These results provide fresh insight into the mechanism of co-treatment with Tongdu Tiaoshen acupuncture and XXMD for PD.展开更多
In recent years,neurodegenerative diseases,such as Parkinson’s or Alzheimer’s diseases,are rapidly rising in prevalence.The main hallmark of Parkinson’s disease is the falling levels of neurotransmitter dopamine in...In recent years,neurodegenerative diseases,such as Parkinson’s or Alzheimer’s diseases,are rapidly rising in prevalence.The main hallmark of Parkinson’s disease is the falling levels of neurotransmitter dopamine in the mid-brain with dopaminergic neurons losing.Typical therapeutic solutions,including drugs,deep brain stimulation,and cell transplantation,can only alleviate the symptoms of Parkinson’s disease.It is a tremendous challenge to reverse the function degeneration of the crucial dopaminergic neurons.Herein,we develop a core-satellite-like nanoassembly(PDA-AFn(by integrating polydopamine nanoparticles and apoferritin))to raise the expression of tyrosine hydroxylase(TH),a rate-limiting enzyme in the formation of the dopamine.Both components in the nanoassembly could cooperate with each other,not only elaborately regulate the iron homeostasis and redox microenvironment,but also utilize excessive reactive oxygen species(ROS)and iron ions in the damaged neurons to supply extra dopamine and enhance TH activity,and consequently restore the function of the degenerated neurons.Remarkably,the nanoassembly-treatment relieves the dyskinesia and dramatical increases the tyrosine hydroxylase and dopamine level in the midbrain of Parkinson’s disease model mice.It is an explicit yet inspiring advance in treatment of the neurodegeneration.展开更多
The positive effect of levodopa in the treatment of Parkinson’s disease,although it is limited in time and has severe side effects,has encouraged the scientific community to look for new drugs that can stop the neuro...The positive effect of levodopa in the treatment of Parkinson’s disease,although it is limited in time and has severe side effects,has encouraged the scientific community to look for new drugs that can stop the neurodegenerative process or even regenerate the neuromelanin-containing dopaminergic nigrostriatal neurons.Successful preclinical studies with coenzyme Q10,mitoquinone,isradipine,nilotinib,TCH346,neurturin,zonisamide,deferiprone,prasinezumab,and cinpanemab prompted clinical trials.However,these failed and after more than 50 years levodopa continues to be the key drug in the treatment of the disease,despite its severe side effects after 4–6 years of chronic treatment.The lack of translated successful results obtained in preclinical investigations based on the use of neurotoxins that do not exist in the human body as new drugs for Parkinson’s disease treatment is a big problem.In our opinion,the cause of these failures lies in the experimental animal models involving neurotoxins that do not exist in the human body,such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 6-hydroxydopamine,that induce a very fast,massive and expansive neurodegenerative process,which contrasts with the extremely slow one of neuromelanin-containing dopaminergic neurons.The exceedingly slow progress of the neurodegenerative process of the nigrostriatal neurons in idiopathic Parkinson’s patients is due to(i)a degenerative model in which the neurotoxic effect of an endogenous neurotoxin affects a single neuron,(ii)a neurotoxic event that is not expansive and(iii)the fact that the neurotoxin that triggers the neurodegenerative process is produced inside the neuromelanin-containing dopaminergic neurons.The endogenous neurotoxin that fits this degenerative model involving one single neuron at a time is aminochrome,since it(i)is generated within neuromelanin-containing dopaminergic neurons,(ii)does not cause an expansive neurotoxic effect and(iii)triggers all the mechanisms involved in the neurodegenerative process of the nigrostriatal neurons in idiopathic Parkinson’s disease.In conclusion,based on the hypothesis that the neurodegenerative process of idiopathic Parkinson’s disease corresponds to a single-neuron neurodegeneration model,we must search for molecules that increase the expression of the neuroprotective enzymes DT-diaphorase and glutathione transferase M2-2.It has been observed that the activation of the Kelch-like ECH-associated protein 1/nuclear factor(erythroid-derived 2)-like 2 pathway is associated with the transcriptional activation of the DT-diaphorase and glutathione transferase genes.展开更多
