Guanosine improves motor behavior, reduces apoptosis, and stimulates neurogenesis in rats with parkinsonism

Parkinson's disease (PD) is characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) caused by an abnormal rate of apoptosis. Endogenous stem cells in the adult mammalian brain indicate an innate potential for regeneration and possible resource for neuroregeneration in PD. We previously showed that guanosine prevents apoptosis even when administered 48 hr after the toxin 1-methyl-4-phenylpyridinium (MPP(+)). Here, we induced parkinsonism in rats with a proteasome inhibitor. Guanosine treatment reduced apoptosis, increased tyrosine hydroxylase-positive dopaminergic neurons and expression of tyrosine hydroxylase in the SNc, increased cellular proliferation in the SNc and subventricular zone, and ameliorated symptoms. Proliferating cells in the subventricular zone were nestin-positive adult neural progenitor/stem cells. Fibroblast growth factor-2-expressing cells were also increased by guanosine. Thus, guanosine protected cells from apoptosis and stimulated "intrinsic" adult progenitor/stem cells to become dopaminergic neurons in rats with proteasome inhibitor-induced PD. The cellular/molecular mechanisms underlying these effects may open new avenues for development of novel therapeutics for PD.     

Mesencephalic neural stem (progenitor) cells develop to dopaminergic neurons more strongly in dopamine-depleted striatum than in intact striatum

Epidermal growth factor (EGF)/fibroblast growth factor (FGF)-responsive stem (progenitor) cells from embryonic brain have self-renewing and multipotent properties and thus are good candidates for donor cells in neural transplantation. However, the survival and differentiation to mature neurons after grafting of stem cells into adult brain are rather poor. We hypothesize that the differentiation of stem cells to mature neurons, such as dopaminergic (DAergic) neurons, is dependent on environmental cues that control the ontogenic development. We compared the survival and differentiation between mesencephalic (MS) and cortical (CTx) stem (progenitor) cells, following grafting into bilateral striata of hemiparkinsonian model rats. MS and CTx stem cells were prepared from E12 rats and proliferated in serum-free medium with EGF or basic FGF for 2 weeks. One day after being primed to differentiate, the cell suspensions of both origins were grafted into the bilateral striata of adult rats that had unilateral 6-OHDA lesions in the substantia nigra. MS cells differentiated to tyrosine hydroxylase (TH)-positive neurons more strongly in DA-depleted striatum than in intact striatum, and methamphetamine-induced rotation was ameliorated in half of the grafted animals. Rosette-like cell aggregation and dysfunction of the blood-brain barrier (BBB) were less in and around the grafts in DA-depleted striatum, suggesting less proliferation and more differentiation of MS stem cells in DA-depleted striatum. Neither TH-positive neurons nor behavioral amelioration were detected following CTx stem (progenitor) cell transplantation in the striata. Data suggest that the DA-depleted striatum offers a suitable environment for MS stem (progenitor) cells to differentiate into mature DAergic neurons.     

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 differen...     

Distribution and localization of fibroblast growth factor-8 in rat brain and nerve cells during neural stem/progenitor cell differentiation

The present study explored the distribution and localization of fibroblast growth factor-8 and its potential receptor,fibroblast growth factor receptor-3,in adult rat brain in vivo and in nerve cells during differentiation of neural stem/progenitor cells in vitro.Immunohistochemistry was used to examine the distribution of fibroblast growth factor-8 in adult rat brain in vivo.Localization of fibroblast growth factor-8 and fibroblast growth factor receptor-3 in cells during neural stem/progenitor cell differentiation in vitro was detected by immunofluorescence.Flow cytometry and immunofluorescence were used to evaluate the effect of an anti-fibroblast growth factor-8 antibody on neural stem/progenitor cell differentiation and expansion in vitro.Results from this study confirmed that fibroblast growth factor-8 was mainly distributed in adult midbrain,namely the substantia nigra,compact part,dorsal tier,substantia nigra and reticular part,but was not detected in the forebrain comprising the caudate putamen and striatum.Unusual results were obtained in retrosplenial locations of adult rat brain.We found that fibroblast growth factor-8 and fibroblast growth factor receptor-3 were distributed on the cell membrane and in the cytoplasm of nerve cells using immunohistochemistry and immunofluorescence analyses.We considered that the distribution of fibroblast growth factor-8 and fibroblast growth factor receptor-3 in neural cells corresponded to the characteristics of fibroblast growth factor-8,a secretory factor.Addition of an anti-fibroblast growth factor-8 antibody to cultures significantly affected the rate of expansion and differentiation of neural stem/progenitor cells.In contrast,addition of recombinant fibroblast growth factor-8 to differentiation medium promoted neural stem/progenitor cell differentiation and increased the final yields of dopaminergic neurons and total neurons.Our study may help delineate the important roles of fibroblast growth factor-8 in brain activities and neural stem/progenitor cell differentiation.     

