Glial Cell Line-derived Neurotrophic Factor Receptor Research Articles

Muscle-resident mesenchymal progenitors sense and repair peripheral nerve injury via the GDNF-BDNF axis.

Fibro-adipogenic progenitors (FAPs) are muscle-resident mesenchymal progenitors that can contribute to muscle tissue homeostasis and regeneration, as well as postnatal maturation and lifelong maintenance of the neuromuscular system. Recently, traumatic injury to the peripheral nerve was shown to activate FAPs, suggesting that FAPs can respond to nerve injury. However, questions of how FAPs can sense the anatomically distant peripheral nerve injury and whether FAPs can directly contribute to nerve regeneration remained unanswered. Here, utilizing single-cell transcriptomics and mouse models, we discovered that a subset of FAPs expressing GDNF receptors Ret and Gfra1 can respond to peripheral nerve injury by sensing GDNF secreted by Schwann cells. Upon GDNF sensing, this subset becomes activated and expresses Bdnf. FAP-specific inactivation of Bdnf (Prrx1Cre; Bdnffl/fl) resulted in delayed nerve regeneration owing to defective remyelination, indicating that GDNF-sensing FAPs play an important role in the remyelination process during peripheral nerve regeneration. In aged mice, significantly reduced Bdnf expression in FAPs was observed upon nerve injury, suggesting the clinical relevance of FAP-derived BDNF in the age-related delays in nerve regeneration. Collectively, our study revealed the previously unidentified role of FAPs in peripheral nerve regeneration, and the molecular mechanism behind FAPs' response to peripheral nerve injury.

Analysis of Acute and Chronic Methamphetamine Treatment in Mice on Gdnf System Expression Reveals a Potential Mechanism of Schizophrenia Susceptibility.

The increase in presynaptic striatal dopamine is the main dopaminergic abnormality in schizophrenia (SCZ). SCZ is primarily treated by modulating the activity of monoamine systems, with a focus on dopamine and serotonin receptors. Glial cell line-derived neurotrophic factor (GDNF) is a strong dopaminergic factor, that recently was shown to correlate with SCZ in human CSF and in striatal tissue. A 2-3-fold increase in GDNF in the brain was sufficient to induce SCZ-like dopaminergic and behavioural changes in mice. Here, we analysed the effect of acute, chronic, and embryonic methamphetamine, a drug known to enhance the risk of psychosis, on Gdnf and its receptors, Gfra1 and Ret, as well as on monoamine metabolism-related gene expression in the mouse brain. We found that acute methamphetamine application increases Gdnf expression in the striatum and chronic methamphetamine decreases the striatal expression of GDNF receptors Gfra1 and Ret. Both chronic and acute methamphetamine treatment upregulated the expression of genes related to dopamine and serotonin metabolism in the striatum, prefrontal cortex, and substantia nigra. Our results suggest a potential mechanism as to how methamphetamine elicits individual psychosis risk in young adults-variation in initial striatal GDNF induction and subsequent GFRα1 and RET downregulation may determine individual susceptibility to psychosis. Our results may guide future experiments and precision medicine development for methamphetamine-induced psychosis using GDNF/GFRa1/RET antagonists.

Differential expression of RET and GDNF family receptor, GFR-α1, between striatum and substantia nigra following nigrostriatal lesion: A case for diminished GDNF-signaling

