Functional integration of grafted neural stem cell-derived dopaminergic neurons monitored by optogenetics in an in vitro Parkinson model

Intrastriatal grafts of stem cell-derived dopamine (DA) neurons induce behavioral recovery in animal models of Parkinson''s disease (PD), but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice were expanded as neurospheres and transplanted into organotypic cultures of wild type mouse striatum. Differentiated GFP-labeled DA neurons in the grafts exhibited mature neuronal properties, including spontaneous firing of action potentials, presence of post-synaptic currents, and functional expression of DA D₂ autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation or inhibition of grafted cells and host neurons using channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), respectively, revealed complex, bi-directional synaptic interactions between grafted cells and host neurons and extensive synaptic connectivity within the graft. Our data demonstrate for the first time using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying behavioral recovery as well as adverse effects following stem cell-based DA cell replacement strategies in PD.     

A subgenomic segment of Theiler's murine encephalomyelitis virus RNA causes demyelination

The DA strain of Theiler's murine encephalomyelitis virus (TMEV) causes a persistent central nervous system (CNS) infection of mice with a restricted virus gene expression and induces an inflammatory demyelinating disease that is thought to be immune mediated and a model of multiple sclerosis (MS). The relative contribution of virus vis-à-vis the immune system in the pathogenesis of DA-induced white matter disease remains unclear, as is also true in MS. To clarify the pathogenesis of DA-induced demyelination, we used Cre/loxP technology to generate a transgenic mouse that has tamoxifen (Tm)-inducible expression of a subgenomic segment of DA RNA in oligodendrocytes and Schwann cells. Tm-treated young transgenic mice developed progressive weakness leading to death, with abnormalities of oligodendrocytes and Schwann cells and demyelination, but without inflammation, demonstrating that DA virus can play a direct pathogenic role in demyelination. Tm treatment of mice at a later age resulted in milder disease, with evidence of peripheral nerve remyelination and focal fur depigmentation; surviving weak mice had persistent expression of the recombined transgene in the CNS, suggesting that the DA subgenomic segment can cause cellular dysfunction but not death, possibly similar to the situation seen during DA virus persistence. These studies demonstrate that DA RNA or a DA protein(s) is toxic to myelin-synthesizing cells. This Cre/loxP transgenic system allows for spatially and temporally controlled expression of the viral transgene and is valuable for clarifying nonimmune (and immune) mechanisms of demyelination induced by TMEV as well as other viruses.     

Apoptotic and antiapoptotic activity of L protein of Theiler's murine encephalomyelitis virus

Cellular apoptosis induced by viral genes can play a critical role in determining virulence as well as viral persistence. This form of cell death has been of interest with respect to Theiler's murine encephalomyelitis virus (TMEV) because the GDVII strain and members of the GDVII subgroup are highly neurovirulent, while the DA strain and members of the TO subgroup induce a chronic progressive inflammatory demyelination with persistence of the virus in the central nervous system. The TMEV L protein has been identified as important in the pathogenesis of Theiler's virus-induced demyelinating disease (TMEV-IDD). We now show that DA L is apoptotic following transfection of L expression constructs or following DA virus infection of HeLa cells; the apoptotic activity depends on the presence of the serine/threonine domain of L, especially a serine at amino acid 57. In contrast, GDVII L has little apoptotic activity following transfection of L expression constructs in HeLa cells and is antiapoptotic following GDVII infection of HeLa cells. Of note, both DA and GDVII L cleave caspase-3 in BHK-21 cells, although neither implements the full apoptotic machinery in this cell type as manifested by the induction of terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. The differences in apoptotic activities of DA and GDVII L in varied cell types may play an important role in TMEV subgroup-specific disease phenotypes.     

Direct and indirect presynaptic control of dopamine release by excitatory amino acids

Summary Dopamine (DA) release from nerve terminals of the nigrostriatal DA neurons not only depends on the activity of nigral DA cells but also on presynaptic regulation. Glutamatergie neurons of cortical origin play a prominent role in these presynaptic regulations. The direct glutamatergic presynaptic control of DA release is mediated by N-methyl-D-aspartate (NMDA) and-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA) receptors, located on DA nerve terminals. In addition, by acting on striatal target cells, these glutamatergic neurons contribute also to indirect regulations of DA release involving several transmitters such as GABA, acetylcholine and neuropeptides. Diffusible messengers such as nitric oxide (NO) or arachidonic acid (AA) which are particularly formed under the stimulation of NMDA receptors may also participate to the regulation of DA release. In the present study, it will be shown that the co-application of NMDA and carbachol synergistically increases the release of [³H]-DA and that this effect is reduced by mepacrine or 4-bromophenacylbromide (10⁷M), two inhibitors of PLA2. Therefore endogenously released AA induced by the co-stimulation of NMDA and cholinergic receptors seems to be involved, at least partly, in the release of DA.     

