The inherent high vulnerability of dopaminergic neurons toward mitochondrial toxins may contribute to the etiology of Parkinson＇s disease

Although the exact mechanism（s）of the degeneration of dopaminergic neurons in Parkinson’s disease（PD）is not well understood,mitochondrial dysfunction is proposed to play a central role.This proposal is strongly strengthened by the findings that compromised mitochondrial functions and/or exposure to mitochondrial toxins such as rotenone,paraquat,or MPTP causes degeneration of the midbrain dopaminergic.

Parkinson's disease and dopaminergic neurons

Gene therapy for Parkinson＇s disease is being explored as an effective strategy to restore and protect the function of neuronal cells in the substantia nigra. Regulation of gene expression is necessary for gene therapy to avoid adverse effects due to excessive synthesis of transgene products. In the present study, to construct a recombinant AAV vector carrying a dopamine synthase gene. The tyrosine hydroxylase gene was inserted using a IoxP fragment that could be regulated by Cre recombinase. The recombinant AAV vector carrying the CreERT2 gene was co-transduced with HEK293 cells and the corpus striatum in a rat model of Parkinson＇s disease, with inducing agent tamoxifen to regulate gene expression. We found that the application of AAV vector-encoded tyrosine hydroxylase gene under the gene regulation system of Cre-ERT2, after tamoxifen treatment, can effectively control the generation of genetically modified products to reduce the production of excessive dopamine in vivo and in vitro. Therefore, this method can increase the safety of gene therapy.

Panax notoginseng saponins influence on transplantation of neural stem cell-derived dopaminergic neurons in a rat model of Parkinson’s disease

BACKGROUND： Dopaminergic neurons differentiated from neural stem cells have been successfully used in the treatment of rat models of Parkinson＇s disease; however, the survival rate of transplanted cells has been low. Most cells die by apoptosis as a result of overloaded intracellular calcium and the formation of oxygen free radicals. OBJECTIVE： To observe whether survival of transplanted cells, transplantation efficacy, and dopaminergic differentiation from neural stem cells is altered by Panax notoginseng saponins （PNS） in a rat model of Parkinson＇s disease. DESIGN, TIME AND SETTING： Cellular and molecular biology experiments with randomized group design. The experiment was performed at the Animal Experimental Center, First Hospital of Sun Yat-sen University from April to October 2007. MATERIALS： Thirty-two adult, healthy, male Sprague Dawley rats, and four healthy Sprague Dawley rat embryos at gestational days 14-15 were selected. The right ventral mesencephalon was injected with 6-hydroxydopamine to establish a model of Parkinson＇s disease. 6-hydroxydopamine and apomorphine were purchased from Sigma, USA. METHODS： Neural stem cells derived from the mesencephalon of embryonic rats were cultivated and passaged in serum-free culture medium. Lesioned animals were randomly divided into four groups （n = 8）： dopaminergic neuron, dopaminergic neuron ＋ PNS, PNS, and control. The dopaminergic neuron group was injected with 3 μL cell suspension containing dopaminergic neurons differentiated from neural stem cells. The dopaminergic neurons ＋ PNS group received 3 μ L dopaminergic cell suspension combined with PNS （250 mg/L）. The PNS group received 3 μL PNS （250 mg/L）, and the control group received 3 μL DMEM/F12 culture medium. MAIN OUTCOME MEASURES： The rats were transcardially perfused with 4% paraformaldehyde at 60 days post-grafting for immunohistochemistry. The rats were intraperitoneally injected with apomorphine （0.5 mg/kg） to induce rotational behavior. RESULTS： Cell counts of tyrosine hydroxylase-positive neurons in the dopaminergic neuron ＋ PNS group were （732±82.6） cells/400-fold field. This was significantly greater than the dopaminergic neuron group [（326 ± 34.8） cells/400-fold field, P 〈 0.01]. Compared to the control group, the rotational asymmetry of rats that received dopaminergic neuron transplants was significantly decreased, beginning at 20 days after operation （P 〈 0.01）. Rotational asymmetry was further reduced between 10-60 days post-surgery in the dopaminergic neuron ＋ PNS group, compared to the dopaminergic neuron group （P 〈 0.01）. CONCLUSION： Panax notoginseng saponins can increase survival and effectiveness of dopaminergic neurons differentiated from neural stem cells for transplantation in a rat model of Parkinson＇s disease.

Stress increases MHC-I expression in dopaminergic neurons and induces autoimmune activation in Parkinson’s disease

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).

