Transcription factor Six2 mediates the protection of GDNF on 6-OHDA lesioned dopaminergic neurons by regulating Smurf1 expression.
ABSTRACT: Glial cell line-derived neurotrophic factor (GDNF) has strong neuroprotective and neurorestorative effects on dopaminergic (DA) neurons in the substantia nigra (SN); however, the underlying molecular mechanisms remain to be fully elucidated. In this study, we found that the expression level of transcription factor Six2 was increased in damaged DA neurons after GDNF rescue in vivo and in vitro. Knockdown of Six2 resulted in decreased cell viability and increased the apoptosis of damaged DA neurons after GDNF treatment in vitro. In contrast, Six2 overexpression increased cell viability and decreased cell apoptosis. Furthermore, genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) indicated that Six2 directly bound to the promoter CAGCTG sequence of smad ubiquitylation regulatory factor 1 (Smurf1). ChIP-quantitative polymerase chain reaction (qPCR) analysis showed that Smurf1 expression was significantly upregulated after GDNF rescue. Moreover, knockdown of Six2 decreased Smurf1 expression, whereas overexpression of Six2 increased Smurf1 expression in damaged DA neurons after GDNF rescue. Meanwhile, knockdown and overexpression of Smurf1 increased and decreased p53 expression, respectively. Taken together, our results from in vitro and in vivo analysis indicate that Six2 mediates the protective effects of GDNF on damaged DA neurons by regulating Smurf1 expression, which could be useful in identifying potential drug targets for injured DA neurons.
Project description:Parkinson's disease (PD) is the second-most-frequent neurodegenerative disorder worldwide. One major hallmark of PD is the degeneration of dopaminergic (DA) neurons in the substantia nigra. Glial cell line-derived neurotrophic factor (GDNF) potently increases DA neuron survival in models of PD; however, the underlying mechanisms are incompletely understood. MicroRNAs (miRNAs) are small, non-coding RNAs that are important for post-transcriptional regulation of gene expression. Using small RNA sequencing, we show that GDNF specifically increases the expression of miR-182-5p and miR-183-5p in primary midbrain neurons (PMNs). Transfection of synthetic miR-182-5p and miR-183-5p mimics leads to increased neurite outgrowth and mediates neuroprotection of DA neurons in vitro and in vivo, mimicking GDNF effects. This is accompanied by decreased expression of FOXO3 and FOXO1 transcription factors and increased PI3K-Akt signaling. Inhibition of endogenous miR-182-5p or miR-183-5p in GDNF-treated PMNs attenuated the pro-DA effects of GDNF. These findings unveil an unknown miR-mediated mechanism of GDNF action and suggest that targeting miRNAs is a new therapeutic avenue to PD phenotypes.
Project description:Salvianolic acid B (SalB), a bioactive compound isolated from the plant-derived medicinal herb Danshen, has been shown to exert various anti-oxidative and anti-inflammatory activities in several neurological disorders. In this study, we sought to investigate the potential protective effects and associated molecular mechanisms of SalB in Parkinson's disease (PD) models. To determine the neuroprotective effects of SalB in vitro, MPP+- or lipopolysaccharide (LPS)-induced neuronal injury was achieved using primary cultures with different compositions of neurons, microglia and astrocytes. Our results showed that SalB reduced both LPS- and MPP+-induced toxicity of dopamine neurons in a dose-dependent manner. Additionally, SalB treatment inhibited the release of microglial pro-inflammatory cytokines and resulted in an increase in the expression and release of glial cell line-derived neurotrophic factor (GDNF) from astrocytes. Western blot analysis illustrated that SalB increased the expression and nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). The knockdown of Nrf2 using specific small interfering RNA (siRNA) partially reversed the SalB-induced GDNF expression and anti-inflammatory activity. Moreover, SalB treatment significantly attenuated dopaminergic (DA) neuronal loss, inhibited neuroinflammation, increased GDNF expression and improved the neurological function in MPTP-treated mice. Collectively, these findings demonstrated that SalB protects DA neurons by an Nrf-2 -mediated dual action: reducing microglia activation-mediated neuroinflammation and inducing astrocyte activation-dependent GDNF expression. Importantly the present study also highlights critical roles of glial cells as targets for developing new strategies to alter the progression of neurodegenerative disorders.