Although glial cell line-derived neurotrophic factor (GDNF) showed efficacy in preclinical and early clinical studies to alleviate parkinsonian signs in Parkinson's disease (PD), later trials did not meet primary endpoints, giving pause to consider further investigation. While GDNF dose and delivery methods may have contributed to diminished efficacy, one crucial aspect of these clinical studies is that GDNF treatment began ∼8 years after PD diagnosis; a time point representing several years after near 100% depletion of nigrostriatal dopamine markers in striatum and at least 50% in substantia nigra (SN), which represents a time point of initiating GDNF treatment later than reported in some preclinical studies. With nigrostriatal terminal loss exceeding 70% at PD diagnosis, we utilized hemiparkinsonian rats to determine if expression of GDNF family receptor, GFR-α1, and receptor tyrosine kinase, RET, differed between striatum and SN at 1 and 4 weeks following a 6-hydroxydopamine (6-OHDA) hemilesion. Whereas GDNF expression changed minimally, GFR-α1 expression decreased progressively in striatum and in tyrosine hydroxylase positive (TH+) cells in SN, correlating with reduced TH cell number. However, in nigral astrocytes, GFR-α1 expression increased. RET expression decreased maximally in striatum by 1 week, whereas in the SN, a transient bilateral increase occurred, returning to control levels by 4 weeks. Expression of brain-derived neurotrophic factor (BDNF) or its receptor, TrkB, were unchanged throughout lesion progression. Together, these results reveal that differential GFR-α1 and RET expression between the striatum and SN, and cell-specific differences in GFR-α1 expression in SN, occur during nigrostriatal neuron loss. Targeting loss of GDNF receptors thus appears critical to enhance GDNF therapeutic efficacy against nigrostriatal neuron loss. Significance statementAlthough preclinical evidence supports that GDNF provides neuroprotection and improves locomotor function in preclinical studies, there is uncertainty if it can alleviate motor impairment in Parkinson's disease patients. Using the established 6-OHDA hemiparkinsonian rat model, we determined whether expression of its cognate receptors, GFR-α1 and RET, were differentially affected between striatum and substantia nigra in a timeline study. In striatum, there was early and significant loss of RET, but a gradual, progressive loss of GFR-α1. In contrast, RET transiently increased in lesioned substantia nigra, but GFR-α1 progressively decreased only in nigrostriatal neurons and correlated with TH cell loss. Our results indicate that direct availability of GFR-α1 may be a critical element that determines GDNF efficacy following striatal delivery.

Opposing Spatially Segregated Function of Endogenous GDNF-RET Signaling in Cocaine Addiction.

Cocaine addiction is a serious condition with potentially lethal complications and no current pharmacological approaches towards treatment. Perturbations of the mesolimbic dopamine system are crucial to the establishment of cocaine-induced conditioned place preference and reward. As a potent neurotrophic factor modulating the function of dopamine neurons, glial cell line-derived neurotrophic factor (GDNF) acting through its receptor RET on dopamine neurons may provide a novel therapeutic avenue towards psychostimulant addiction. However, current knowledge on endogenous GDNF and RET function after the onset of addiction is scarce. Here, we utilized a conditional knockout approach to reduce the expression of the GDNF receptor tyrosine kinase RET from dopamine neurons in the ventral tegmental area (VTA) after the onset of cocaine-induced conditioned place preference. Similarly, after establishing cocaine-induced conditioned place preference, we studied the effect of conditionally reducing GDNF in the ventral striatum nucleus accumbens (NAc), the target of mesolimbic dopaminergic innervation. We find that the reduction of RET within the VTA hastens cocaine-induced conditioned place preference extinction and reduces reinstatement, while the reduction of GDNF within the NAc does the opposite: prolongs cocaine-induced conditioned place preference and increases preference during reinstatement. In addition, the brain-derived neurotrophic factor (BDNF) was increased and key dopamine-related genes were reduced in the GDNF cKO mutant animals after cocaine administration. Thus, RET antagonism in the VTA coupled with intact or enhanced accumbal GDNF function may provide a new approach towards cocaine addiction treatment.

Endogenous Inflammatory Mediators Produced by Injury Activate TRPV1 and TRPA1 Nociceptors to Induce Sexually Dimorphic Cold Pain That Is Dependent on TRPM8 and GFRα3.