Parkinson’s disease(PD)is a multifactorial disorder of the nervous system where a progressive loss of dopaminergic neurons exist.However,the pathogenesis of PD remains undefined,which becomes the main limitation for ...Parkinson’s disease(PD)is a multifactorial disorder of the nervous system where a progressive loss of dopaminergic neurons exist.However,the pathogenesis of PD remains undefined,which becomes the main limitation for the development of clinical PD treatment.Demethylenetetrahydroberberine(DMTHB)is a novel derivative of natural product berberine.This study was aimed to explore the neuroprotective effects and pharmacological mechanism of DMTHB on Parkinson’s disease using C57BL/6 mice.A PD model of mice was induced by administration of MPTP(20 mg·kg^(-1))and probenecid(200 mg·kg^(-1))twice per week for five weeks.The mice were administered with DMTHB daily by gavage at the dose of 5 and 50 mg·kg^(-1)for one-week prophylactic treatment and five-week theraputic treatment.The therapeutic effects of DMTHB were evaluated by behavior tests(the open field,rotarod and pole tests),immunohistochemical staining of tyrosine hydroxylase(TH),Nissl staining and biochemical assays.The molecular mechanisms of DMTHB on the key biomarkers of PD pathological states were analyzed by Western blot(WB)and qRT-PCR.DMTHB treatment alleviated the behavioral disorder induced by MPTP-probenecid.Nissl staining and TH staining showed that the damage of dopaminergic neurons in the substantia nigra was remarkably suppressed by DMTHB treatment.Western blot results showed that the ratio of Bcl-2/Bax and TH increased,but the level ofα-synuclein(α-syn)was remarkably reduced,which indicated that the apoptosis of dopaminergic neurons in mice was significantly reduced.The protein phosphorylation of p-PI3K,p-AKT and p-mTOR also increased about 2-fold,compared with the model group.Furthermore,qRT-PCR results demonstrated that the mRNA levels of pro-inflammatory cytokines,IL-1βand TNF-α,were reduced,but the level of anti-inflammatory cytokine IL-10 increased after DMTHB treatment.Finally,the cellular assay displayed that DMTHB was also a strong antioxidant to protect neuron cell line PC12 by scavenging ROS.In this study,we demonstrated DMTHB alleviates the behavioral disorder and protects dopaminergic neurons through multiple-target effects includubg anti-apoptotic,anti-inflammatory and antioxidant effects.展开更多
Protein O-GlcNAcylation is a post-translational modification that links environmental stimuli with changes in intracellular signal pathways,and its disturbance has been found in neurodegenerative diseases and metaboli...Protein O-GlcNAcylation is a post-translational modification that links environmental stimuli with changes in intracellular signal pathways,and its disturbance has been found in neurodegenerative diseases and metabolic disorders.However,its role in the mesolimbic dopamine(DA)system,especially in the ventral tegmental area(VTA),needs to be elucidated.Here,we found that injection of Thiamet G,an O-GlcNAcase(OGA)inhibitor,in the VTA and nucleus accumbens(NAc)of mice,facilitated neuronal O-GlcNAcylation and decreased the operant response to sucrose as well as the latency to fall in rotarod test.Mice with DAergic neuron-specific knockout of O-GlcNAc transferase(OGT)displayed severe metabolic abnormalities and died within 4–8 weeks after birth.Furthermore,mice specifically overexpressing OGT in DAergic neurons in the VTA had learning defects in the operant response to sucrose,and impaired motor learning in the rotarod test.Instead,overexpression of OGT in GABAergic neurons in the VTA had no effect on these behaviors.These results suggest that protein O-GlcNAcylation of DAergic neurons in the VTA plays an important role in regulating the response to natural reward and motor learning in mice.展开更多