Project description:Glial cell line-derived neurotrophic factor (GDNF) has been shown to protect and restore dopamine (DA) neurons in injury models and is being evaluated for the treatment of Parkinson's disease. Nevertheless, little is known of its physiological role. We have shown that GDNF suppresses apoptosis in DA neurons of the substantia nigra (SN) postnatally both in vitro and during their first phase of natural cell death in vivo. Furthermore, intrastriatal injection of neutralizing antibodies augments cell death, suggesting that endogenous GDNF plays a role as a target-derived factor. Such a role would predict that overexpression of GDNF in striatum would increase the surviving number of SN DA neurons. To test this hypothesis, we used the tetracycline-dependent transcription activator (tTA)/tTA-responsive promoter system to create mice that overexpress GDNF selectively in the striatum, cortex, and hippocampus. These mice demonstrate an increased number of SN DA neurons after the first phase of natural cell death. However, this increase does not persist into adulthood. As adults, these mice also do not have increased dopaminergic innervation of the striatum. They do, however, demonstrate increased numbers of ventral tegmental area (VTA) neurons and increased innervation of the cortex. This morphologic phenotype is associated with an increased locomotor response to amphetamine. We conclude that striatal GDNF is necessary and sufficient to regulate the number of SN DA neurons surviving the first phase of natural cell death, but it is not sufficient to increase their final adult number. GDNF in VTA targets, however, is sufficient to regulate the adult number of DA neurons.
Project description:Parkinson's disease (PD) is characterized by the selective and progressive loss of dopaminergic (DA) neurons in the midbrain substantia nigra. Currently, available treatment is unable to alter PD progression. Previously, we demonstrated that valproic acid (VPA), a mood stabilizer, anticonvulsant and histone deacetylase (HDAC) inhibitor, increases the expression of glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) in astrocytes to protect DA neurons in midbrain neuron-glia cultures. The present study investigated whether these effects are due to HDAC inhibition and histone acetylation. Here, we show that two additional HDAC inhibitors, sodium butyrate (SB) and trichostatin A (TSA), mimic the survival-promoting and protective effects of VPA on DA neurons in neuron-glia cultures. Similar to VPA, both SB and TSA increased GDNF and BDNF transcripts in astrocytes in a time-dependent manner. Furthermore, marked increases in GDNF promoter activity and promoter-associated histone H3 acetylation were noted in astrocytes treated with all three compounds, where the time-course for acetylation was similar to that for gene transcription. Taken together, our results indicate that HDAC inhibitors up-regulate GDNF and BDNF expression in astrocytes and protect DA neurons, at least in part, through HDAC inhibition. This study indicates that astrocytes may be a critical neuroprotective mechanism of HDAC inhibitors, revealing a novel target for the treatment of psychiatric and neurodegenerative diseases.
Project description: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.
Project description:Support of ageing neurons by endogenous neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) may determine whether the neurons resist or succumb to neurodegeneration. GDNF has been tested in clinical trials for the treatment of Parkinson disease (PD), a common neurodegenerative disorder characterized by the loss of midbrain dopaminergic (DA) neurons. BDNF modulates nigrostriatal functions and rescues DA neurons in PD animal models. The physiological roles of GDNF and BDNF signaling in the adult nigrostriatal DA system are unknown. We generated mice with regionally selective ablations of the genes encoding the receptors for GDNF (Ret) and BDNF (TrkB). We find that Ret, but not TrkB, ablation causes progressive and adult-onset loss of DA neurons specifically in the substantia nigra pars compacta, degeneration of DA nerve terminals in striatum, and pronounced glial activation. These findings establish Ret as a critical regulator of long-term maintenance of the nigrostriatal DA system and suggest conditional Ret mutants as useful tools for gaining insights into the molecular mechanisms involved in the development of PD.
Project description:Prenatal systemic inflammation has been implicated in neurological diseases, but optimal animal models have not been developed. We investigated whether a partial genetic deletion of glial cell line-derived neurotrophic factor (Gdnf(+/-)) increased vulnerability of dopamine (DA) neurons to prenatal lipopolysaccharide (LPS). LPS [0.01 mg/kg intraperitoneal (i.p.)] or saline was administered to wild-type (WT) or Gdnf(+/-) pregnant mice on gestational day 9.5. Male offspring were examined at 3 weeks, 3 and 12 months of age. There was a progressive degeneration of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra (SN) with age in Gdnf(+/-) but not in WT mice, with no observed effects on locus coeruleus (LC) noradrenergic neurons or DA neurons of the ventral tegmental area. Inflammatory markers were elevated in SN of LPS treated offspring, with exacerbation in Gdnf(+/-) mice. Intracellular accumulation of ?-synuclein (?-syn) immunoreactivity in DA neurons of SN was observed in all groups of Gdnf(+/-) and in WT mice with prenatal LPS, with altered distribution between pars reticulata (pr) and pars compacta (pc). The findings suggest that prenatal LPS leads to accelerated neuropathology in the SN with age, and that a partial loss of GDNF exacerbates these effects, providing a novel model for age-related neuropathology of the nigrostriatal DA system.
