Absence of Ret signaling in mice causes progressive and late degeneration of the nigrostriatal system.
ABSTRACT: 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:Parkinson's disease (PD) is the second most common neurodegenerative disease, which is clinically and pathologically characterized by motor dysfunction and the loss of dopaminergic neurons in the substantia nigra, respectively. PD treatment with stem cells has long been studied by researchers; however, no adequate treatment strategy has been established. The results of studies so far have suggested that stem cell transplantation can be an effective treatment for PD. However, PD is a progressively deteriorating neurodegenerative disease that requires long-term treatment, and this has been insufficiently studied. Thus, we aimed to investigate the therapeutic potential of human adipose-derived stem cells (hASC) for repeated vein transplantation over long-term in an animal model of PD. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model mice, hASCs were administered on the tail vein six times at two-week intervals. After the last injection of hASCs, motor function significantly improved. The number of dopaminergic neurons present in the nigrostriatal pathway was recovered using hASC transplantation. Moreover, the administration of hASC restored altered dopamine transporter expression and increased neurotrophic factors, such as brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF), in the striatum. Overall, this study suggests that repeated intravenous transplantation of hASC may exert therapeutic effects on PD by restoring BDNF and GDNF expressions, protecting dopaminergic neurons, and maintaining the nigrostriatal pathway.
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:Parkinson's disease (PD) is an incurable neurodegenerative disorder affecting up to 10 million people in the world. Diagnostic motor symptoms of PD appear as a result of progressive degeneration and death of nigrostriatal dopamine neurons. Current PD treatments only relieve symptoms without halting the progression of the disease, and their use is complicated by severe adverse effects emerging as the disease progresses. Therefore, there is an urgent need for new therapies for PD management. We developed a small molecule compound, BT13, targeting receptor tyrosine kinase RET. RET is the signalling receptor for a known survival factor for dopamine neurons called glial cell line-derived neurotrophic factor (GDNF). Previously we showed that BT13 prevents the death of cultured dopamine neurons, stimulates dopamine release and activates pro-survival signalling cascades in naïve rodent brain. In the present study, we evaluate the effects of BT13 on motor imbalance and nigrostriatal dopamine neurons in a unilateral 6-hydroxydopamine rat model of PD. We show that BT13 alleviates motor dysfunction in experimental animals. Further studies are needed to make a conclusion whether BT13 can protect the integrity of the nigrostriatal dopamine system since even the positive control, GDNF protein, was unable to produce a clear neuroprotective effect in the model used in the present work. In contrast to GDNF, BT13 is able to cross the blood-brain barrier, which together with the ability to reduce motor symptoms of the disease makes it a valuable lead for further development as a potential disease-modifying agent to treat PD.
Project description:Skin is a major source of secretion of the neurotrophic factors nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and glial-derived neurotrophic factor (GDNF) controlling cutaneous sensory innervation. Beside their neuronal contribution, we hypothesized that neurotrophic factors also modulate the cutaneous microvascular network. First, we showed that NGF, BDNF, NT-3, and GDNF were all expressed in the epidermis, while only NGF and NT-3 were expressed by cultured fibroblasts, and BDNF by human endothelial cells. We demonstrated that these peptides are highly potent angiogenic factors using a human tissue-engineered angiogenesis model. A 40% to 80% increase in the number of capillary-like tubes was observed after the addition of 10?ng/mL of NGF, 0.1?ng/mL of BDNF, 15?ng/mL of NT-3, and 50?ng/mL of GDNF. This is the first characterization of the direct angiogenic effect of NT-3 and GDNF. This angiogenic effect was mediated directly through binding with the neurotrophic factor receptors tropomyosin-receptor kinase A (TrkA), TrkB, GFR?-1 and c-ret that were all expressed by human endothelial cells, while this effect was blocked by addition of the Trk inhibitor K252a. Thus, if NGF, BDNF, NT-3, and GDNF may only moderately regulate the microvascular network in normal skin, they might have the potential to greatly increase angiogenesis in pathological situations.
Project description:Glial cell line-derived neurotrophic factor (GDNF) is the most potent neuroprotective agent tested in cellular and animal models of Parkinson's disease (PD). However, CNS delivery of GDNF is restricted by the blood-brain barrier (BBB). Using total body irradiation as transplant preconditioning, we previously reported that hematopoietic stem cell (HSC) transplantation (HSCT)-based macrophage-mediated gene therapy could deliver GDNF to the brain to prevent degeneration of nigrostriatal dopamine (DA) neurons in an acute murine neurotoxicity model. Here, we validate this therapeutic approach in a chronic progressive PD model - the MitoPark mouse, with head shielding to avoid inducing neuroinflammation and compromising BBB integrity. Bone marrow HSCs were transduced ex vivo with a lentiviral vector expressing macrophage promoter-driven GDNF and transplanted into MitoPark mice exhibiting well developed PD-like impairments. Transgene-expressing macrophages infiltrated the midbrains of MitoPark mice, but not normal littermates, and delivered GDNF locally. Macrophage GDNF delivery markedly improved both motor and non-motor symptoms, and dramatically mitigated the loss of both DA neurons in the substantia nigra and tyrosine hydroxylase-positive axonal terminals in the striatum. Our data support further development of this HSCT-based macrophage-mediated GDNF delivery approach in order to address the unmet need for a disease-modifying therapy for PD.