This study aimed to investigate the protective effect of nicotine on dopaminergic neurons and its mechanisms in mice with Parkinson disease(PD)induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP).C57BL/6J mic...This study aimed to investigate the protective effect of nicotine on dopaminergic neurons and its mechanisms in mice with Parkinson disease(PD)induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP).C57BL/6J mice were injected with MPTP for 8 days to establish a PD model.Nicotine was given for 10 days in the nicotine therapeutic group.Animals were examined behaviorally with the pole test and traction test.Tyrosine hydroxylase(TH)andγ-aminobutyric acid(GABA)were determined by using the immunocytochem-istry(ICC)method.The ultrastructural changes of the caudate nucleus(CN)were observed under electron microscopy.The results showed that pretreatment with nicotine could improve the dyskinesia of PD mice markedly.Simultaneously,TH-positive(P<0.01)neurons and GABA-positive(P<0.05)neurons in the nicotine therapeutic group were significantly more than those in the model group.The ultrastructural injury of the nicotine therapeutic group was also ameliorated.Nicotine has protective effects on theγ-aminobutyric acid neurons and dopaminergic neurons in the MPTP-treated mice.展开更多
A preferential dysfunction/loss of dopaminergic(DA)neurons in the substantia nigra pars compacta(SNpc)accounts for the main motor symptoms of Parkinson’s disease(PD),the most common degenerative movement disorder.How...A preferential dysfunction/loss of dopaminergic(DA)neurons in the substantia nigra pars compacta(SNpc)accounts for the main motor symptoms of Parkinson’s disease(PD),the most common degenerative movement disorder.However,the neuronal loss is not stochastic,but rather displays regionally selectivity,indicating the existence of different DA subpopulations in the SNpc.To identify the underlying molecular determinants is thereby instrumental in understanding the pathophysiological mechanisms of PD-related neuron dysfunction/loss and offering new therapeutic targets.Recently,we have demonstrated that aldehyde dehydrogenase 1(ALDH1A1)is one such molecular determinant that defines and protects an SNpc DA neuron subpopulation preferentially affected in PD.In this review,we provide further analysis and discussion on the roles of ALDH1A1 in the function and survival of SNpc DA neurons in both rodent and human brains.We also explore the feasibility of ALDH1A1 as a potential biomarker and therapeutic target for PD.展开更多
Background:α–synuclein(α–syn)is the main component of intracytoplasmic inclusions deposited in the brains of patients with Parkinson’s disease(PD)and certain other neurodegenerative disorders.Recent studies have ...Background:α–synuclein(α–syn)is the main component of intracytoplasmic inclusions deposited in the brains of patients with Parkinson’s disease(PD)and certain other neurodegenerative disorders.Recent studies have explored the ability ofα–syn to propagate between or across neighboring neurons and supposedly“infect”them with a prion–like mechanism.However,much of this research has used stereotaxic injections of heterologousα–syn fibrils to induce the spreading of inclusions in the rodent brains.Whetherα–syn is able to transmit from the host cells to their neighboring cells in vivo is unclear.Methods:Using immunestaining,we examined the potential propagation ofα–syn into nigrostriatal dopaminergic(DA)neurons in three lines of transgenic mice that overexpress human wild–typeα–syn(hα–syn)in different neuron populations.Results:After testing for three different routes by which hα–syn propagation might occur,we were unable to find any evidence that hα–syn behaved like a prion and could be transmitted overtime into the DA neurons initially lack of hα–syn expression.Conclusions:In transgenic mice hα–syn does not have the ability to propagate at pathologically significant levels between or across neurons.It must be noted that these observations do not disprove the studies that show its prion–like qualities,but rather that propagation is not detectable in transgenic models that do not use any injections of heterologous proteins or viral vectors to induce a spreading state.展开更多