Tyrosine hydroxylase and Parkinson's disease

A consistent neurochemical abnormality in Parkinson's disease (PD) is degeneration of dopaminergic neurons in substantia nigra, leading to a reduction of striatal dopamine (DA) levels. As tyrosine hydroxylase (TH) catalyses the formation ofl-DOPA, the rate-limiting step in the biosynthesis of DA, the disease can be considered as a TH-deficiency syndrome of the striatum. Similarly, some patients with hereditaryl-DOPA-responsive dystonia, a neurological disorder with clinical similarities to PD, have mutations in the TH gene and decreased TH activity and/or stability. Thus, a logical and efficient treatment strategy for PD is based on correcting or bypassing the enzyme deficiency by treatment withl-DOPA, DA agonists, inhibitors of DA metabolism, or brain grafts with cells expressing TH. A direct pathogenetic role of TH has also been suggested, as the enzyme is a source of reactive oxygen species (ROS) in vitro and a target for radical-mediated oxidative injury. Recently, it has been demonstrated thatl-DOPA is effectively oxidized by mammalian TH in vitro, possibly contributing to the cytotoxic effects of DOPA. This enzyme may therefore be involved in the pathogenesis of PD at several different levels, in addition to being a promising candidate for developing new treatments of this disease.     

Co-Transplantation of GDNF-Overexpressing Neural Stem Cells and Fetal Dopaminergic Neurons Mitigates Motor Symptoms in a Rat Model of Parkinson’s Disease

Striatal transplantation of dopaminergic (DA) neurons or neural stem cells (NSCs) has been reported to improve the symptoms of Parkinson’s disease (PD), but the low rate of cell survival, differentiation, and integration in the host brain limits the therapeutic efficacy. We investigated the therapeutic effects of intracranial co-transplantation of mesencephalic NSCs stably overexpressing human glial-derived neurotrophic factor (GDNF-mNSCs) together with fetal DA neurons in the 6-OHDA rat model of PD. Striatal injection of mNSCs labeled by the contrast enhancer superparamagnetic iron oxide (SPIO) resulted in a hypointense signal in the striatum on T2-weighted magnetic resonance images that lasted for at least 8 weeks post-injection, confirming the long-term survival of injected stem cells in vivo. Co-transplantation of GDNF-mNSCs with fetal DA neurons significantly reduced apomorphine-induced rotation, a behavioral endophenotype of PD, compared to sham-treated controls, rats injected with mNSCs expressing empty vector (control mNSCs) plus fetal DA neurons, or rats injected separately with either control mNSCs, GDNF-mNSCs, or fetal DA neurons. In addition, survival and differentiation of mNSCs into DA neurons was significantly greater following co-transplantation of GDNF-mNSCs plus fetal DA neurons compared to the other treatment groups as indicated by the greater number of cell expressing both the mNSCs lineage tracer enhanced green fluorescent protein (eGFP) and the DA neuron marker tyrosine hydroxylase. The success of cell-based therapies for PD may be greatly improved by co-transplantation of fetal DA neurons with mNSCs genetically modified to overexpress trophic factors such as GDNF that support differentiation into DA cells and their survival in vivo.     

体外诱导人骨髓间充质干细胞向多巴胺神经元分化的研究

通过体外诱导人骨髓间充质干细胞(bone marrow mesenchymal stemcells,BMSCs)向多巴胺(dopamine,DA)神经元分化,探讨人BMSCs来源的DA神经元的功能特征及其分化机制,为临床上细胞移植替代治疗诸如帕金森氏病(parkinson’sdisease,PD)等神经精神性疾病提供一种理想的细胞来源。通过密度梯度离心获取人骨髓中的单个核细胞,贴壁培养纯化BMSCs。50μmol/L脑源性神经营养因子(brain derivedneurotrophy factor,BDNF),10μmol/Lforskolin(FSK)和10μmol/LDA联合对BMSCs进行诱导。电子显微镜观察诱导2周后细胞是否具有神经元的超微结构特点;免疫细胞化学染色和RT-PCR检测DA神经元分化过程中的标志物酪氨酸羟化酶(tyrosine hydroxylase,TH)的表达以及转录因子Nurr1、Ptx3和Lmx1b的表达;高效液相色谱(highperformance liquid chromatogram,HPLC)检测诱导2周后的细胞多巴胺的释放水平。结果表明,诱导2周后,电镜下细胞胞浆中有大量密集的呈扁平囊状的粗面内质网及其间的一些游离核糖体以及神经微丝的形成。RT-PCR结果显示NSE(neuron specificenolase)、Nurr1、Ptx3、Lmx1b和TH的mRNA均有表达;免疫细胞化学染色结果表明诱导2周后TH阳性细胞(24·80±3·36)%的表达较诱导3d后(3·77±1·77)%明显提高(P<0·01);HPLC检测到诱导2周后的细胞DA释放水平[(1·22±0·36)μg/mL(n=6)]高于未经诱导的细胞[(0·75±0·22)μg/mL(n=6)(t=-2·79,P=0·038)]。由此得出,BDNF、FSK和DA可以在体外诱导人BMSCs向DA神经元分化,并具有DA神经元的功能特征,是临床用于治疗神经精神性疾病的理想细胞来源。     