Project description:Glial cell line-derived neurotrophic factor (GDNF) is a potent survival and regeneration-promoting factor for dopaminergic neurons in cell and animal models of Parkinson disease (PD). GDNF is currently tested in clinical trials on PD patients with so far inconclusive results. The receptor tyrosine kinase Ret is the canonical GDNF receptor, but several alternative GDNF receptors have been proposed, raising the question of which signaling receptor mediates here the beneficial GDNF effects. To address this question we overexpressed GDNF in the striatum of mice deficient for Ret in dopaminergic neurons and subsequently challenged these mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Strikingly, in this established PD mouse model, the absence of Ret completely abolished GDNF's neuroprotective and regenerative effect on the midbrain dopaminergic system. This establishes Ret signaling as absolutely required for GDNF's effects to prevent and compensate dopaminergic system degeneration and suggests Ret activation as the primary target of GDNF therapy in PD.
Project description:To investigate the therapeutic effect of microencapsulated porcine retinal pigmented epithelial cells (RPE-M) transplantation on rat model of Parkinson's disease (PD).Primary porcine RPE cells were harvested by enzyme digestion and expanded in culture medium. Determine the levels of dopamine (DA) and homovanillic acid (HVA) by high performance liquid chromatography electrochemical (HPLC) assay, and the levels of brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) were detected by ELISA. Alginate-polylysine-alginate (APA) microencapsulated cells were produced by using a high voltage electrostatic system. PD rat model was established by unilateral injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB). After that, the RPE-M was transplanted into the corpus striatum of PD rat, and then the rotation test scores were recorded and biochemical changes of the corpus striatum were tested.The levels of DA, HVA, BDNF and GDNF secreted by RPE were stable in the RPE culture supernatant and were not changed by the microencapsulation. Eighty-three percent rats developed PD by unilateral lesion of 6-OHDA in the MFB. The RPE-M transplantation had therapeutic effect on 33% PD rats.Porcine RPE cells grow actively in vitro and could secrete DA, HVA, BDNF, and GDNF constantly, which does not be affected by the passage culture and the APA miroencapsulation. RPE-M transplantation of may be a curative therapy for PD.
Project description:The overall goal of our research is to establish a preformed molecular guidance pathway to direct the growth of dopaminergic axons from embryonic ventral mesencephalon (VM), tissue placed within the substantia nigra (SN), into the striatum to reconstruct the nigrostriatal pathway in a hemi-Parkinson's disease rat model. Guidance pathways were prepared by injecting lentivirus encoding either GFP or a combination of glial-cell-line-derived neurotrophic factor (GDNF) with either GDNF family receptor ?1 (GFR?1) or netrin1. In another cohort of animals, adeno-associated virus (AAV) encoding brain-derived neurotrophic factor (BDNF) was injected within the striatum after guidance pathway formation. GDNF combined with either GFR?1 or netrin significantly increased growth of dopaminergic axons out of transplants and along the pathway, resulting in a significant reduction in the number of amphetamine-induced rotations. Retrograde tract tracing showed that the dopaminergic axons innervating the striatum were from A9 neurons within the transplant. Increased dopaminergic innervation of the striatum and improved behavioral recovery were observed with the addition of BDNF. Preformed guidance pathways using a combination of GDNF and netrin1 can be used to reconstruct the nigrostriatal pathway and improve motor recovery.
Project description:The striatum integrates motor behavior using a well-defined microcircuit whose individual components are independently affected in several neurological diseases. The glial cell line-derived neurotrophic factor (GDNF), synthesized by striatal interneurons, and Sonic hedgehog (Shh), produced by the dopaminergic neurons of the substantia nigra (DA SNpc), are both involved in the nigrostriatal maintenance but the reciprocal neurotrophic relationships among these neurons are only partially understood. To define the postnatal neurotrophic connections among fast-spiking GABAergic interneurons (FS), cholinergic interneurons (ACh), and DA SNpc, we used a genetically induced mouse model of postnatal DA SNpc neurodegeneration and separately eliminated Smoothened (Smo), the obligatory transducer of Shh signaling, in striatal interneurons. We show that FS postnatal survival relies on DA SNpc and is independent of Shh signaling. On the contrary, Shh signaling but not dopaminergic striatal innervation is required to maintain ACh in the postnatal striatum. ACh are required for DA SNpc survival in a GDNF-independent manner. These data demonstrate the existence of three parallel but interdependent neurotrophic relationships between SN and striatal interneurons, partially defined by Shh and GDNF. The definition of these new neurotrophic interactions opens the search for new molecules involved in the striatal modulatory circuit maintenance with potential therapeutic value.