The detection of environmental temperatures is critical for survival, yet inappropriate responses to thermal stimuli can have a negative impact on overall health. The physiological effect of cold is distinct among somatosensory modalities in that it is soothing and analgesic, but also agonizing in the context of tissue damage. Inflammatory mediators produced during injury activate nociceptors to release neuropeptides, such as calcitonin gene-related peptide (CGRP) and substance P, inducing neurogenic inflammation, which further exasperates pain. Many inflammatory mediators induce sensitization to heat and mechanical stimuli but, conversely, inhibit cold responsiveness, and the identity of molecules inducing cold pain peripherally is enigmatic, as are the cellular and molecular mechanisms altering cold sensitivity. Here, we asked whether inflammatory mediators that induce neurogenic inflammation via the nociceptive ion channels TRPV1 (vanilloid subfamily of transient receptor potential channel) and TRPA1 (transient receptor potential ankyrin 1) lead to cold pain in mice. Specifically, we tested cold sensitivity in mice after intraplantar injection of lysophosphatidic acid or 4-hydroxy-2-nonenal, finding that each induces cold pain that is dependent on the cold-gated channel transient receptor potential melastatin 8 (TRPM8). Inhibition of CGRP, substance P, or toll-like receptor 4 (TLR4) signaling attenuates this phenotype, and each neuropeptide produces TRPM8-dependent cold pain directly. Further, the inhibition of CGRP or TLR4 signaling alleviates cold allodynia differentially by sex. Last, cold pain induced by both inflammatory mediators and neuropeptides requires TRPM8, as well as the neurotrophin artemin and its receptor GDNF receptor α3 (GFRα3). These results are consistent with artemin-induced cold allodynia requiring TRPM8, demonstrating that neurogenic inflammation alters cold sensitivity via localized artemin release that induces cold pain via GFRα3 and TRPM8.SIGNIFICANCE STATEMENT The cellular and molecular mechanisms that generate pain are complex with a diverse array of pain-producing molecules generated during injury that act to sensitize peripheral sensory neurons, thereby inducing pain. Here we identify a specific neuroinflammatory pathway involving the ion channel TRPM8 (transient receptor potential cation channel subfamily M member 8) and the neurotrophin receptor GFRα3 (GDNF receptor α3) that leads to cold pain, providing select targets for potential therapies for this pain modality.

GDNF Increases Inhibitory Synaptic Drive on Principal Neurons in the Hippocampus via Activation of the Ret Pathway.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to counteract seizures when overexpressed or delivered into the brain in various animal models of epileptogenesis or chronic epilepsy. The mechanisms underlying this effect have not been investigated. We here demonstrate for the first time that GDNF enhances GABAergic inhibitory drive onto mouse pyramidal neurons by modulating postsynaptic GABAA receptors, particularly in perisomatic inhibitory synapses, by GFRα1 mediated activation of the Ret receptor pathway. Other GDNF receptors, such as NCAM or Syndecan3, are not contributing to this effect. We observed similar alterations by GDNF in human hippocampal slices resected from epilepsy patients. These data indicate that GDNF may exert its seizure-suppressant action by enhancing GABAergic inhibitory transmission in the hippocampal network, thus counteracting the increased excitability of the epileptic brain. This new knowledge can contribute to the development of novel, more precise treatment strategies based on a GDNF gene therapy approach.

Reawakening GDNF's regenerative past in mice and humans.

The ability of an animal to regenerate lost tissue and body parts has obviously life-saving implications. Understanding how this ability became restricted or active in specific animal lineages will help us understand our own regeneration. According to phylogenic analysis, the glial cell line-derived neurotrophic factor (GDNF) signaling pathway, but not other family members, is conserved in axolotls, a salamander with remarkable regenerative capacity. Furthermore, comparing the pro-regenerative Spiny mouse to its less regenerative descendant, the House mouse, revealed that the GDNF signaling pathway, but not other family members, was induced in regenerating Spiny mice. According to GDNF receptor expression analysis, GDNF may promote hair follicle neogenesis – an important feature of skin regeneration – by determining the fate of dermal fibroblasts as part of new hair follicles. These findings support the idea that GDNF treatment will promote skin regeneration in humans by demonstrating the GDNF signaling pathway's ancestral and cellular nature.

Decreased Expression of Glial-Derived Neurotrophic Factor Receptors in Glaucomatous Human Retinas

Purpose The purpose of this study was to examine the expression of glial-derived neurotrophic factor (GDNF), the GDNF receptors GFRα1 and GFRα2, ciliary neurotrophic factor (CNTF), and the CNTF receptor CNTFRα in normal and glaucomatous human tissue. Methods Human retinas were collected from 8 donors that had been clinically diagnosed and treated for glaucoma, and also from 9 healthy control donors. Immunohistochemical analysis for each trophic factor and receptor was performed. The percent of each retinal section labeled with each antibody was quantified for the total retinal thickness, and separately for the retinal ganglion cell (RGC) complex + retinal nerve fiber layer (RNFL). The expression of each protein was correlated with measures of the subject’s ocular histories. Results The percentage area immunopositive for GFRα2 was significantly decreased in the total retinal thickness containing all retinal layers and in the combined RGC complex + RNFL in glaucomatous eyes in both the peripapillary region and more peripheral retinal locations. We also observed a decrease in GFRα1 expression in the peripapillary RGC Complex + RNFL in glaucoma patients compared to healthy control patients. We also observed a relationship between GDNF and its receptors with several outcomes obtained from the medical record. No differences in CNTF or CNTFR labeling were observed. Conclusion Decreases in GDNF receptor expression in glaucomatous tissue may limit the potential for neuroprotective therapy by supplementation with GDNF.