Parkinson’s disease is typically characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta.Many studies have been performed based on the supplementation of lost dopaminergic ...Parkinson’s disease is typically characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta.Many studies have been performed based on the supplementation of lost dopaminergic neurons to treat Parkinson’s disease.The initial strategy for cell replacement therapy used human fetal ventral midbrain and human embryonic stem cells to treat Parkinson’s disease,which could substantially alleviate the symptoms of Parkinson’s disease in clinical practice.However,ethical issues and tumor formation were limitations of its clinical application.Induced pluripotent stem cells can be acquired without sacrificing human embryos,which eliminates the huge ethical barriers of human stem cell therapy.Another widely considered neuronal regeneration strategy is to directly reprogram fibroblasts and astrocytes into neurons,without the need for intermediate proliferation states,thus avoiding issues of immune rejection and tumor formation.Both induced pluripotent stem cells and direct reprogramming of lineage cells have shown promising results in the treatment of Parkinson’s disease.However,there are also ethical concerns and the risk of tumor formation that need to be addressed.This review highlights the current application status of cell reprogramming in the treatment of Parkinson’s disease,focusing on the use of induced pluripotent stem cells in cell replacement therapy,including preclinical animal models and progress in clinical research.The review also discusses the advancements in direct reprogramming of lineage cells in the treatment of Parkinson’s disease,as well as the controversy surrounding in vivo reprogramming.These findings suggest that cell reprogramming may hold great promise as a potential strategy for treating Parkinson’s disease.展开更多
Endoplasmic reticulum stress and mitochondrial dysfunction play important roles in Parkinson s disease,but the regulato ry mechanism remains elusive.Prohibitin-2(PHB2)is a newly discove red autophagy receptor in the m...Endoplasmic reticulum stress and mitochondrial dysfunction play important roles in Parkinson s disease,but the regulato ry mechanism remains elusive.Prohibitin-2(PHB2)is a newly discove red autophagy receptor in the mitochondrial inner membrane,and its role in Parkinson’s disease remains unclear.Protein kinase R(PKR)-like endoplasmic reticulum kinase(PERK)is a factor that regulates cell fate during endoplasmic reticulum stress.Parkin is regulated by PERK and is a target of the unfolded protein response.It is unclear whether PERK regulates PHB2-mediated mitophagy thro ugh Parkin.In this study,we established a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced mouse model of Parkinson’s disease.We used adeno-associated virus to knockdown PHB2 expression.Our res ults showed that loss of dopaminergic neurons and motor deficits were aggravated in the MPTP-induced mouse model of Parkinson’s disease.Ove rexpression of PHB2 inhibited these abnormalities.We also established a 1-methyl-4-phenylpyridine(MPP+)-induced SH-SY5Y cell model of Parkinson’s disease.We found that ove rexpression of Parkin increased co-localization of PHB2 and microtubule-associated protein 1 light chain 3,and promoted mitophagy.In addition,MPP+regulated Parkin involvement in PHB2-mediated mitophagy through phosphorylation of PERK.These findings suggest that PHB2 participates in the development of Parkinson’s disease by intera cting with endoplasmic reticulum stress and Parkin.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.