Protective effects of a polysaccharide from Spirulina platensis on dopaminergic neurons in an MPT-Pinduced Parkinson's disease model in C57BL/6J mice

The present study aimed to determine whether a polysaccharide obtained fromSpirulina platensis shows protective effects on dopaminergic neurons. A Parkinson’s disease model was established through the intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyr-idine （MPTP） in C57BL/6J mice. Prior to the MPTP injection, some mice were pretreated with intraperitoneal injections of a polysaccharide derived fromSpirulina platensis once daily for 10 days. The results showed that the immunoreactive staining and mRNA expression of the dopa-mine transporter and tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, in the substantia nigra, were signiifcantly increased in mice pretreated with 800 mg/kg of the poly-saccharide compared with those in MPTP-treated mice. The activities of superoxide dismutase and glutathione peroxidase in the serum and midbrain were also increased signiifcantly in mice injected with MPTP after pretreatment with the polysaccharide fromSpirulina platensis. By con-trast, the activity of monoamine oxidase B in serum and midbrain maintained unchanged. These experimental ifndings indicate that the polysaccharide obtained fromSpirulina platensis plays a protective role against the MPTP-induced loss of dopaminergic neurons in C57BL/6J mice, and that the antioxidative properties of this polysaccharide likely underlie its neuroprotective effect.

Brain-derived neurotrophic factor and substantia nigra dopaminergic neurons in Parkinson’s disease

Parkinson＇s disease （PD） is a chronic, progressive neurodegenerative central nervous system disease which occurs in the substantia nigra-corpus striatum system. The main pathological feature of PD is selective dopaminergic neuronal loss with distinctive Lewy bodies in populations of surviving dopaminergic neurons. In the clinical and neuropathological diagnosis of PD, brain-derived neurotrophic factor mRNA expression in the substantia nigra pars compacta is reduced by 70%, and surviving dopaminergic neurons in the PD substantia nigra pars compacta express less brain-derived neurotrophic factor （BDNF） mRNA （20%） than their normal counterparts. In recent years, knowledge surrounding the relationship between neurotrophic factors and PD has increased, and detailed pathogenesis of the role of neurotrophic factors in PD becomes more important.

Thrombin-induced microglial activation contributes to the degeneration of nigral dopaminergic neurons in vivo

Objective To evaluate the role of thrombin-activated microglia in the neurodegeneration of nigral dopaminergic neurons in the rat substantia nigra （SN） in vivo. Methods After stereotaxic thrombin injection into unilateral SN of rats, immunostaining, reverse transcription polymerase chain reaction （RT-PCR） and biochemical methods were used to observe tyrosine hydroxylase （TH） irnmunoreactive positive cells, microglia activation, nitric oxide （NO） amount and inducible nitricoxide synthase （iNOS） expression. Results （1） Selective damage to dopaminergic neurons was produced after thrombin injection, which was evidenced by loss of TH imrnunostaining in time-dependent manner; （2） Strong microglial activation was observed in the SN; （3） RT-PCR demonstrated the early and transient expression of neurotoxic factors iNOS mRNA in the SN. Immunofluorescence results found that thrombin induced expression of iNOS in microglia. The NO production in the thrombininjected rats was significantly higher than that of controls （P 〈 0.05）. Conclusion Thrombin intranigral injection can injure the dopaminergic neurons in the SN. Thrombin-induced microglia activation precedes dopaminergic neuron degeneration, which suggest that activation of microglia and release of NO may play important roles in dopaminergic neuronal death in the SN.

Protective effects of nicotine on gamma-aminobutyricacid neurons and dopaminergic neurons in micewith Parkinson disease

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.

Effect of rifampicin pre-and post-treatment on rotenone-induced dopaminergic neuronal apoptosis and alpha-synuclein expression

BACKGROUND： Rifampicin inhibits the formation of a-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 SE＇B＇ING： 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 3^rd 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 expression 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.

Astrocytes protect dopaminergic neurons against aminochrome neurotoxicity

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.

Ginsenoside Rb1 protects dopaminergic neurons from inflammatory injury induced by intranigral lipopolysaccharide injection

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).

Nigral dopaminergic neuron replenishment in adult mice through VE-cadherin-expressing neural progenitor cells

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 CREERT2：TIlox/Tlox 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.

C.elegans as a model system for Parkinson disease

Parkinson disease(PD) is characterized by the selective loss of dopaminergic neurons in the substantia nigra. Although investigation in mammalian animal models of PD has enhanced our understanding of PD, the complexity of the mammalian nervous system and our inability to visualize DA neurons in vivo restricts the advances in elucidating the molecular mechanisms of PD. Conservation between C. elegans and mammals in genomic, biosynthetic and metabolic pathways as well as the advantages of observing DA neurons morphology in vivo and the ease of transgenic and genetic manipulation make C. elegans an excellent model organism for PD.

Tyrosine hydroxylase-positive cells and dopaminergic neuronal function in human embryonic stem cells: An electrophysiological validation

BACKGROUND： Induced differentiation strategies and cytochemical properties of human embryonic stem ceils （hESCs） have been investigated. However, the electrophysiological functions of tyrosine hydroxylase （TH）-positive cells dedved 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, nestino Cell karyotype analysis demonstrated that all of the hESCs had a stable and normal karyotype （46, XX） after differentiation. 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/pE 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.