GDNF neurotrophic factor signalling determines the fate of dermal fibroblasts in wound-induced hair neogenesis and skin regeneration.

We propose that GDNF, a glial cell line‐derived neurotrophic factor, can promote hair follicle neogenesis and skin regeneration after wounding by directing the fate of dermal fibroblasts. Our hypothesis is largely based on detailed GDNF and receptor analysis during skin regenerative stages, as well as the induction of GDNF receptors after wounding between the pro‐regenerative spiny mouse (genus Acomys) and its less‐regenerative descendant, the house mouse (Mus musculus). To characterize the GDNF‐target cells, we will conduct a series of lineage‐tracing experiments in conjunction with single‐cell RNA and assay for transposase‐accessible chromatin sequencing experiments. The heterogenetic dynamics of skin regeneration have yet to be fully defined, and this research will help to advance the fields of regenerative medicine and biology. Finally, we believe that stimulating the GDNF signalling pathway in fibroblasts from less‐regenerative animals, such as humans, will promote skin regeneration, morphogenesis and scarless wound healing.

Edaravone activates the GDNF/RET neurotrophic signaling pathway and protects mRNA-induced motor neurons from iPS cells

BackgroundSpinal cord motor neurons (MNs) from human iPS cells (iPSCs) have wide applications in disease modeling and therapeutic development for amyotrophic lateral sclerosis (ALS) and other MN-associated neurodegenerative diseases. We need highly efficient MN differentiation strategies for generating iPSC-derived disease models that closely recapitulate the genetic and phenotypic complexity of ALS. An important application of these models is to understand molecular mechanisms of action of FDA-approved ALS drugs that only show modest clinical efficacy. Novel mechanistic insights will help us design optimal therapeutic strategies together with predictive biomarkers to achieve better efficacy.MethodsWe induce efficient MN differentiation from iPSCs in 4 days using synthetic mRNAs coding two transcription factors (Ngn2 and Olig2) with phosphosite modification. These MNs after extensive characterization were applied in electrophysiological and neurotoxicity assays as well as transcriptomic analysis, to study the neuroprotective effect and molecular mechanisms of edaravone, an FDA-approved drug for ALS, for improving its clinical efficacy.ResultsWe generate highly pure and functional mRNA-induced MNs (miMNs) from control and ALS iPSCs, as well as embryonic stem cells. Edaravone alleviates H2O2-induced neurotoxicity and electrophysiological dysfunction in miMNs, demonstrating its neuroprotective effect that was also found in the glutamate-induced miMN neurotoxicity model. Guided by the transcriptomic analysis, we show a previously unrecognized effect of edaravone to induce the GDNF receptor RET and the GDNF/RET neurotrophic signaling in vitro and in vivo, suggesting a clinically translatable strategy to activate this key neuroprotective signaling. Notably, edaravone can replace required neurotrophic factors (BDNF and GDNF) to support long-term miMN survival and maturation, further supporting the neurotrophic function of edaravone-activated signaling. Furthermore, we show that edaravone and GDNF combined treatment more effectively protects miMNs from H2O2-induced neurotoxicity than single treatment, suggesting a potential combination strategy for ALS treatment.ConclusionsThis study provides methodology to facilitate iPSC differentiation and disease modeling. Our discoveries will facilitate the development of optimal edaravone-based therapies for ALS and potentially other neurodegenerative diseases.Graphical abstract

Glial cell line derived neurotrophic factor in brain repair after focal ischemic stroke.

Glial cell line derived neurotrophic factor in brain repair after focal ischemic stroke.

