Inhibition of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor expression reduces dopaminergic sprouting in the injured striatum

After striatal injury, sprouting dopaminergic fibres grow towards and intimately surround wound macrophages which, together with microglia, express the dopaminergic neurotrophic factors glial cell line-derived neurotrophic factor (GDNF) and brain derived neurotrophic factor (BDNF). To evaluate the importance of these endogenously secreted neurotrophic factors in generating striatal peri-wound dopaminergic sprouting, the peri-wound expression of BDNF or GDNF was inhibited by intrastriatal infusion of antisense oligonucleotides for 2 weeks in mice. Knock-down of both BDNF and GDNF mRNA and protein levels in the wounded striatum were confirmed by in situ hybridization and enzyme-linked immunosorbent assay, respectively. Dopamine transporter immunohisto-chemistry revealed that inhibition of either BDNF or GDNF expression resulted in a marked decrease in the intensity of peri-wound sprouting. Quantification of this effect using [H3]-mazindol autoradiography confirmed that peri-wound sprouting was significantly reduced in mice receiving BDNF or GDNF antisense infusions whilst control infusions of buffered saline or sense oligonucleotides resulted in the pronounced peri-wound sprouting response normally associated with striatal injury. BDNF and GDNF thus appear to be important neurotrophic factors inducing dopaminergic sprouting after striatal injury.     

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-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were purchased from Sigma Company (USA); LEICAQWin image processing and analytical system. METHODS: The experiments were carried 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 μL GDNF solution (20 μg/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 12th 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.     

Dental pulp stem cells and their potential roles in central nervous system regeneration and repair

Functional recovery from injuries to the brain or spinal cord represents a major clinical challenge. The transplantation of stem cells, traditionally isolated from embryonic tissue, may help to reduce damage following such events and promote regeneration and repair through both direct cell replacement and neurotrophic mechanisms. However, the therapeutic potential of using embryonic stem/progenitor cells is significantly restricted by the availability of embryonic tissues and associated ethical issues. Populations of stem cells reside within the dental pulp, representing an alternative source of cells that can be isolated with minimal invasiveness, and thus should illicit fewer moral objections, as a replacement for embryonic/fetal‐derived stem cells. Here we discuss the similarities between dental pulp stem cells (DPSCs) and the endogenous stem cells of the central nervous system (CNS) and their ability to differentiate into neuronal cell types. We also consider in vitro and in vivo studies demonstrating the ability of DPSCs to help protect against and repair neuronal damage, suggesting that dental pulp may provide a viable alternative source of stem cells for replacement therapy following CNS damage. © 2013 Wiley Periodicals, Inc.     

Grafts of fetal central nervous system tissue rescue axotomized clarke's nucleus neurons in adult and neonatal operates

Many conditions are thought to contribute to neuron death after axotomy, including immaturity of the cell at the time of injury, inability to reestablish or maintain target contact, and dependence on trophic factors produced by targets. Exogenous application of neurotrophic factors and transplants of peripheral nerve and embryonic central nervous system (CNS) tissue temporarily rescue axotomized CNS neurons, but permanent rescue may require transplants that are normal targets of the injured neurons. We examined the requirements for survival of axotomized Clarke's nucleus (CN) neurons. Two months after hemisection of the spinal cord at the T8 segment, there was an ipsilateral 30% loss of neurons at the L1 segment in adult operates and a 40% loss in neonates. Transplants of embryonic spinal cord, cerebellum, and neocortex inserted into the T8 segment at the time of hemisection prevented virtually all of the cell death in both adults and neonates, but transplants of embryonic striatum were ineffective. None of the grafts prevented the somal atrophy of CN neurons caused by axotomy. Retrograde transport of fluoro-gold from the cerebellum demonstrated that 33% of all CN neurons at L1 project to the cerebellum, 50% of these died following a T8 hemisection, but all these projection neurons were rescued by a transplant of embryonic spinal cord. These results suggest that the rescue of axotomized CN neurons is relatively specific for the normal target areas of these neurons, but this specificity is not absolute and may depend on the distribution and synthesis of particular neurotrophic agents. © 1994 Wiley-Liss, Inc.     