Peptide hormone exendin-4 stimulates subventricular zone neurogenesis in the adult rodent brain and induces recovery in an animal model of Parkinson's disease

We investigated the effects of exendin-4 on neural stem/progenitor cells in the subventricular zone of the adult rodent brain and its functional effects in an animal model of Parkinson's disease. Our results showed expression of GLP-1 receptor mRNA or protein in the subventricular zone and cultured neural stem/progenitor cells isolated from this region. In vitro, exendin-4 increased the number of neural stem/progenitor cells, and the number of cells expressing the neuronal markers microtubule-associated protein 2, β-III-tubulin, and neuron-specific enolase. When exendin-4 was given intraperitoneally to naïve rodents together with bromodeoxyuridine, a marker for DNA synthesis, both the number of bromodeoxyuridine-positive cells and the number of neuronal precursor cells expressing doublecortin were increased. Exendin-4 was tested in the 6-hydroxydopamine model of Parkinson's disease to investigate its possible functional effects in an animal model with neuronal loss. After unilateral lesion and a 5-week stabilization period, the rats were treated for 3 weeks with exendin-4. We found a reduction of amphetamine-induced rotations in animals receiving exendin-4 that persisted for several weeks after drug administration had been terminated. Histological analysis showed that exendin-4 significantly increased the number of both tyrosine hydroxylase- and vesicular monoamine transporter 2-positive neurons in the substantia nigra. In conclusion, our results show that exendin-4 is able to promote adult neurogenesis in vitro and in vivo, normalize dopamine imbalance, and increase the number of cells positive for markers of dopaminergic neurons in the substantia nigra in a model of Parkinson's disease. © 2007 Wiley-Liss, Inc.     

Dopaminergic nigrostriatal projections regulate neural precursor proliferation in the adult mouse subventricular zone

An understanding of the regulators of neurogenesis in the normal and diseased brain is necessary in order to recruit endogenously produced neural precursors for cell replacement in neurodegenerative disorders such as Parkinson's disease. The location of dopaminergic projections from the midbrain to the neostriatum and nucleus accumbens overlaps with the most active region of neurogenesis in the adult brain, the subventricular zone of the anterior lateral ventricle. This suggests that dopamine may contribute to regulation of the subventricular niche of adult neurogenesis. Here, we show in adult mice that destruction of the dopaminergic neurons in the substantia nigra and ventral tegmental area in a 6-hydroxydopamine model of Parkinson's disease reduced the number of proliferating neural precursors in the subventricular zone of the anterior lateral ventricle by approximately 40%. The effect on neural precursor proliferation correlated with the extent of dopaminergic denervation in the neighboring neostriatum. This identifies dopamine as one of the few known endogenous regulators of adult neurogenesis with implications for the potential use of endogenous neural precursors in cell replacement strategies for Parkinson's disease.     

X-box-binding protein 1-modified neural stem cells for treatment of Parkinson’s disease

X-box-binding protein 1-transfected neural stem cells were transplanted into the right lateral ventricles of rats with rotenone-induced Parkinson’s disease. The survival capacities and differentiation rates of cells expressing the dopaminergic marker tyrosine hydroxylase were higher in X-box-binding protein 1-transfected neural stem cells compared to non-transfected cells. Moreover, dopamine and 3,4-dihydroxyphenylacetic acid levels in the substantia nigra were significantly increased, α-synuclein expression was decreased, and neurological behaviors were significantly ameliorated in rats following transplantation of X-box-binding protein 1-transfected neural stem cells. These results indicate that transplantation of X-box-binding protein 1-transfected neural stem cells can promote stem cell survival and differentiation into dopaminergic neurons, increase dopamine and 3,4-dihydroxyphenylacetic acid levels, reduce α-synuclein aggregation in the substantia nigra, and improve the symptoms of Parkinson’s disease in rats.     