Stem cell potential in Parkinson's disease and molecular factors for the generation of dopamine neurons

Parkinson's disease (PD) involves the loss of dopamine (DA) neurons, making it the most expected neurodegenerative disease to be treated by cell replacement therapy. Stem cells are a promising source for cell replacement therapy due to their ability to self-renew and their pluripotency/multipotency that allows them to generate various types of cells. However, it is challenging to derive midbrain DA neurons from stem cells. Thus, in this review, I will discuss the molecular factors that are known to play critical roles in the generation and survival of DA neurons. The developmental process of DA neurons and functions of extrinsic soluble factors and homeodomain proteins, forkhead box proteins, proneural genes, Nurr1 and genes involved in epigenetic control are discussed. In addition, different types of stem cells that have potential for future cell replacement therapy are reviewed.     

A comparative study of proteasomal inhibition and apoptosis induced in N27 mesencephalic cells by dopamine and MG132

Dopamine (DA) and its metabolites have been implicated in the pathogenesis of Parkinson's disease. DA can produce reactive-oxygen species and DA-derived quinones such as aminochrome can induce proteasomal inhibition. We therefore examined the ability of DA and MG132 to induce apoptosis and proteasomal inhibition in N27 rat dopaminergic cells. DA (0-500 micromol/L, 0-24 h) and MG132 (0-5 micromol/L, 0-24 h) treated N27 cells resulted in time- and concentration-dependent apoptosis. To better define DA and MG132-induced apoptosis, the activation of initiator caspases 2 and caspase 9 and the executioner caspase 3 was investigated. Activation of caspase 2, caspase 9, and caspase 3 occurred early and prior to cell death. In addition, N-acetylcysteine (NAC) blocked DA but not MG132-induced apoptosis and mitochondrial membrane potential loss. NAC can react with both reactive-oxygen and quinoid metabolites and its inhibitory activity suggests a role for reactive species in DA-induced apoptosis. Proteasomal inhibition was detected after DA treatment in N27 cells which occurred prior to cell death and was abrogated by NAC. Our results implicate DA-derived reactive species in proteasomal inhibition and caspase-dependent apoptosis in N27 cells. The ability of endogenous DA-derived metabolites to induce proteasomal inhibition and apoptosis may contribute to the selective loss of dopaminergic neurons in Parkinson's disease.     

Human embryonic stem cell-derived neural precursors as a continuous, stable, and on-demand source for human dopamine neurons

Human embryonic stem (hES) cells can be guided to differentiate into ventral midbrain-type neural precursor (NP) cells that proliferate in vitro by specific mitogens. We investigated the potential of these NP cells derived from hES cells (hES-NP) for the large-scale generation of human dopamine (DA) neurons for functional analyses and therapeutic applications. To address this, hES-NP cells were expanded in vitro for 1.5 months with six passages, and their proliferation and differentiation properties determined over the NP passages. Interestingly, the total hES-NP cell number was increased by > 2 × 10⁴-folds over the in vitro period without alteration of phenotypic gene expression. They also sustained their differentiation capacity toward neuronal cells, exhibiting in vitro pre-synaptic DA neuronal functionality. Furthermore, the hES-NP cells can be cryopreserved without losing their proliferative and developmental potential. Upon transplantation into a Parkinson's disease rat model, the multi-passaged hES-NP cells survived, integrated into the host striatum, and differentiated toward the neuronal cells expressing DA phenotypes. A significant reduction in the amphetamine-induced rotation score of Parkinson's disease rats was observed by the cell transplantation. Taken together, these findings indicate that hES-NP cell expansion is exploitable for a large-scale generation of experimental and transplantable DA neurons of human-origin.     