基金Supported by National Natural Science Foundation of China:Study on the Mechanism of Regulating miR-124 by Tongdu Tiaoshen Acupuncture to Promote Neuroprotection in Cerebral Ischemia Reperfusion Injury(No.81973933)Natural Science Fund for Colleges and Universities in Anhui Province:Study on the Regulatory Mechanism of Tongdu Tiaoshen Acupuncture on Dopaminergic Neurons in Parkinson’s Disease Model Mice Based on Mitochondrial Autophagy(No.KJ2019A0475)。
文摘OBJECTIVE:To explore the possible mechanism of Tongdu Tiaoshen acupuncture combined with Xiaoxuming decoction(小续命汤,XXMD)in the treatment of Parkinson’s disease(PD).METHODS:C57BL/6 mice were randomly divided into eight groups(n=12),including blank group,model group,medication group,acupuncture group,high-dose XXMD group(XXMD-H),low-dose XXMD group(XXMD-L),acupuncture combined with high-dose XXMD group(A+H),and acupuncture combined with low-dose XXMD group(A+L).After treatment for 6 weeks,dopamine(DA)neurons and the pathological changes of tyrosine hydroxylase(TH)positive cells were observed.The enzyme-linked immunosorbent assay(ELISA)was used to measure the content of DA and the level of interleukin-1β(IL-1β),interleukin-6(IL-6),interleukin-10(IL-10)and tumor necrosis factor alpha(TNF-α).The m RNA level of PINK1 and Parkin and the protein expression of Nix,PINK1 and Parkin in the substantia nigra were also detected.RESULTS:Combination treatment effectively ameliorated the symptoms of PD.Compared with model group,combined treatment significantly up-regulated the protein expression of Nix,Parkin and PINK1 and the m RNA levels of PINK1 and Parkin in the substantia nigra(P<0.0001,P<0.001,P<0.01 or P<0.05).Furthermore,the levels of pro-inflammation cytokines were obviously decreased after combination therapy,while IL-10 content was increased remarkably(P<0.01).CONCLUSION:Compared with each treatment alone,combination therapy improved the pathological damage of DA neurons of PD mice more effectively.The possible mechanism may be attributed to the up-regulated level of mitochondrial autophagy and improved mitochondrial function.These results provide fresh insight into the mechanism of co-treatment with Tongdu Tiaoshen acupuncture and XXMD for PD.
基金This work was supported by National Natural Science Foundation of China(Nos.22175085 and 21875101)National Key Research and Development Program of China(No.2017YFA0701301)State Key Laboratory of Analytical Chemistry for Life Science(No.SKLACLS2219).
文摘In recent years,neurodegenerative diseases,such as Parkinson’s or Alzheimer’s diseases,are rapidly rising in prevalence.The main hallmark of Parkinson’s disease is the falling levels of neurotransmitter dopamine in the mid-brain with dopaminergic neurons losing.Typical therapeutic solutions,including drugs,deep brain stimulation,and cell transplantation,can only alleviate the symptoms of Parkinson’s disease.It is a tremendous challenge to reverse the function degeneration of the crucial dopaminergic neurons.Herein,we develop a core-satellite-like nanoassembly(PDA-AFn(by integrating polydopamine nanoparticles and apoferritin))to raise the expression of tyrosine hydroxylase(TH),a rate-limiting enzyme in the formation of the dopamine.Both components in the nanoassembly could cooperate with each other,not only elaborately regulate the iron homeostasis and redox microenvironment,but also utilize excessive reactive oxygen species(ROS)and iron ions in the damaged neurons to supply extra dopamine and enhance TH activity,and consequently restore the function of the degenerated neurons.Remarkably,the nanoassembly-treatment relieves the dyskinesia and dramatical increases the tyrosine hydroxylase and dopamine level in the midbrain of Parkinson’s disease model mice.It is an explicit yet inspiring advance in treatment of the neurodegeneration.