Glial cell line‐derived neurotrophic factor enhances in vitro differentiation of mid‐/hindbrain neural progenitor cells to dopaminergic‐like neurons

Parkinson's disease (PD) affects the motor system through the degeneration of the dopaminergic neurons of the substantia nigra. The use of human embryonic stem cell (hESC)‐derived human neural progenitor (hNP) cells provides a potential cell source for cell therapies and drug screens for future treatments. Glial cell line‐derived neurotrophic factor (GDNF) is a known dopaminergic neuroprotectant agent; however, its potential role in neural differentiation remains largely unknown. Addition of 25 ng/ml GDNF to hNP cell differentiation media, over a 21‐day period, induced a significantly (P < 0.05) greater portion of hNP cells to differentiate into dopaminergic neurons than non‐GDNF cultures, 50% compared with 2.9% of cells expressing tyrosine hydroxylase (TH), respectively. The hNP cells exposed to GDNF selectively expressed dopamine receptors 1, 4, and 5 and were evoked to release dopamine with KCl. This is the first report of GDNF and leukemia inhibitory factor enriching hESC‐derived hNP cells toward dopaminergic‐like neurons. © 2010 Wiley‐Liss, Inc.     

Characterization of a monoclonal antibody, Namu mAb, which reacts to the subventricular zone in mouse brain

The lateral ventricle in adult mammalian brain is widely acknowledged as one of the areas that undifferentiated neural cells such as neural stem cells and neural progenitor cells inhabit. However, immunological aspects of neural stem cells in the lateral ventricle are still under debate. Here, we report the generation and characterization of a novel monoclonal antibody (mAb), called Namu mAb, which stains the subventricular zone in the lateral ventricle of adult mouse brain. Namu mAb reacted to the cells in the subventricular zone and never reacted to differentiated neural cells such as neurons and glial cells such as astrocytes and oligodendrocytes. Its reaction pattern for the subventricular zone and the neurospheres was similar to that of Nestin and glial fibrillary acidic protein mAbs. Namu mAb recognition molecule, Namu antigen, was a 50 kDa protein present in the cytoplasmic fraction of mouse brain, and its expression was clearly observed in neurospheres cultured in the presence of epidermal growth factor, but it was never or only weakly induced in the presence of basic fibroblast growth factor or leukemia inhibitory factor. Collectively, it is concluded that Namu mAb specifically reacts to undifferentiated neural cells in mouse brain.     

Therapeutic potentials of human embryonic stem cells in Parkinson’s disease

The loss of dopaminergic neurons of the substantia nigra is the pathological hallmark characteristic of Parkinson’s disease (PD). The strategy of replacing these degenerating neurons with other cells that produce dopamine has been the main approach in the cell transplantation field for PD research. The isolation, differentiation, and long-term cultivation of human embryonic stem cells and the therapeutic research discovery made in relation to the beneficial properties of neurotrophic and neural growth factors has advanced the transplantation field beyond dopamine-producing cells. The present review addresses recent advances in human embryonic stem cell experimentation in relation to treating PD, as well as cell transplantation techniques in conjunction with alternative therapeutics.     

Self-renewal and differentiation of reactive astrocyte-derived neural stem/progenitor cells isolated from the cortical peri-infarct area after stroke