Intrastriatal grafts of mesenchymal stem cells in adult intact rats can elevate tyrosine hydroxylase expression and dopamine levels

MSCs (mesenchymal stem cells) derived from the bone marrow have shown to be a promising source of stem cells in a therapeutic strategy of neurodegenerative disorder. Also, MSCs can enhance the TH (tyrosine hydroxylase) expression and DA (dopamine) content in catecholaminergic cells by in vitro co‐culture system. In the present study, we investigated the effect of intrastriatal grafts of MSCs on TH protein and gene levels and DA content in adult intact rats. When MSCs were transplanted into the striatum of normal rats, the grafted striatum not only had significantly higher TH protein and mRNA levels, but also significantly higher DA content than the untransplanted striatum. Meanwhile, the grafted MSCs differentiated into neurons, astrocytes and oligodendrocytes; however, TH‐positive cells could not be detected in our study. These experimental results offer further evidence that MSCs are a promising candidate for treating neurodegenerative diseases such as Parkinson's disease.     

Involvement of dopaminergic neuronal cystatin C in neuronal injury-induced microglial activation and neurotoxicity

Factors released from injured dopaminergic (DA) neurons may trigger microglial activation and set in motion a vicious cycle of neuronal injury and inflammation that fuels progressive DA neurodegeneration in Parkinson's disease. In this study, using proteomic and immunoblotting analysis, we detected elevated levels of cystatin C in conditioned media (CM) from 1-methyl-4-phenylpyridinium and dieldrin-injured rat DA neuronal cells. Immunodepletion of cystatin C significantly reduced the ability of DA neuronal CM to induce activation of rat microglial cells as determined by up-regulation of inducible nitric oxide synthase, production of free radicals and release of proinflammatory cytokines as well as activated microglia-mediated DA neurotoxicity. Treatment of the cystatin C-containing CM with enzymes that remove O- and sialic acid-, but not N-linked carbohydrate moieties markedly reduced the ability of the DA neuronal CM to activate microglia. Taken together, these results suggest that DA neuronal cystatin C plays a role in the neuronal injury-induced microglial activation and neurotoxicity. These findings from the rat DA neuron-microglia in vitro model may help guide continued investigation to define the precise role of cystatin C in the complex interplay among neurons and glia in the pathogenesis of Parkinson's disease.     

Development of an In Vitro Model to Evaluate the Regenerative Capacity of Adult Brain-Derived Tyrosine Hydroxylase-Expressing Dopaminergic Neurons

The loss of nigral dopaminergic (DA) neurons is the disease-defining pathological change responsible for progressive motor dysfunction in Parkinson’s disease. In this study, we sought to establish a culture method for adult rat tyrosine hydroxylase (TH)-immunoreactive DA neurons. In this context, we investigated the role of fibroblast growth factor 2 (FGF2), brain-derived neurotrophic factor (BDNF), transforming growth factor-β3 (TGF-β3), glial-derived neurotrophic factor (GDNF) and dibutyryl-cyclic AMP (dbcAMP) in these cultures. Culturing in the presence of FGF2, BDNF and GDNF enhanced the survival of DA neurons by 15-fold and promoted neurite growth. In contrast, dbcAMP promoted neurite growth in all neurons but did not enhance DA cell survival. This study demonstrates that long-term cultures of DA neurons can be established from the mature rat brain and that survival and regeneration of DA neurons can be manipulated by epigenetic factors such as growth factors and intracellular cAMP pathways.     

Conjugates of Catecholamines with Cysteine and GSH in Parkinson's Disease: Possible Mechanisms of Formation Involving Reactive Oxygen Species

Abstract: Oxidation of l -3,4-dihydroxyphenylalanine ( l -DOPA) and dopamine (DA) to generate semiquinones/quinones, oxygen radicals, and other reactive oxygen species may play a role in neuronal cell death in Parkinson's disease (PD). In particular, semiquinones/quinones can form conjugates with thiol compounds such as GSH and cysteine. Exposure of l -DOPA, DA, and other catecholamines to a system generating O₂^•− radical led to O₂^•−-dependent depletion of added GSH (or cysteine), accompanied by the formation of thiol-DA or -DOPA adducts as detected by HPLC. Superoxide could additionally cause destruction of these adducts. Iron or copper ions could also promote conjugate formation between GSH or cysteine and DA and l -DOPA, especially if H₂O₂ was present. We applied HPLC to measure glutathionyl and cysteinyl conjugates of l -DOPA, DA, and 3,4-dihydroxyphenylacetic acid (DOPAC) in postmortem brain samples from PD patients and normal control subjects. Conjugates were detected in most brain areas examined, but levels were highest in the substantia nigra and putamen. In most regions, adduct levels were lower in PD, but there were significant increases in cysteinyl adducts of l -DOPA, DA, and DOPAC in PD substantia nigra, suggesting that acceleration of l -DOPA/DA oxidation occurs in PD, although we cannot say if this is a primary feature of the disease or if it is related to therapy with l -DOPA. In vitro, conjugate formation could be inhibited by the dithiol dihydrolipoate but not by its oxidised form, lipoic acid.     