Interactions of cyclic adenosine monophosphate,brain-derived neurotrophic factor,and glial cell line-derived neurotrophic factor treatment on the survival and growth of postnatal mesencephalic dopamine neurons in vitro

The survival of rat postnatal mesencephalic dopamine (DA) neurons in dissociated cell cultures was studied by examining the combinatorial effects of dibutyryl cyclic adenosine monophosphate (db-cAMP), glial cell line-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF), as well as selective inhibitors of protein kinase A (PKA), and mitogen-activated protein kinase (MAPK). Postnatal DA neurons were maintained for 14 days in vitro, and were identified by immunohistochemistry using tyrosine hydroxylase antibody. The survival and growth of DA neurons was significantly increased by the inclusion of either >100 microM db-cAMP or 10 microM Forskolin plus 100 microM IBMX in the culture medium. Neither 10-50 ng/ml GDNF nor 50 ng/ml BDNF alone significantly increased DA neuron survival in vitro. However, the combined use of GDNF and BDNF did increase DA neuron survival, and the addition of either db-cAMP or IBMX/Forskolin to media containing these neurotrophins markedly increased DA neuron survival and growth. The cAMP inhibitor Rp-cAMP, the cAMP-dependent protein kinase A inhibitor H89, and the MAP kinase (MAPK) pathway inhibitor PD98059 significantly reduced the survival of DA neurons when applied alone in the absence of added growth factors. Application of GDNF plus BDNF, or db-cAMP significantly protected the DA neurons from the deleterious effects on survival of either 20 microM H89 or 20 microM PD 98059. The results suggest that BDNF, GDNF, and cAMP produce convergent signals to activate PKA and MAPK pathways which are involved in the survival of postnatal mesencephalic DA neurons in vitro.     

常氧及低氧条件下胶质源性神经营养因子体外诱导中脑神经干细胞向多巴胺能神经元的分化

背景：神经干细胞的定向诱导分化和扩增受细胞自身基因和外来信号的调控。 目的：观察中脑源性神经干细胞在常氧、低氧和胶质源性神经营养因子诱导下向多巴胺能神经元的分化情况。 方法：无菌条件下分离E12小鼠胚胎腹侧中脑组织,胰酶消化和机械吹打制成单细胞悬液,在无血清培养基中培养扩增;Nestin免疫细胞化学染色方法鉴定神经干细胞。在有血清培养基中对纯化神经干细胞自然分化;神经元特异性烯醇化酶和胶质纤维酸性蛋白免疫细胞化学染色方法分别鉴定神经元和星形胶质细胞。建立常氧和低氧环境,设置常氧组、常氧+胶质源性神经营养因子组、低氧组、低氧+胶质源性神经营养因子组,按实验分组在有血清条件下诱导分化。 结果与结论：在低氧条件下,中脑神经干细胞向多巴胺能神经元分化均高于常氧组;尤其是低氧环境和胶质源性神经营养因子诱导下向多巴胺能神经元分化比例更高,表型更成熟。说明低氧环境下胶质源性神经营养因子可明显促进中脑神经干细胞分化为数量足够、形态及功能成熟的多巴胺能神经元。     

Modification of the brain-derived neurotrophic factor gene: a portal to transform mesenchymal stem cells into advantageous engineering cells for neuroregeneration and neuroprotection

Multipotential mesenchymal stem cells (MSCs) are ideal seed cells for recruiting the loss of neural cells due to their strong proliferative capacity, easy acquisition, and considerable tolerance of genetic modifications. After transduction of brain-derived neurotrophic factor (BDNF) gene via recombinant retroviral vectors into the human MSCs, nearly 100% of cells expressed BDNF (which were therefore transformed into BNDF-MSCs) as detected by immunocytochemistry, and the quantity of BDNF in the culture medium was increased by approximately 20,000-fold. In spite of the genomic integration of an exogenous gene, BDNF-MSCs did not present any structural aberration in the chromosomes. All-trans-retinoic acid (RA) induction caused the BDNF-MSCs to differentiate into neural cells with significantly increased expressions of such neural-specific proteins as nestin, NeuN, O4, and glial fibrillary acidic protein (GFAP). The voltage-dependent K+/Ca2+ currents were recorded from the induced BDNF-MSCs using patch-clamp technique. Compared with the MSCs induced by both RA and BDNF, BDNF-MSCs survived in significantly greater number in the induction medium, and also more cells were induced into neuron-like cells (NeuN, P < 0.01) and oligodendrocyte-like cells (O4, P < 0.05). We suppose that, once engrafted into human central nervous system, the BDNF-MSCs would not only recruit the neuronal losses, but also provide, by way of paracrine, large quantities of BDNF that effectively perform the functions of neuroprotection and neuroregeneration, promoting the activation of endogenous neural stem/progenitor cells and their chemotactic migration. On the other hand, the BDNF-MSCs that can survive in the host environment and differentiate subsequently into functional mature cells may also serve as specifically targeting vectors for ex vivo gene therapy.     