LPS0induced Degeneration of Dopaminergic Neurons of Substantia Nigra in Rats

In order to investigate the neurotoxicity of lipopolysaccharide (LPS) on the dopaminergic neurons of substantia nigra and the pathogenesis of Parkinson disease, LPS was stereotaxically infused into substantia nigra (SN). At different dosages and different time points with 5 μg LPS, the damage of the dopaminergic neurons in SN was observed by using tyrosine hydroxylase (TH) immunohistochemical staining. The results showed that 14 days after injection of 0.1 μg to 10 μg LPS into the rat SN, TH positive (TH + ) neurons in the SN were decreased by 5 %, 15 %, 20 %, 45 %, 96 % and 99 % respectively. After injection of 5 μg LPS, as compared with the control groups, TH + neurons began to decrease at 3rd day and obviously decrease at 14 th day, only 5 % of total cells, and almost disappeared 30 days later. The results suggested that LPS could induce the degeneration of dopaminergic neurons in the SN in a dose and time dependent manner.

Protective effect of alpha-synuclein knockdown on methamphetamine-induced neurotoxicity in dopaminergic neurons

The over-expression of α-synuclein is a major factor in the death of dopaminergic neurons in a methamphetamine-induced model of Parkinson’s disease. In the present study, α-synuclein knockdown rats were created by injecting α-synuclein-shRNA lentivirus stereotaxically into the right striatum of experimental rats. At 2 weeks post-injection, the rats were injected intraper-itoneally with methamphetamine to establish the model of Parkinson’s disease. Expression of α-synuclein mRNA and protein in the right striatum of the injected rats was significantly down-regulated. Food intake and body weight were greater in α-synuclein knockdown rats, and water intake and stereotyped behavior score were lower than in model rats. Striatal dopamine and tyrosine hydroxylase levels were significantly elevated in α-synuclein knockdown rats. Moreover, superoxide dismutase activity was greater in α-synuclein knockdown rat striatum, but the levels of reactive oxygen species, malondialdehyde, nitric oxide synthase and nitrogen monoxide were lower compared with model rats. We also found that α-synuclein knockdown inhibited metham-phetamine-induced neuronal apoptosis. These results suggest that α-synuclein has the capacity to reverse methamphetamine-induced apoptosis of dopaminergic neurons in the rat striatum by inhibiting oxidative stress and improving dopaminergic system function.

Protective effect of bone marrow-derived mesenchymal stem cells on dopaminergic neurons against 1-methyl-4-phenylpyridinium ion-induced neurotoxicity in rat brain slices

BACKGROUND： To date, the use of bone marrow-derived mesenchymal stem cells （MSCs） for the treatment of Parkinson’s disease have solely focused on in vivo animal models. Because of the number of influencing factors, it has been difficult to determine a consistent outcome. OBJECTIVE： To establish an injury model in brain slices of substantia nigra and striatum using 1-methyl-4-phenylpytidinium ion （MPP＋）, and to investigate the effect of MSCs on dopaminergic neurons following MPP＋ induced damage. DESIGN, TIME AND SETTING： An in vitro, randomized, controlled, animal experiment using I mmunohistochemistry was performed at the Laboratory of the Department of Anatomy, Fudan University between January 2004 and December 2006. MATERIALS： Primary MSC cultures were obtained from femurs and tibias of adult Sprague Dawley rats. Organotypic brain slices were isolated from substantia nigra and striatum of 1-day-old Sprague Dawley rat pups. Monoclonal antibodies for tyrosine hydroxylase （TH, 1：5 000） were from Santa Cruz （USA）; goat anti-rabbit IgG antibodies labeled with FITC were from Boster Company （China）. METHODS： Organotypic brain slices were cultured for 5 days in whole culture medium supplemented with 50% DMEM, 25% equine serum, and 25% Tyrode’s balanced salt solution. The medium was supplemented with 5 μg/mL Ara-C, and the culture was continued for an additional 5 days. The undergrowth of brain slices was discarded at day 10. Eugonic brain slices were cultured with basal media for an additional 7 days. The brain slices were divided into three groups： control, MPP＋ exposure, and co-culture. For the MPP＋ group, MPP＋ （30 μmol/L） was added to the media at day 17 and brain slices were cultured for 4 days, followed by control media. For the co-culture group, the MPP＋ injured brain slices were placed over MSCs in the well and were further cultured for 7 days. MAIN OUTCOME MEASURES： After 28 days in culture, neurite outgrowth was examined in the brain slices under phase-contrast microscopy. The percent of area containing dead cells in each brain slice was calculated with the help of propidium iodide fluorescence. Brain slices were stained with antibodies for TH to indicate the presence of dopaminergic neurons. Transmission electron microscopy was applied to determine the effect of MSCs on neuronal ultrastructure. RESULTS： Massive cell death and neurite breakage was observed in the MPP＋ group. In addition, TH expression was significantly reduced, compared to the control group （P 〈 0.01）. After 7 days in culture with MSCs, the co-culture group presented with less cell damage and reduced neurite breakage, and TH expression was increased. However, these changes were not significantly different from the MPP＋ group （P 〈 0.01）. Electron microscopy revealed reduced ultrastructural injury to cells in the brain slices. However, vacuoles were present in cells, with some autophagic vacuoles. CONCLUSION： Bone marrow-derived MSCs can promote survival of dopaminergic neurons following MPP＋-induced neurotoxicity in co-cultures with substantia nigra and striatum brain slices.