Valproic Acid Increases the Level of Expression of BDNF, GDNF and THEIR Receptors in the Embryonic Brain of Mice and Inducts the Stereotype Behavior

Valproic Acid Increases the Level of Expression of BDNF, GDNF and THEIR Receptors in the Embryonic Brain of Mice and Inducts the Stereotype Behavior

GDNF to the rescue: GDNF delivery effects on motor neurons and nerves, and muscle re-innervation after peripheral nerve injuries.

Peripheral nerve injuries commonly occur due to trauma, like a traffic accident. Peripheral nerves get severed, causing motor neuron death and potential muscle atrophy. The current golden standard to treat peripheral nerve lesions, especially lesions with large (≥ 3 cm) nerve gaps, is the use of a nerve autograft or reimplantation in cases where nerve root avulsions occur. If not tended early, degeneration of motor neurons and loss of axon regeneration can occur, leading to loss of function. Although surgical procedures exist, patients often do not fully recover, and quality of life deteriorates. Peripheral nerves have limited regeneration, and it is usually mediated by Schwann cells and neurotrophic factors, like glial cell line-derived neurotrophic factor, as seen in Wallerian degeneration. Glial cell line-derived neurotrophic factor is a neurotrophic factor known to promote motor neuron survival and neurite outgrowth. Glial cell line-derived neurotrophic factor is upregulated in different forms of nerve injuries like axotomy, sciatic nerve crush, and compression, thus creating great interest to explore this protein as a potential treatment for peripheral nerve injuries. Exogenous glial cell line-derived neurotrophic factor has shown positive effects in regeneration and functional recovery when applied in experimental models of peripheral nerve injuries. In this review, we discuss the mechanism of repair provided by Schwann cells and upregulation of glial cell line-derived neurotrophic factor, the latest findings on the effects of glial cell line-derived neurotrophic factor in different types of peripheral nerve injuries, delivery systems, and complementary treatments (electrical muscle stimulation and exercise). Understanding and overcoming the challenges of proper timing and glial cell line-derived neurotrophic factor delivery is paramount to creating novel treatments to tend to peripheral nerve injuries to improve patients’ quality of life.

Neuro-mesenchymal units control ILC2 and obesity via a brain-adipose circuit.

Signals from sympathetic neurons and immune cells regulate adipocytes and thereby contribute to fat tissue biology. Interactions between the nervous and immune systems have recently emerged as important regulators of host defence and inflammation1-4. Nevertheless, it is unclear whether neuronal and immune cells co-operate in brain-body axes to orchestrate metabolism and obesity. Here we describe a neuro-mesenchymal unit that controls group 2 innate lymphoid cells (ILC2s), adipose tissue physiology, metabolism and obesity via a brain-adipose circuit. We found that sympathetic nerve terminals act on neighbouring adipose mesenchymal cells via the β2-adrenergic receptor to control the expression of glial-derived neurotrophic factor (GDNF) and the activity of ILC2s in gonadal fat. Accordingly, ILC2-autonomous manipulation of the GDNF receptor machinery led to alterations in ILC2 function, energy expenditure, insulin resistance and propensity to obesity. Retrograde tracing and chemical, surgical and chemogenetic manipulations identified a sympathetic aorticorenal circuit that modulates ILC2s in gonadal fat and connects to higher-order brain areas, including the paraventricular nucleus of the hypothalamus. Our results identify a neuro-mesenchymal unit that translates cues from long-range neuronal circuitry into adipose-resident ILC2 function, thereby shaping host metabolism and obesity.

3D-printed nerve guidance conduits multi-functionalized with canine multipotent mesenchymal stromal cells promote neuroregeneration after sciatic nerve injury in rats