In response to stroke, subpopulations of cortical reactive astrocytes proliferate and express proteins commonly associated with neural stem/progenitor cells such as glial fibrillary acidic protein (GFAP) and Nestin. To examine the stem cell-related properties of cortical reactive astrocytes after injury, we generated GFAP-CreER(TM);tdRFP mice to permanently label reactive astrocytes. We isolated cells from the cortical peri-infarct area 3 d after stroke, and cultured them in neural stem cell medium containing epidermal growth factor and basic fibroblast growth factor. We observed tdRFP-positive neural spheres in culture, suggestive of tdRFP-positive reactive astrocyte-derived neural stem/progenitor cells (Rad-NSCs). Cultured Rad-NSCs self-renewed and differentiated into neurons, astrocytes, and oligodendrocytes. Pharmacological inhibition and conditional knock-out mouse studies showed that Presenilin 1 and Notch 1 controlled neural sphere formation by Rad-NSCs after stroke. To examine the self-renewal and differentiation potential of Rad-NSCs in vivo, Rad-NSCs were transplanted into embryonic, neonatal, and adult mouse brains. Transplanted Rad-NSCs were observed to persist in the subventricular zone and secondary Rad-NSCs were isolated from the host brain 28 d after transplantation. In contrast with neurogenic postnatal day 4 NSCs and adult NSCs from the subventricular zone, transplanted Rad-NSCs differentiated into astrocytes and oligodendrocytes, but not neurons, demonstrating that Rad-NSCs had restricted differentiation in vivo. Our results indicate that Rad-NSCs are unlikely to be suitable for neuronal replacement in the absence of genetic or epigenetic modification.     

Brain-derived neurotrophic factor-transduced fibroblasts: Production of BDNF and effects of grafting to the adult rat brain

Local delivery of brain-derived neurotrophic factor (BDNF) by genetically modified cells provides the unique opportunity to examine the effects of BDNF on adult dopaminergic and cholinergic neurons in vivo. Primary rat fibroblasts were genetically engineered to produce BDNF. Conditioned media from BDNF-transduced fibroblasts supported embryonic chick dorsal root ganglion neurons as well as rat fetal mesencephalic neurons. BDNF-transduced fibroblasts grafted to the rat brain survived and showed continued mRNA production for at least 2 weeks. The effects of BDNF-transduced fibroblast grafts on the dopaminergic and cholinergic systems were then assessed. BDNF-transduced fibroblasts grafted into the normal intact substantia nigra induced sprouting of tyrosine hydroxylase- and neurofilamentimmunoreactive fibers into the graft. Fibroblast grafts implanted into the normal intact striatum and midbrain as well as the 6-hydroxydopamine-lesioned brain did not induce sprouting of dopaminergic fibers; neither did they affect drug-induced rotational behavior. BDNF-transduced fibroblasts did, however, significantly increase the homovanillic acid/dopamine ratio when grafted into the normal midbrain. Following transection of the fimbriafornix, BDNF-transduced fibroblasts grafted into the septum were unable to rescue the septal cholinergic population, as did nerve growth factor-producing fibroblast grafts. Genetically modified fibroblast grafts may provide an effective, localized method of BDNF delivery in vivo to test biological effects of this factor on the central nervous system. © 1995 Wiley-Liss, Inc.     

Basic FGF is present in dopaminergic neurons of the ventral midbrain of the rat.

Immunocytochemistry procedures and 6-hydroxy-dopamine-induced degeneration of dopamine nerve cells provided evidence that practically all tyrosine hydroxylase immunoreactive (IR) neurons of the substantia nigra and ventral tegmental area contain cytoplasmic basic fibroblast growth factor immunoreactivity (bFGF IR), while many astroglial cells in the neostriatum and substantia nigra contain nuclear bFGF IR. RNA blot analysis demonstrated strong expression of a major 3.7 kb bFGF mRNA in the substantia nigra and ventral tegmental area. These results indicate that bFGF may be a significant growth factor in the DA neurons.     

Dopaminergic neurons in rat ventral midbrain express brain-derived neurotrophic factor and neurotrophin-3 mRNAs