Project description:Glial cell line-derived neurotrophic factor (GDNF) has potent neurotrophic effects and is known to promote the dopaminergic (DA) neuronal survival in cellular and animal models of Parkinson's disease (PD). However, long-term ectopic GDNF delivery is associated with long lasting adverse side effects in PD patients. Therefore, finding safer and effective ways to elevate endogenous GDNF levels is an active area of research. This study underlines the importance of sodium benzoate (NaB), a metabolite of commonly-used spice cinnamon, a food-additive and an FDA-approved drug against hyperammonemia, in stimulating GDNF in primary mouse and human astrocytes. Presence of cAMP response element (CRE) in the Gdnf gene promoter, recruitment of CREB to the Gdnf promoter by NaB and abrogation of NaB-mediated GDNF expression by siRNA knockdown of CREB suggest that NaB induces the transcription of Gdnf via CREB. Finally, oral administration of NaB and cinnamon itself increased the level of GDNF in vivo in the substantia nigra pars compacta (SNpc) of normal as well as MPTP-intoxicated mice. Accordingly, cinnamon and NaB treatment protected tyrosine hydroxylase positive neurons in the SNpc and fibers in the striatum, normalized striatal neurotransmitters, and improved locomotor activities in MPTP-intoxicated Gfapcre mice, but not Gdnf?astro mice lacking GDNF in astrocytes. These findings highlight the importance of astroglial GDNF in cinnamon- and NaB-mediated protection of the nigrostriatum in MPTP mouse model of PD and suggest possible therapeutic potential of cinnamon and NaB in PD patients. Graphical abstract Cinnamon metabolite sodium benzoate (NaB) activates cAMP-response element-binding (CREB) via protein kinase A (PKA) in astrocytes. Activated CREB then binds to cAMP-response element (CRE) present in GDNF gene promoter to stimulate the transcription of GDNF in astrocytes. This astrocytic GDNF leads to nigral trophism and protects dopaminergic neurons from MPTP insult.
Project description:Spontaneous firing of ventral tegmental area (VTA) dopamine (DA) neurons provides ambient levels of DA in target areas such as the nucleus accumbens (NAc) and the prefrontal cortex (PFC). Here we report that the glial cell line-derived neurotrophic factor (GDNF), produced in one target region, the NAc, is retrogradely transported by DA neurons to the VTA where the growth factor positively regulates the spontaneous firing activity of both NAc- and PFC-projecting DA neurons in a mechanism that requires the activation of the mitogen-activated protein kinase (MAPK) pathway. We also show that the consequence of GDNF-mediated activation of the MAPK signaling cascade in the VTA is an increase in DA overflow in the NAc. Together, these results demonstrate that NAc-produced GDNF serves as a retrograde enhancer that upregulates the activity of the mesocorticolimbic DA system.
Project description:Glial cell line-derived neurotrophic factor (GDNF) protects dopamine (DA) neurons from 6-hydroxydopamine (6-OHDA) toxicity. We have now explored this protection over 8 weeks following toxin administration. Infusion of Fluoro-Gold (FG) into the striatum was followed 1 week later by GDNF (9?g) or its vehicle. Six hours later, animals received 6-OHDA (4 ?g) into the same site. 6-OHDA caused a loss of cells in the substantia nigra that expressed both FG and tyrosine hydroxylase (TH) and striatal terminals expressing TH, the high affinity dopamine transporter (DAT), and the vesicular monoamine transporter 2 (VMAT2) as assessed 2-8 weeks later. Loss of FG(+) cells, and striatal DA was completely blocked by GDNF by 2 weeks. In contrast, GDNF only slightly attenuated the loss of TH, DAT, or VMAT2 in the striatum at 2 weeks, but had restored these markers by 4-8 weeks. Thus, GDNF prevents DA cell death and loss of striatal DA content, but several weeks are required to fully restore the dopaminergic phenotype. These results provide insight into the mechanism of GDNF protection of DA neurons, and may help avoid incorrect interpretations of temporary phenotypic changes.