BackgroundNerve injuries are debilitating, leading to long-term motor deficits. Remyelination and axonal growth are supported and enhanced by growth factor and cytokines. Combination of nerve guidance conduits (NGCs) with adipose-tissue-derived multipotent mesenchymal stromal cells (AdMSCs) has been performing promising strategy for nerve regeneration.Methods3D-printed polycaprolactone (PCL)-NGCs were fabricated. Wistar rats subjected to critical sciatic nerve damage (12-mm gap) were divided into sham, autograft, PCL (empty NGC), and PCL + MSCs (NGC multi-functionalized with 106 canine AdMSCs embedded in heterologous fibrin biopolymer) groups. In vitro, the cells were characterized and directly stimulated with interferon-gamma to evaluate their neuroregeneration potential. In vivo, the sciatic and tibial functional indices were evaluated for 12 weeks. Gait analysis and nerve conduction velocity were analyzed after 8 and 12 weeks. Morphometric analysis was performed after 8 and 12 weeks following lesion development. Real-time PCR was performed to evaluate the neurotrophic factors BDNF, GDNF, and HGF, and the cytokine and IL-10. Immunohistochemical analysis for the p75NTR neurotrophic receptor, S100, and neurofilament was performed with the sciatic nerve.ResultsThe inflammatory environment in vitro have increased the expression of neurotrophins BDNF, GDNF, HGF, and IL-10 in canine AdMSCs. Nerve guidance conduits multi-functionalized with canine AdMSCs embedded in HFB improved functional motor and electrophysiological recovery compared with PCL group after 12 weeks. However, the results were not significantly different than those obtained using autografts. These findings were associated with a shift in the regeneration process towards the formation of myelinated fibers. Increased immunostaining of BDNF, GDNF, and growth factor receptor p75NTR was associated with the upregulation of BDNF, GDNF, and HGF in the spinal cord of the PCL + MSCs group. A trend demonstrating higher reactivity of Schwann cells and axonal branching in the sciatic nerve was observed, and canine AdMSCs were engrafted at 30 days following repair.Conclusions3D-printed NGCs multi-functionalized with canine AdMSCs embedded in heterologous fibrin biopolymer as cell scaffold exerted neuroregenerative effects. Our multimodal approach supports the trophic microenvironment, resulting in a pro-regenerative state after critical sciatic nerve injury in rats.

Intestinal Epithelial Barrier Maturation by Enteric Glial Cells Is GDNF-Dependent.

Enteric glial cells (EGCs) of the enteric nervous system are critically involved in the maintenance of intestinal epithelial barrier function (IEB). The underlying mechanisms remain undefined. Glial cell line-derived neurotrophic factor (GDNF) contributes to IEB maturation and may therefore be the predominant mediator of this process by EGCs. Using GFAPcre x Ai14floxed mice to isolate EGCs by Fluorescence-activated cell sorting (FACS), we confirmed that they synthesize GDNF in vivo as well as in primary cultures demonstrating that EGCs are a rich source of GDNF in vivo and in vitro. Co-culture of EGCs with Caco2 cells resulted in IEB maturation which was abrogated when GDNF was either depleted from EGC supernatants, or knocked down in EGCs or when the GDNF receptor RET was blocked. Further, TNFα-induced loss of IEB function in Caco2 cells and in organoids was attenuated by EGC supernatants or by recombinant GDNF. These barrier-protective effects were blunted when using supernatants from GDNF-deficient EGCs or by RET receptor blockade. Together, our data show that EGCs produce GDNF to maintain IEB function in vitro through the RET receptor.

Synergistic interaction of nerve growth factor and glial cell-line derived neurotrophic factor in muscular mechanical hyperalgesia in rats.

Nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) are essential for neuronal development and survival in embryo. However, after birth they play pivotal roles in the generation of hyperalgesia in many painful conditions. Both factors are believed to act on different groups of primary afferents, but interaction between them has not yet been studied. Here we show a synergism between the two factors. Intramuscular injection of a mixture of both factors at a low concentration, each of which alone had no effect, induced a significant muscular mechanical hyperalgesia in rats. We show that synergism occurs in the primary afferent neurons and find that about 25% primary afferents innervating the muscle express both TrkA (NGF receptor) and GFRα1 (GDNF receptor). We show by pharmacological means that afferent neurons with TrkA and GFRα1 express both TRPV1 and ASICs. Our data establish a basis for synergism between NGF and GDNF. In some inflammatory conditions both nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) are upregulated and play pivotal roles in inducing hyperalgesia. However, their interaction has not been studied. We examined whether and where interaction between both neurotrophic factors occurs in SD rats. Intramuscular injection to gastrocnemius muscle (GC) of a mixture of NGF (0.1µm) and GDNF (0.008µm), which alone had no effect, induced a significant mechanical hyperalgesia (F(6,30) =13.62, P=0.0001), demonstrating synergism between the two factors. Phosphorylated extracellular signal-regulated kinase (pERK) immunoreactivity in dorsal root ganglia (DRGs) induced by compression of GC increased after injection of the mixture (P=0.028, compared with PBS); thus the interaction of NGF and GDNF could occur at the primary afferent level. An in situ hybridization study (n=4) demonstrated that 23.7-29.2% of GC-innervating DRG neurons coexpressed TrkA (NGF receptor) and GFRα1 (GDNF receptor). The cell size of the coexpressing GC DRG neurons showed no skewing towards the small size range but was distributed widely from the small to the large size ranges. Therefore, some of the coexpressing neurons with thin axons are thought to contribute to this mechanical hyperalgesia. The hyperalgesia was reversed by both amiloride (F(1,13) =5.056, P=0.0425, compared with PBS) and capsazepine (F(1,10) =8.402, P=0.0159, compared with DMSO), suggesting that the primary afferents sensitized by the mixture express both TRPV1 and ASICs. These results showed a basis of synergism between NGF and GDNF.