Studies of the trophic activities of brain-derived neurotrophic factor and neurotrophin-3 indicate that both molecules support the survival of a number of different embryonic cell types in culture. We have shown that mRNAs for brain-derived neurotrophic factor and neurotrophin-3 are localized to specific ventral mesencephalic regions containing dopaminergic cell bodies, including the substantia nigra and ventral tegmental area. In the present study, in situ hybridization with ³⁵S-labeled cRNA probes for the neurotrophin mRNAs was combined with neurotoxin lesions or with immunocytochemistry for the catecholamine-synthesizing enzyme tyrosine hydroxylase to determine whether the dopaminergic neurons, themselves, synthesize the neurotrophins in adult rat midbrain. Following unilateral destruction of the midbrain dopamine cells with 6-hydroxydopamine, a substantial, but incomplete, depletion of brain-derived neurotrophic factor and neurotrophin-3 mRNA-containing cells was observed in the ipsilateral substantia nigra pars compacta and ventral tegmental area. In other rats, combined in situ hybridization and tyrosine hydroxylase immunocytochemistry demonstrated that the vast majority of the neurotrophin mRNA-containing neurons in the substantia nigra and ventral tegmental area were tyrosine hydroxylase immunoreactive. Of the total population of tyrosine hydroxylase-positive cells, double-labeled neurons constituted 25–50% in the ventral tegmental area and 10–30% in the substantia nigra pars compacta, with the proportion being greater in medial pars compacta. In addition, tyrosine hydroxylase/neurotrophin mRNA coexistence was observed in neurons in other mesencephalic regions including the retrorubral field, interfascicular nucleus, rostral and central linear nuclei, dorsal raphe nucleus, and supramammillary region. The present results demonstrate brain-derived neurotrophic factor and neurotrophin-3 expression by adult midbrain dopamine neurons and support the suggestion that these neurotrophins influence dopamine neurons via autocrine or paracrine mechanisms. These data raise the additional possibility that inappropriate expression of the neurotrophins by dopaminergic neurons could contribute to the neuropathology of disease states such as Parkinson's disease and schizophrenia. © 1994 Wiley-Liss, Inc.     

Neural stem cells: isolation and differentiation into cholinergic neurons

This investigation aimed to isolate neural stem cells from neonatal hippocampus and induce them to differentiate into cholinergic neurons. The isolated neural stem cells were incubated in serum-free Dulbecco's modified Eagle medium/F12 medium added with 20 ng/ml basic fibroblast growth factor and B27. The cell line isolated from the hippocampal formation of neonatal rats expressed nestin and had the potency to form clones and differentiate into neurons, astrocytes and oligodendrocytes. Embryonic chick skeletal muscle extract was used to induce the differentiation of the neural stem cells into cholinergic neurons. Immunocytochemistry was used to detect the choline acetyltransferase antigen of cholinergic neurons for confirmation. The results showed that embryonic chick skeletal muscle extract could induce isolated neural stem cell to differentiate into a significantly larger number of cholinergic neurons than controls.     

Anatomical and functional reconstruction of the nigrostriatal pathway by intranigral transplants

The main transplantation strategy in Parkinson's disease has been to place dopaminergic grafts not in their ontogenic site, the substantia nigra, but in their target area, the striatum with contrasting results. Here we have used green fluorescent protein transgenic mouse embryos as donors of ventral mesencephalic cells for transplantation into the pre-lesioned substantia nigra of an adult wild-type host. This allows distinguishing the transplanted cells and their projections from those of the host. Grafted cells integrated within the host mesencephalon and expressed the dopaminergic markers tyrosine hydroxylase, vesicular monoamine transporter 2 and dopamine transporter. Most of the dopaminergic cells within the transplant expressed the substantia nigra marker Girk2 while a lesser proportion expressed the ventral tegmental area marker calbindin. Mesencephalic transplants developed projections through the medial forebrain bundle to the striatum, increased striatal dopamine levels and restored normal behavior. Interestingly, only mesencephalic transplants were able to restore the nigrostriatal projections as dopamine neurons originating from embryonic olfactory bulb transplants send projections only in the close vicinity of the transplantation site that did not reach the striatum. Our results show for the first time the ability of intranigral foetal dopaminergic neurons grafts to restore the damaged nigrostriatal pathway in adult mice. Together with our previous findings of efficient embryonic transplantation within the pre-lesioned adult motor cortex, these results demonstrate that the adult brain is permissive to specific and long distance axonal growth. They further open new avenues in cell transplantation therapies applied for the treatment of neurodegenerative disorders such as Parkinson's disease.     