Anatomical and functional reconstruction of the nigrostriatal system in vitro: selective innervation of the striatum by dopaminergic neurons.

To study development of the nigrostriatal pathway in an in vitro model system, organotypic slices obtained from rat pups (P4) and containing the striatum and the cortex were grown together with apposed embryonic (E13.5) mesencephalic blocks according to the static slice culture method of Stoppini et al. (1991; J. Neurosci. Methods 37:173-182). Under these conditions, mesencephalic dopaminergic (DA) fibers rapidly grow through the slice, preferentially its striatal portion. This innervation provides a true synaptic innervation to the striatum, as shown by the presence of DA terminals on striatal neurons. DA fibers are able to exert a functional influence, as seen by their ability to modulate c-Fos expression in striatal neurons in the same way as in vivo. Thus, blockade, under basal conditions, of the effect of spontaneously released dopamine by the D2 receptor antagonist haloperidol leads to the activation of c-Fos expression in the striatum. Furthermore, stimulation of DA release by amphetamine induces striatal c-Fos expression in a D1 receptor-dependent manner. Next, the mechanisms of the selective striatal innervation were examined. Indeed, DA fibers innervated specifically the striatum, avoiding the cortical portion of the slice. This selectivity seems to be specific for DA neurons; no selectivity could be observed when noradrenergic neurons were substituted for DA neurons. Short-term cocultures in a collagen gel of mesencephalic blocks with striatal blocks failed to reveal any oriented outgrowth of DA fibers from the mesencephalon, suggesting that the selective innervation observed in the organotypic slices results from some contact-dependent, presumably adhesive interactions rather than from the presence of some diffusible substance orienting the growth of DA fibers towards the striatum. On the other hand, DA neurons seeded onto striatal slices did not attach selectively onto the striatal portion of the slice, indicating that the putative specific adhesive interactions governing the selective striatal innervation are not the same as those determining the adhesion of the DA neurons. These results show that cocultures of cortex-striatum and mesencephalic slices result in a system that displays a number of the morphological and functional traits of the normal nigrostriatal system and that can be relied on as a good in vitro model of in vivo development.     

Transplantation of glial cell line-derived neurotrophic factor-expressing cells into the striatum and nucleus accumbens attenuates acquisition of cocaine self-administration in rats

Neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), may play a role in drug-induced biochemical and behavioural adaptations that characterize addiction. We found that GDNF mRNA levels are lower in the striatum of rats that chronically self-administered cocaine. Therefore, we examined the effect of transplanted cells used as a biodelivery system for GDNF on cocaine self-administration in rats. A human astrocyte-like cell line, which produces and excretes GDNF, was transplanted into the striatum and nucleus accumbens of rats. These rats showed a significantly lower number of active lever presses in the cocaine self-administration paradigm compared with control rats. Moreover, rats that received a chronic infusion of GDNF via a micro-osmotic pump also exhibited weak cocaine self-administration. Therefore, we conclude that exogenous augmentation of GDNF repositories may be useful in suppressing cocaine self-administration.     

Cyclic AMP, but not basic FGF, increases the in vitro survival of mesencephalic dopaminergic neurons and protects them from MPP(+)-induced degeneration.

