Multiple factors to assist human-derived induced pluripotent stem cells to efficiently differentiate into midbrain dopaminergic neurons

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.

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.

Differentiation of embryonic versus adult rat neural stem cells into dopaminergic neurons in vitro

BACKGROUND：It has been reported that the conversion of neural stem cells into dopaminergic neurons in vitro can be increased through specific cytokine combinations. Such neural stem cell-derived dopaminergic neurons could be used for the treatment of Parkinson’s disease. However, little is known about the differences in dopaminergic differentiation between neural stem cells derived from adult and embryonic rats. OBJECTIVE： To study the ability of rat adult and embryonic-derived neural stem cells to differentiate into dopaminergic neurons in vitro. DESIGN： Randomized grouping design. SETTING： Department of Neurosurgery in the First Affiliated Hospital of Sun Yat-sen University. MATERIALS： This experiment was performed at the Surgical Laboratory in the First Affiliated Hospital of Sun Yat-sen University （Guangzhou, Guangdong, China） from June to December 2007. Eight, adult, male, Sprague Dawley rats and eight, pregnant, Sprague Dawley rats （embryonic day 14 or 15） were provided by the Experimental Animal Center of Sun Yat-sen University. METHODS： Neural stem cells derived from adult and embryonic rats were respectively cultivated in serum-free culture medium containing epidermal growth factor and basic fibroblast growth factor. After passaging, neural stem cells were differentiated in medium containing interleukin-1α, interleukin-11, human leukemia inhibition factor, and glial cell line-derived neurotrophic factor. Six days later, cells were analyzed by immunocytochemistry and flow cytometry. MAIN OUTCOME MEASURES： Alterations in cellular morphology after differentiation of neural stem cells derived from adult and embryonic rats; and percentage of tyrosine hydroxylase-positive neurons in the differentiated cells. RESULTS： Neural stem cells derived from adult and embryonic rats were cultivated in differentiation medium. Six days later, differentiated cells were immunoreactive for tyrosine hydroxylase. The percentage of tyrosine hydroxylase positive neurons was （5.6 ± 2.8）% and （17.8 ± 4.2）% for adult and embryonic cells, respectively, with a significant difference between the groups （P 〈 0.01）. CONCLUSION： Neural stem cells from embryonic rats have a higher capacity to differentiate into dopaminergic neurons than neural stem cells derived from adult rats.

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.

Ketamine enhances structural plasticity in human dopaminergic neurons:possible relevance for treatment-resistant depression

Depression refers to a series of mental health issues characterized by loss of interest and enjoyment in everyday life,low mood and selected emotional,cognitive,physical and behavioral symptoms.Depression is a common disorder,affecting 5–15%of the general population.When diagnosed as major depressive disorder（MDD）,patients are currentlytreated with pharmacological agents such as serotonin or noradren- aline uptake inhibitors （SSRI or SNRI） or tricyclics.

Intrastriatal glial cell line-derived neurotrophic factors for protecting dopaminergic neurons in the substantia nigra of mice with Parkinson disease

BACKGROUND： Substantia nigra is deep in position and limited in range, the glial cell line-derived neurotrophic factor （GDNF） injection directly into substantia nigra has relatively greater damages with higher difficulty. GDNF injection into striatum, the target area of dopaminergic neuron, may protect the dopaminergic neurons in the compact part of substantia nigra through retrograde transport. OBJECTIVE： To investigate the protective effect of intrastriatal GDNF on dopaminergic neurons in the substantia nigra of mice with Parkinson disease （PD）, and analyze the action pathway. DESIGN： A controlled observation. SETTING： Neurobiological Laboratory of Xuzhou Medical College. MATERIALS： Twenty-four male Kunming mice of 7 - 8 weeks old were used. GDNF, 1-methy1-4-pheny1-1,2,3,6-tetrahydropyridine （MPTP） were purchased from Sigma Company （USA）; LEICAQWin image processing and analytical system. METHODS： The experiments were carded out in the Neurobiological Laboratory of Xuzhou Medical College from September 2005 to October 2006. The PD models were established in adult KunMing mice by intraperitoneal injection of MPTP. The model mice were were randomly divided into four groups with 6 mice in each group： GDNF 4-day group, phosphate buffer solution （PSB） 4-day group, GDNF 6-day group and PSB 6-day group. Mice in the GDNF 4 and 6-day groups were administrated with 1 μ L GDNF solution （20 μ g/L, dispensed with 0.01 mol/L PBS） injected into right striatum at 4 and 6 days after model establishment. Mice in the PSB 4 and 6-day groups were administrated with 0.01 mol/L PBS of the same volume to the same injection at corresponding time points. ② On the 12^th day after model establishment, the midbrain tissue section of each mice was divided into 3 areas from rostral to caudal sides. The positive neurons of tyroxine hydroxylase （TH） and calcium binding protein （CB） with obvious nucleolus and clear outline were randomly selected for the measurement, and the number of positive neurons in unit area was counted. MAIN OUTCOME MEASURES： Number of positive neurons of TH and CB in midbrain substantia nigra of mice in each group. RESULTS： All the 24 mice were involved in the analysis of results. The numbers of TH^＋ and CB^＋ neurons in the GDNF 4-day group （54.33±6.92, 46.33±5.54） were obviously more than those in the PBS 4-day group （27.67±5.01, 21.50±5.96, P 〈 0.01）. The numbers of TH^＋ and CB^＋ neurons in the GDNF 6-day group （75.67±5.39, 69.67±8.69） were obviously more than those in the PBS 6-day group （27.17±4.50, 21.33 ±5.72, P 〈 0.01） and those in the GDNF 4-day group （P 〈 0.01 ）. CONCLUSION： Intrastriatal GDNF can protect dopaminergic neurons in substantia nigra of PD mice, and it may be related to the increase of CB expression.