Rapid and efficient differentiation of dopaminergic neurons from mouse embryonic stem cells

We have developed a fast and effective method for the differentiation of dopaminergic neurons from mouse embryonic stem cells. Neuronal precursors are obtained by formation of embryonic bodies or neural stem spheres via free-floating culture in the presence of the mitogens basic fibroblast growth factor and epidermal growth factor together with L-ascorbic acid. Subsequent culturing of the precursor cells in medium containing epidermal growth factor, FGF8b, SHH and ascorbic acid induces cell proliferation, following withdrawal of the growth factors leads differentiation into predominantly dopaminergic neurons. Mature neurons are obtained within 10 days of replacing the proliferation to differentiation medium. Embryonic stem-derived dopaminergic neurons are purified by cell sorting and may serve as a convenient source for the study of molecular, genetic and cellular properties of dopaminergic neurons.     

细胞因子联合纹状体条件培养液定向诱导间充质干细胞体外分化为多巴胺能神经元

背景：在众多体外诱导间充质干细胞向多巴胺能神经元的诱导分化研究中，诱导阳性率仍不理想。 目的：实验应用碱性成纤维细胞生长因子、表皮生长因子和纹状体条件培养液定向诱导大鼠骨髓间充质干细胞分化为多巴胺能神经元，拟探讨提高诱导阳性率的方法。 设计、时间及地点：以细胞为对象的对照观察细胞学实验，于2006-07/2007-12在山东大学齐鲁儿童医院和山东大学第二医院血液实验室完成。 材料：健康成年Wistar大鼠用于骨髓间充质干细胞的分离，新生Wistar大鼠用于纹状体条件培养液的制备。 方法：采用贴壁法分离纯化健康成年Wistar大鼠骨髓间充质干细胞进行传代培养。取出生24 h内新生Wistar大鼠，完整剥离其大脑组织制备纹状体条件培养液。取体外培养的第5代间充质干细胞，用含碱性成纤维细胞生长因子和表皮生长因子的预诱导液进行预诱导，24 h后去除预诱导液，换用纹状体条件培养液进行诱导。 主要观察指标：倒置显微镜下观察细胞形态变化，并应用细胞免疫化学技术检测细胞内神经元特异烯醇化酶和酪氨酸羟化酶表达。 结果：大鼠骨髓间充质干细胞经碱性成纤维细胞生长因子、表皮生长因子和纹状体条件培养液诱导后细胞胞体逐渐回缩成团，形成梭形，部分细胞可见突起伸出，类似神经元。细胞免疫化学检测，诱导后细胞神经元特异烯醇化酶阳性表达率为( 72.70±14.81)%，酪氨酸羟化酶阳性表达率为(34.50±15.93)%。 结论：应用碱性成纤维细胞生长因子、表皮生长因子联合纹状体条件培养液诱导分化体系，获得了高比例的神经元，其中包括较多的多巴胺能神经元。     

Basic fibroblast growth factor stimulates the proliferation and differentiation of neural stem cells in neonatal rats after ischemic brain injury

A little is known about the proliferation and fate of neural stem cells in the subventricular zone (SVZ) after cerebral ischemia. However, how endogenous neural stem cells are activated in the premature brain is not clear, although basic fibroblast growth factor (bFGF) is important in neurogenesis. To investigate the effect of bFGF on the proliferation and differentiation of neural stem cells after brain ischemia, we observed cellular changes in the subventricular zone (SVZ) of 3-day-old rats (approximately equivalent to premature infants) using immunofluorescence assays, Western blot analysis, and real-time quantitative PCR methods. The bilateral common carotid artery (BCCA) was occluded in 108 animals, then half received bFGF 10ng/g. Besides, 54 rats without ischemia as normal control. Proliferating cells were labeled by bromodeoxyuridine (BrdU) through intraperitoneal injection in a pulsed or a cumulative protocol. Rats were killed at 4, 7, and 14 days after ischemic injury. The number of proliferating cells in the SVZ in bFGF-treated rats was higher than that in untreated rats; bFGF also promoted neural stem cell differentiation into neurons, astrocytes, and oligodendrocytes. Western blot analysis and real-time quantitative PCR assays confirmed these results. We suggest that bFGF promotes the repair of ischemia brain injury through increasing the proliferation of neural stem cells and their differentiation into neurons, astrocytes, and oligodendrocytes.