文摘The positive effect of levodopa in the treatment of Parkinson’s disease,although it is limited in time and has severe side effects,has encouraged the scientific community to look for new drugs that can stop the neurodegenerative process or even regenerate the neuromelanin-containing dopaminergic nigrostriatal neurons.Successful preclinical studies with coenzyme Q10,mitoquinone,isradipine,nilotinib,TCH346,neurturin,zonisamide,deferiprone,prasinezumab,and cinpanemab prompted clinical trials.However,these failed and after more than 50 years levodopa continues to be the key drug in the treatment of the disease,despite its severe side effects after 4–6 years of chronic treatment.The lack of translated successful results obtained in preclinical investigations based on the use of neurotoxins that do not exist in the human body as new drugs for Parkinson’s disease treatment is a big problem.In our opinion,the cause of these failures lies in the experimental animal models involving neurotoxins that do not exist in the human body,such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 6-hydroxydopamine,that induce a very fast,massive and expansive neurodegenerative process,which contrasts with the extremely slow one of neuromelanin-containing dopaminergic neurons.The exceedingly slow progress of the neurodegenerative process of the nigrostriatal neurons in idiopathic Parkinson’s patients is due to(i)a degenerative model in which the neurotoxic effect of an endogenous neurotoxin affects a single neuron,(ii)a neurotoxic event that is not expansive and(iii)the fact that the neurotoxin that triggers the neurodegenerative process is produced inside the neuromelanin-containing dopaminergic neurons.The endogenous neurotoxin that fits this degenerative model involving one single neuron at a time is aminochrome,since it(i)is generated within neuromelanin-containing dopaminergic neurons,(ii)does not cause an expansive neurotoxic effect and(iii)triggers all the mechanisms involved in the neurodegenerative process of the nigrostriatal neurons in idiopathic Parkinson’s disease.In conclusion,based on the hypothesis that the neurodegenerative process of idiopathic Parkinson’s disease corresponds to a single-neuron neurodegeneration model,we must search for molecules that increase the expression of the neuroprotective enzymes DT-diaphorase and glutathione transferase M2-2.It has been observed that the activation of the Kelch-like ECH-associated protein 1/nuclear factor(erythroid-derived 2)-like 2 pathway is associated with the transcriptional activation of the DT-diaphorase and glutathione transferase genes.
基金supported by the National Natural Science Foundation of China(No.81573484)the Postgraduate Research&Practice Innovation Program of Jiangsu Province(KYCX17_0705)+1 种基金the Opening Project of Shanghai Key Laboratory of Complex Prescription(Shanghai University of Traditional Chinese Medicine)(14DZ2271000)a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD)。
文摘Parkinson’s disease(PD)is a multifactorial disorder of the nervous system where a progressive loss of dopaminergic neurons exist.However,the pathogenesis of PD remains undefined,which becomes the main limitation for the development of clinical PD treatment.Demethylenetetrahydroberberine(DMTHB)is a novel derivative of natural product berberine.This study was aimed to explore the neuroprotective effects and pharmacological mechanism of DMTHB on Parkinson’s disease using C57BL/6 mice.A PD model of mice was induced by administration of MPTP(20 mg·kg^(-1))and probenecid(200 mg·kg^(-1))twice per week for five weeks.The mice were administered with DMTHB daily by gavage at the dose of 5 and 50 mg·kg^(-1)for one-week prophylactic treatment and five-week theraputic treatment.The therapeutic effects of DMTHB were evaluated by behavior tests(the open field,rotarod and pole tests),immunohistochemical staining of tyrosine hydroxylase(TH),Nissl staining and biochemical assays.The molecular mechanisms of DMTHB on the key biomarkers of PD pathological states were analyzed by Western blot(WB)and qRT-PCR.DMTHB treatment alleviated the behavioral disorder induced by MPTP-probenecid.Nissl staining and TH staining showed that the damage of dopaminergic neurons in the substantia nigra was remarkably suppressed by DMTHB treatment.Western blot results showed that the ratio of Bcl-2/Bax and TH increased,but the level ofα-synuclein(α-syn)was remarkably reduced,which indicated that the apoptosis of dopaminergic neurons in mice was significantly reduced.The protein phosphorylation of p-PI3K,p-AKT and p-mTOR also increased about 2-fold,compared with the model group.Furthermore,qRT-PCR results demonstrated that the mRNA levels of pro-inflammatory cytokines,IL-1βand TNF-α,were reduced,but the level of anti-inflammatory cytokine IL-10 increased after DMTHB treatment.Finally,the cellular assay displayed that DMTHB was also a strong antioxidant to protect neuron cell line PC12 by scavenging ROS.In this study,we demonstrated DMTHB alleviates the behavioral disorder and protects dopaminergic neurons through multiple-target effects includubg anti-apoptotic,anti-inflammatory and antioxidant effects.
基金the National Natural Science Foundation of China(31871021,82021002,31930046,and 32000671)the China Postdoctoral Science Foundation(2020M670978 and 2021T140127)the Shanghai Municipal Science and Technology Major Project(2018SHZDZX01),and ZhangJiang Lab.