Lineage restriction of adult human olfactory-derived progenitors to dopaminergic neurons

Human adult olfactory epithelium contains neural progenitors (hONPs) which replace damaged cellular components throughout life. Methods to isolate and expand the hONPs which form neurospheres in vitro have been developed in our laboratory. In response to morphogens, the hONPs differentiate along several neural lineages. This study optimized conditions for the differentiation of hONPs towards dopaminergic neurons. The hONPs were treated with Sonic hedgehog (Shh), in the presence or absence of retinoic acid (RA) and/or forskolin (FN). Transcription factors (nurr1, pitx3 and lmx1a) that promote embryonic mouse or chicken dopaminergic development were employed to determine if they would modulate lineage restriction of these adult human progenitors. Four expression vectors (pIRES-pitx3-nurr1, pLN-CX2-pitx3, pLNCX2-nurr1 and pLNCX2-lmx1a) were transfected into the hONPs, respectively. Transcription factor expression and the rate-limiting enzyme in dopamine synthesis tyrosine hydroxylase (TH) were detected in the transfected cells after 4 month-selection with G418, indicating transfected hONPs were stably restricted towards a dopaminergic lineage. Furthermore, a dopamine enzyme immunoassay (EIA) was employed to detect the synthesis and release of dopamine. The most efficient transfection paradigm was determined. Several neurotrophic factors were detected in the pre-transfected hONPs which have potential roles in the maintenance, survival and proliferation of dopaminergic neurons. Therefore the effect of transfection on the neurotrophin synthesis was examined. Transfection did not alter synthesis. The use of olfactory progenitors as a cell-based therapy for Parkinson’s disease (PD) would allow harvest without invasive surgery, provide an autologous cell population, eliminate need for immunosuppression and avoid the ethical concerns associated with embryonic tissues. This study suggests that specific transcription factors and treatment with morphogens can restrict human adult olfactory-derived progenitors to a dopaminergic neuronal lineage. Future studies will evaluate the utility of these unique cells in cell-replacement paradigms for the treatment of PD like animal models.

Genetic analysis of transcription factors in dopaminergic neuronal development in Parkinson’s disease

Background:Genetic variants of dopaminergic transcription factor-encoding genes are suggested to be Parkinson’s disease(PD)risk factors;however,no comprehensive analyses of these genes in patients with PD have been undertaken.Therefore,we aimed to genetically analyze 16 dopaminergic transcription factor genes in Chinese patients with PD.Methods:Whole-exome sequencing(WES)was performed using a Chinese cohort comprising 1917 unrelated patients with familial or sporadic early-onset PD and 1652 controls.Additionally,whole-genome sequencing(WGS)was performed using another Chinese cohort comprising 1962 unrelated patients with sporadic late-onset PD and 1279 controls.Results:We detected 308 rare and 208 rare protein-altering variants in the WES and WGS cohorts,respectively.Gene-based association analyses of rare variants suggested that MSX1 is enriched in sporadic late-onset PD.However,the significance did not pass the Bonferroni correction.Meanwhile,72 and 1730 common variants were found in the WES and WGS cohorts,respectively.Unfortunately,single-variant logistic association analyses did not identify significant associations between common variants and PD.Conclusions:Variants of 16 typical dopaminergic transcription factors might not be major genetic risk factors for PD in Chinese patients.However,we highlight the complexity of PD and the need for extensive research elucidating its etiology.

Modulation of the activity of dopaminergic neurons by SK channels:a potential target for the treatment of Parkinson's disease?

SK channels are small conductance calcium-activated potassium channels that are widely expressed in different neurons with distinct subtypes.They play an important role in modulating synaptic plasticity,dopaminergic neurotransmission, and learning and memory.The present review was mainly focused on the recent findings on the contradictory roles of SK channels in modulating dopaminergic neurons in substantia nigra and in the pathogenesis of Parkinson＇s disease （PD） . Besides,whether modulation of SK channels could be a potential target for PD treatment was also discussed.