Approaches to Prevent Dopamine Quinone-Induced Neurotoxicity

Dopamine (DA) and its metabolites containing two hydroxyl residues exert cytotoxicity in dopaminergic neuronal cells, primarily due to the generation of highly reactive DA and DOPA quinones. Quinone formation is closely linked to other representative hypotheses such as mitochondrial dysfunction, inflammation, oxidative stress, and dysfunction of the ubiquitin-proteasome system, in the pathogenesis of neurodegenerative diseases such as Parkinson’s disease and methamphetamine-induced neurotoxicity. Therefore, pathogenic effects of the DA quinone have focused on dopaminergic neuron-specific oxidative stress. Recently, various studies have demonstrated that some intrinsic molecules and several drugs exert protective effects against DA quinone-induced damage of dopaminergic neurons. In this article, we review recent studies on some neuroprotective approaches against DA quinone-induced dysfunction and/or degeneration of dopaminergic neurons. Special issue article in honor of Dr. Akitane Mori.     

Effects of GABA and GABA receptor inhibition on differentiation of mesencephalic precursors into dopaminergic neurons in vitro

Neurotransmitters have been shown to control CNS neurogenesis, and GABA-mediated signaling is thought to be involved in the regulation of nearly all key developmental stages. Generation of dopaminergic (DA) neurons from stem/precursor cells for cell therapy in Parkinson's disease has become a major focus of research. However, the possible effects of GABA on generation of DA neurons from proliferating neurospheres of mesencephalic precursors have not been studied. In the present study, GABA(A), and GABA(B) receptors were found to be located in DA cells. Treatment of cultures with GABA did not cause significant changes in generation of DA cells from precursors. However, treatment with the GABA(A) receptor antagonist bicuculline (10(-5) M) led to a significant increase in the number DA cells, and treatment with the GABA(B) receptor antagonist CGP 55845 (10(-5) M) to a significant decrease. Simultaneous treatment with bicuculline and CGP 55845 did not induce significant changes. Apoptotic cell death studies and bromodeoxyuridine immunohistochemistry indicated that the aforementioned differences in generation of DA neurons are not due to changes in survival or proliferation of DA cells, but rather to increased or decreased differentiation of mesencephalic precursors towards the DA phenotype. The results suggest that these effects are exerted via GABA receptors located on DA precursors, and are not an indirect consequence of effects on the serotonergic or glial cell population. Administration of GABA(A) receptor antagonists in the differentiation medium may help to obtain higher rates of DA neurons for potential use in cell therapy for Parkinson's disease.     

Dopamine modulates mitochondrial function in viable SH-SY5Y cells possibly via its interaction with complex I: relevance to dopamine pathology in schizophrenia

Deleterious effects of dopamine (DA) involving mitochondrial dysfunction have an important role in DA-associated neuronal disorders, including schizophrenia and Parkinson's disease. DA detrimental effects have been attributed to its ability to be auto-oxidized to toxic reactive oxygen species. Since, unlike Parkinson's disease, schizophrenia does not involve neurodegenerative processes, we suggest a novel mechanism by which DA impairs mitochondrial function without affecting cell viability. DA significantly dissipated mitochondrial membrane potential (delta psi m) in SH-SY5Y cells. Bypassing complex I prevented the DA-induced depolarization. Moreover, DA inhibited complex I but not complex II activity in disrupted mitochondria, suggesting complex I participation in DA-induced mitochondrial dysfunction. We further demonstrated that intact mitochondria can accumulate DA in a saturated manner, with an apparent Km=122.1+/-28.6 nM and Vmax=1.41+/-0.15 pmol/mg protein/min, thereby enabling the interaction between DA and complex I. DA accumulation was an energy and Na+-dependent process. The pharmacological profile of mitochondrial DA uptake differed from that of other characterized DA transporters. Finally, relevance to schizophrenia is demonstrated by an abnormal interaction between DA and complex I in schizophrenic patients. These results suggest a non-lethal interaction between DA and mitochondria possibly via complex I, which can better explain DA-related pathological processes observed in non-degenerative disorders, such as schizophrenia.