We studied how stimulation of protein kinase C and cAMP-dependent protein kinases affect the development of mesencephalic dopaminergic neurons in primary cell cultures derived from fetal rats at embryonic day E14. The effects of compounds which activate these second messenger systems were compared to those of basic fibroblast growth factor (bFGF) and insulin-like growth factor I (IGF-I). In mesencephalic cultures, there was a continuous loss of dopaminergic neurons. Despite this decline in cell number, neurotransmitter uptake per neuron increased with time, indicating that the surviving dopaminergic neurons continued their biochemical differentiation while others degenerated. IGF-I and bFGF did not affect the number of dopaminergic neurons. However, dopamine uptake per neuron was significantly higher in bFGF and IGF-I treated cultures, suggesting that these factors stimulated differentiation. Protein kinase C and cAMP-dependent protein kinases were not involved in mediating the effects of bFGF and IGF-I. Treatment of cultures with phorbol esters did not affect dopamine uptake, whereas elevated levels of intracellular cAMP resulted in an increase in dopamine uptake which was additive to that elicited by bFGF or IGF-I. Further analysis revealed that exposure of mesencephalic cultures to dibutyryl cAMP (dbcAMP) during the first 3 days after plating increased the survival of dopaminergic neurons, whereas prolonged treatment attenuated the development of the dopamine uptake system. Moreover, cyclic AMP, but not bFGF, was able to prevent the degeneration of dopaminergic neurons induced by 1-methyl-4-phenyl-pyridinium ion (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The results suggest that increased intracellular levels of cAMP protect dopaminergic neurons in situations of stress like the process of dissociation and plating or the exposure to neurotoxic compounds. Our results reveal novel possibilities for the treatment of Parkinson's disease.     

Olfactory ensheathing cells represent an optimal substrate for hippocampal neurons: an in vitro study

Olfactory ensheathing cells (OECs) are cells that display Schwann cell or astrocyte-like properties. They are a source of growth factors and adhesion molecules which play a very important role as neuronal support enhancing cellular survival. Over the past 10 years, OECs have emerged as a leading reparative candidate, when transplanted into the injured spinal cord, having shown significant promise in the regeneration of spinal cord lesions. In this study we assessed the efficacy of OECs on the survival and neurite outgrowth of hippocampal neurons in vitro. Co-cultures of OECs and hippocampal of postnatal rats were successfully established and cells were immunocytochemically characterized. Some hippocampal cultures were added with growth factors, as bFGF, NGF and GDNF. Furthermore, conditioned medium from OECs cultures was used to feed some hippocampal neurons coverslips. Our results show that in co-cultures of hippocampal neurons and OECs the number of neurons and their neurite outgrowth were significantly increased in comparison with controls. Moreover, we showed that NGF and GDNF promoted a more positive effect in both neuronal survival and neurite outgrowth than bFGF. OEC-conditioned media stimulated both the neuronal survival and dense neurite outgrowth. These data indicate that OECs, as a source of growth factors, can promote the survival and the neurite outgrowth of hippocampal neurons in vitro and that bFGF, NGF and GDNF support them differently. Therefore, as OECs and their secreted growth factors appear to exert a neuroprotective effect for functional restoration and for neural plasticity in neurodegenerative disorders, they might be considered an approach for functional recovery.     

FGF/FGFR system in the central nervous system demyelinating disease: Recent progress and implications for multiple sclerosis

Background

With millions of victims worldwide, multiple sclerosis is the second most common cause of disability among young adults. Although formidable advancements have been made in understanding the disease, the neurodegeneration associated with multiple sclerosis is only partially counteracted by current treatments, and effective therapy for progressive multiple sclerosis remains an unmet need. Therefore, new approaches are required to delay demyelination and the resulting disability and to restore neural function by promoting remyelination and neuronal repair.

Aims

The article reviews the latest literature in this field.

Materials and methods

The fibroblast growth factor (FGF) signaling pathway is a promising target in progressive multiple sclerosis.

Discussion

FGF signal transduction contributes to establishing the oligodendrocyte lineage, neural stem cell proliferation and differentiation, and myelination of the central nervous system. Furthermore, FGF signaling is implicated in the control of neuroinflammation. In recent years, interventions targeting FGF, and its receptor (FGFR) have been shown to ameliorate autoimmune encephalomyelitis symptoms in multiple sclerosis animal models moderately.

Conclusion

Here, we summarize the recent findings and investigate the role of FGF/FGFR signaling in the onset and progression, discuss the potential therapeutic advances, and offer fresh insights into managing multiple sclerosis.     

A protocol for the differentiation of human embryonic stem cells into dopaminergic neurons using only chemically defined human additives: Studies in vitro and in vivo

Our ability to use human embryonic stem (hES) cells in cell replacement therapy for Parkinson's disease depends on the discovery of ways to simply and reliably differentiate a dopaminergic (DA) phenotype in these cells. Although several protocols exist for the differentiation of DA traits in hES, they involve the prolonged use of complex media with undefined components, cell conditioned media and/or co-culture with various cells, usually of animal origin. In this study, several well-characterized (H9, BG01) and several new uncharacterized (HUES7, HUES8) hES cell lines were studied for their capacity to differentiate into DA neurons in culture using a novel rapid protocol which uses only chemically-defined human-derived media additives and substrata. Within 3 weeks, cells from all 4 cell lines progressed from the undifferentiated state to beta-tubulin III positive cells expressing DA markers in vitro. Moreover, transplantation of these cells into the striata of 6-hydroxydopamine-treated rats at the neuronal progenitor stage resulted in the appearance of differentiated DA traits in vivo 2-3 weeks later.     