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.

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.

ATP13A2 protects dopaminergic neurons in Parkinson's disease:from biology to pathology

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.

Mammalian target of rapamycin complex 1 as an inducer of neurotrophic factors in dopaminergic neurons

The defining neuropathological feature of Parkinson＇s disease （PD） is the loss of nigrostriatal dopaminergic （DA） projections. This results in striatal dopamine levels and a biochemical reduction of movement disorders, such as a tremor at rest, rigidity of the limbs, bradykinesia, and postural instability （Kim et al., 2011; Kim et al., 2012; Burke and O＇Malley, 2013; Leem et al., 2014; Namet al., 2014）.

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.

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 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.

Exploring pre-degenerative alterations in humans using induced pluripotent stem cell-derived dopaminergic neurons

Understanding the cellular and molecular mechanisms underlying human neurological disorders is hindered by both the complexity of the disorders and the lack of suitable experimental models recapitulating key pathological features of the disease.This is a crucial issue since a limited understanding of pathogenic mechanisms precludes the development of drugs counteracting the progression of the disease.Among neurological disorders,

Directional induction of dopaminergic neurons from neural stem cells using substantia nigra homogenates and basic fibroblast growth factor

To date, complex components of available reagents have been used for directional induction of neural stem cells into dopaminergic neurons, resulting in a poor ability to repeat experiments. This study sought to investigate whether a homogenate of the substantia nigra of adult rats and/or basic fibroblast growth factor could directionally induce neural stem cells derived from the subventricular zone of embryonic rats to differentiate into dopaminergic neurons. Tyrosine hydroxylase-positive cells were observed exclusively after induction with the homogenate supernatant of the substantia nigra from adult rats and basic fibroblast growth factor for 48 hours in vitro. However, in the groups treated with homogenate supernatant or basic fibroblast growth factor alone, tyrosine hydroxylase expression was not observed. Moreover, the content of dopamine in the culture medium of subventricular zone neurons was significantly increased at 48 hours after induction with the homogenate supernatant of the substantia nigra from adult rats and basic fibroblast growth factor. Experimental findings indicate that the homogenate supernatant of the substantia nigra from adult rats and basic fibroblast growth factor could directionally induce neural stem cells derived from the subventricular zone of embryonic rats to differentiate into dopaminergic neurons in the substantia nigra with the ability to secrete dopamine.

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.

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.

Embryonic stem cell-derived neural progenitors as non-tumorigenic source for dopaminergic neurons

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.

Loss of Hrh2 on dopaminergic neurons leads to mania-like behavior in mice

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.

The study on neuroprotective mechanism of water extract of Fomitopsis Pinicola on mesencephala dopaminergic neurons induced by MPP+

Objective: To observe the neuroprotective mechanism of water extract of Fomito^p^is Pinicola on MPP+ induced apoptosis of mesencephala dopaminergic cells in vitro. Methods: The antioxidant activity of fungi was determined by FRAP method. The anti-inflammatory activity of the fungi was detected by LPS-induced NO release method. Mesencephalic dopaminergic neurons were labeled by TH staining to observe the survival of THir neurons. Results: In the anti-oxidant activity assay, the Trolox equivalent anti-oxidant capacity (TEAC) of water extract of Fomitopsis Pinicola was determined to be ( 165.80±7.13 )μmol Trolox/g extract. Water extracts o f Fomitopsis Pinicola treatment(100, 5 0 ,2 5 , 12.5^g/ml) decreased NO formation significantly. MPP+ induced significant chromatin condensation in the nuclei of mesencephala dopaminergic neurons with nuclear lysis, the mitochondrial membrane potential decreased remarkably, and ROS production increased significantly. Compared with the MPP+ control group, the morphological changes of cell nuclei after apoptosis was reversed by water extract of Fomitopsis Pinicola. Water extract of Fomitopsis Pinicola treatment (50,25,12.5^g/ml) dramatically increased relative mitochondrial membrane potential compared with MPP+ control respectively. Compared with the MPP+ control, water extract of Fomitopsis Pinicola treatment (50, 25^g/ml) significantly decreased relative ROS formation respectively. Conclusions: Water extract of Fomitopsis Pinicola showed significant neuroprotective effect on mesencephala dopaminergic cells induced by MPP+. The water extract of Fomitopsis Pinicola showed antioxidant and anti-inflammatory activities. The mechanism of neuroprotective effect of water extract of Fomitopsis Pinicola may be related to inhibitory on mitochondrial oxidative stress.