文摘Protein O-GlcNAcylation is a post-translational modification that links environmental stimuli with changes in intracellular signal pathways,and its disturbance has been found in neurodegenerative diseases and metabolic disorders.However,its role in the mesolimbic dopamine(DA)system,especially in the ventral tegmental area(VTA),needs to be elucidated.Here,we found that injection of Thiamet G,an O-GlcNAcase(OGA)inhibitor,in the VTA and nucleus accumbens(NAc)of mice,facilitated neuronal O-GlcNAcylation and decreased the operant response to sucrose as well as the latency to fall in rotarod test.Mice with DAergic neuron-specific knockout of O-GlcNAc transferase(OGT)displayed severe metabolic abnormalities and died within 4–8 weeks after birth.Furthermore,mice specifically overexpressing OGT in DAergic neurons in the VTA had learning defects in the operant response to sucrose,and impaired motor learning in the rotarod test.Instead,overexpression of OGT in GABAergic neurons in the VTA had no effect on these behaviors.These results suggest that protein O-GlcNAcylation of DAergic neurons in the VTA plays an important role in regulating the response to natural reward and motor learning in mice.
基金supported by the Foundation of Education Department of Liaoning Province,China(No.20060211).
文摘This study aimed to investigate the protective effect of nicotine on dopaminergic neurons and its mechanisms in mice with Parkinson disease(PD)induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP).C57BL/6J mice were injected with MPTP for 8 days to establish a PD model.Nicotine was given for 10 days in the nicotine therapeutic group.Animals were examined behaviorally with the pole test and traction test.Tyrosine hydroxylase(TH)andγ-aminobutyric acid(GABA)were determined by using the immunocytochem-istry(ICC)method.The ultrastructural changes of the caudate nucleus(CN)were observed under electron microscopy.The results showed that pretreatment with nicotine could improve the dyskinesia of PD mice markedly.Simultaneously,TH-positive(P<0.01)neurons and GABA-positive(P<0.05)neurons in the nicotine therapeutic group were significantly more than those in the model group.The ultrastructural injury of the nicotine therapeutic group was also ameliorated.Nicotine has protective effects on theγ-aminobutyric acid neurons and dopaminergic neurons in the MPTP-treated mice.
基金by the Intramural Research Program of National Institute on Aging,National Institutes of Health(AG000959-07 and AG000945-03).
文摘A preferential dysfunction/loss of dopaminergic(DA)neurons in the substantia nigra pars compacta(SNpc)accounts for the main motor symptoms of Parkinson’s disease(PD),the most common degenerative movement disorder.However,the neuronal loss is not stochastic,but rather displays regionally selectivity,indicating the existence of different DA subpopulations in the SNpc.To identify the underlying molecular determinants is thereby instrumental in understanding the pathophysiological mechanisms of PD-related neuron dysfunction/loss and offering new therapeutic targets.Recently,we have demonstrated that aldehyde dehydrogenase 1(ALDH1A1)is one such molecular determinant that defines and protects an SNpc DA neuron subpopulation preferentially affected in PD.In this review,we provide further analysis and discussion on the roles of ALDH1A1 in the function and survival of SNpc DA neurons in both rodent and human brains.We also explore the feasibility of ALDH1A1 as a potential biomarker and therapeutic target for PD.
基金the intramural research program of National Institute on Aging(HC:AG-000928,929).