Nurr1对小胶质细胞联合神经干细胞共培养促进神经干细胞向多巴胺神经元分化作用研究

目的探讨联合过表达核受体相关因子1(Nurr1)基因的小胶质细胞(MG)和神经干细胞(NSC)共培养对神经干细胞向多巴胺神经元分化的影响。方法原代培养SD大鼠神经干细胞和小胶质细胞,并过表达Nurr1基因。CCK-8法检测Nurr1过表达对神经干细胞以及小胶质细胞活率的影响。Transwell系统共培养神经干细胞和小胶质细胞,实验分为NSC组、NSC+MG组和N(NSC+MG)组。ELISA检测共培养后第3天、第6天和第9天各组脑源性神经营养因子(BDNF)、血小板源性神经营养因子(PDNF)和胶质细胞源性神经营养因子(GDNF)表达变化;RT-PCR和Western Blot检测各组第9天酪氨酸羟化酶(TH)、多巴胺转运蛋白(DAT)DAT和Nurr1的表达变化;细胞免疫荧光鉴定神经干细胞的分化,并对TH和DAT阳性细胞计数,计算各组神经干细胞向多巴胺神经元的分化效率。结果原代培养小胶质细胞以及神经干细胞并成功过表达Nurr1基因。CCK-8法检测结果表明,Nurr1过表达对神经干细胞以及小胶质细胞活率无明显影响。ELISA检测结果表明,N(NSC+MG)组在不同时间点神经营养因子(BDNF、PDNF和GDNF)表达量明显高于其他各组(P0.05)。RT-PCR和Westen Blot检测结果表明,N(NSC+MG)组TH、DAT和Nurr1的表达水平明显高于其他各组(P0.05)。细胞免疫荧光鉴定结果表明,N(NSC+MG)组TH阳性细胞率明显高于其他各组(P0.05)。结论Nurr1基因可促进神经干细胞和小胶质细胞共培养系统神经营养因子的分泌。过表达Nurr1基因的神经干细胞和小胶质细胞共培养可促进神经干细胞向多巴胺神经元的分化。     

Lentiviral gene delivery of GDNF into the striatum of R6/2 Huntington mice fails to attenuate behavioral and neuropathological changes

Transgenic R6/2 mice, which express exon 1 of the human mutant Huntington disease gene, develop behavioral and neuropathological changes that bear some resemblance to the human disease. Several studies have shown that elevated glial cell line-derived neurotrophic factor (GDNF) levels can exert neuroprotective effects in animal models of Huntington disease that are based on intrastriatal injections of excitotoxins. Therefore, the aim of the present study was to examine whether intrastriatal delivery of the GDNF gene by lentivirus (LV-GDNF) could provide structural and functional protection in R6/2 transgenic mice. Four- to 5-week-old mice were left untreated or alternatively received intrastriatal injections of either LV-GDNF or the same viral vector encoding green fluorescent protein (GFP) (LV-GFP) as a control. During the 4-week follow-up period, there was the expected deterioration in performance of the R6/2 mice in paw clasping, rotarod, and open field tests, and the LV-GDNF treated mice showed no improvement over controls. ELISA showed that the LV-GDNF-injected animals had a significant increase in GDNF level in the striatum, and immunohistochemical analysis revealed that GDNF was also overexpressed in brain regions receiving striatal projections. However, GDNF overexpression had no effect on the neuropathological changes examined. Thus, there were no significant differences in the number of EM-48-positive intraneuronal huntingtin inclusions, number of BrdU-positive cells and size of striatal neuronal cross-sectional area. These results suggest that intrastriatal lentiviral vector transfer of GDNF, performed at 5 weeks of age, does not ameliorate neurological and behavioral impairments in the R6/2 transgenic mice model of HD. Further studies are, however, needed to investigate if GDNF given at earlier time points is beneficial.