文摘Background:α–synuclein(α–syn)is the main component of intracytoplasmic inclusions deposited in the brains of patients with Parkinson’s disease(PD)and certain other neurodegenerative disorders.Recent studies have explored the ability ofα–syn to propagate between or across neighboring neurons and supposedly“infect”them with a prion–like mechanism.However,much of this research has used stereotaxic injections of heterologousα–syn fibrils to induce the spreading of inclusions in the rodent brains.Whetherα–syn is able to transmit from the host cells to their neighboring cells in vivo is unclear.Methods:Using immunestaining,we examined the potential propagation ofα–syn into nigrostriatal dopaminergic(DA)neurons in three lines of transgenic mice that overexpress human wild–typeα–syn(hα–syn)in different neuron populations.Results:After testing for three different routes by which hα–syn propagation might occur,we were unable to find any evidence that hα–syn behaved like a prion and could be transmitted overtime into the DA neurons initially lack of hα–syn expression.Conclusions:In transgenic mice hα–syn does not have the ability to propagate at pathologically significant levels between or across neurons.It must be noted that these observations do not disprove the studies that show its prion–like qualities,but rather that propagation is not detectable in transgenic models that do not use any injections of heterologous proteins or viral vectors to induce a spreading state.
基金supported by the National Natural Science Foundation of China,No.31960120Yunnan Science and Technology Talent and Platform Plan,No.202105AC160041(both to ZW).
文摘Parkinson’s disease is typically characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta.Many studies have been performed based on the supplementation of lost dopaminergic neurons to treat Parkinson’s disease.The initial strategy for cell replacement therapy used human fetal ventral midbrain and human embryonic stem cells to treat Parkinson’s disease,which could substantially alleviate the symptoms of Parkinson’s disease in clinical practice.However,ethical issues and tumor formation were limitations of its clinical application.Induced pluripotent stem cells can be acquired without sacrificing human embryos,which eliminates the huge ethical barriers of human stem cell therapy.Another widely considered neuronal regeneration strategy is to directly reprogram fibroblasts and astrocytes into neurons,without the need for intermediate proliferation states,thus avoiding issues of immune rejection and tumor formation.Both induced pluripotent stem cells and direct reprogramming of lineage cells have shown promising results in the treatment of Parkinson’s disease.However,there are also ethical concerns and the risk of tumor formation that need to be addressed.This review highlights the current application status of cell reprogramming in the treatment of Parkinson’s disease,focusing on the use of induced pluripotent stem cells in cell replacement therapy,including preclinical animal models and progress in clinical research.The review also discusses the advancements in direct reprogramming of lineage cells in the treatment of Parkinson’s disease,as well as the controversy surrounding in vivo reprogramming.These findings suggest that cell reprogramming may hold great promise as a potential strategy for treating Parkinson’s disease.
基金supported by the Key Science and Technology Research of Henan Province,No.222102310351(to JW)Luoyang 2022 Medical and Health Guiding Science and Technology Plan Project,No.2022057Y(to JY)Henan Medical Science and Technology Research Program Province-Ministry Co-sponsorship,No.SBGJ202002099(to JY)。
文摘Endoplasmic reticulum stress and mitochondrial dysfunction play important roles in Parkinson s disease,but the regulato ry mechanism remains elusive.Prohibitin-2(PHB2)is a newly discove red autophagy receptor in the mitochondrial inner membrane,and its role in Parkinson’s disease remains unclear.Protein kinase R(PKR)-like endoplasmic reticulum kinase(PERK)is a factor that regulates cell fate during endoplasmic reticulum stress.Parkin is regulated by PERK and is a target of the unfolded protein response.It is unclear whether PERK regulates PHB2-mediated mitophagy thro ugh Parkin.In this study,we established a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced mouse model of Parkinson’s disease.We used adeno-associated virus to knockdown PHB2 expression.Our res ults showed that loss of dopaminergic neurons and motor deficits were aggravated in the MPTP-induced mouse model of Parkinson’s disease.Ove rexpression of PHB2 inhibited these abnormalities.We also established a 1-methyl-4-phenylpyridine(MPP+)-induced SH-SY5Y cell model of Parkinson’s disease.We found that ove rexpression of Parkin increased co-localization of PHB2 and microtubule-associated protein 1 light chain 3,and promoted mitophagy.In addition,MPP+regulated Parkin involvement in PHB2-mediated mitophagy through phosphorylation of PERK.These findings suggest that PHB2 participates in the development of Parkinson’s disease by intera cting with endoplasmic reticulum stress and Parkin.