Chronic 2-Fold Elevation of Endogenous GDNF Levels Is Safe and Enhances Motor and Dopaminergic Function in Aged Mice

Glial cell line-derived neurotrophic factor (GDNF) supports function and survival of dopamine neurons that degenerate in Parkinson’s disease (PD). Ectopic delivery of GDNF in clinical trials to treat PD is safe but lacks significant therapeutic effect. In pre-clinical models, ectopic GDNF is effective but causes adverse effects, including downregulation of tyrosine hydroxylase, only a transient boost in dopamine metabolism, aberrant neuronal sprouting, and hyperactivity. Hindering development of GDNF mimetic increased signaling via GDNF receptor RET by activating mutations results in cancer. Safe and effective mode of action must be defined first in animal models to develop successful GDNF-based therapies. Previously we showed that about a 2-fold increase in endogenous GDNF expression is safe and results in increased motor and dopaminergic function and protection in a PD model in young animals. Recently, similar results were reported using a novel Gdnf mRNA-targeting strategy. Next, it is important to establish the safety of a long-term increase in endogenous GDNF expression. We report behavioral, dopamine system, and cancer analysis of five cohorts of aged mice with a 2-fold increase in endogenous GDNF. We found a sustained increase in dopamine levels, improvement in motor learning, and no side effects or cancer. These results support the rationale for further development of endogenous GDNF-based treatments and GDNF mimetic.

Fisetin Prevents HT22 Cells From High Glucose-Induced Neurotoxicity via PI3K/Akt/CREB Signaling Pathway.

Hyperglycemia has been widely considered as a key risk factor for diabetic encephalopathy which can cause neuronal apoptosis and cognitive deficits. The flavonoid compound, fisetin, possesses potential neuroprotective effects and also enhances learning and memory. However, the role of fisetin in hyperglycemia-induced neuronal cytotoxicity has not been fully elucidated. In the present study, HT22 murine hippocampal neuronal cell line was used to establish the injured cell model. Cell proliferation and cytotoxicity assay, Hoechst 33258 staining, qRT-PCR, western blot analysis, and specific inhibitor were used to investigate the effect and molecular mechanisms of fisetin on high glucose (HG)-induced neurotoxicity in HT22 cells. Our results showed that 125 μM and 48 h of treatment was identified as optimal damage parameter of HG. Fisetin significantly improved HG-inhibited cell viability. The levels of LDH, malondialdehyde (MDA), and superoxide dismutase (SOD) were noticeably modulated by fisetin, which alleviated HG-induced HT22 cell oxidative damage. Besides, the apoptosis of HT22 cells was rescued by fisetin pretreatment. In addition, fisetin also prevented HG-induced downregulation of the mRNA expression of Bdnf, Gdnf, synaptophysin (Syp), and glutamate ionotropic receptor AMPA type subunit 1 (Gria1) in cells. More importantly, the decreased phosphorylation of phosphoinositide 3 kinase (PI3K), Akt, and cAMP-response element binding protein (CREB) was rescued by fisetin treatment and that neuroprotective effect of fisetin was partially blocked by PI3K inhibitor, LY294002. These findings indicate that fisetin has potent neuroprotective effect and prevents HG-induced neurotoxicity by activation of PI3K/Akt/CREB pathway.

Glial cell line-derived neurotrophic factor receptor Rearranged during transfection agonist supports dopamine neurons in Vitro and enhances dopamine release In Vivo.

BackgroundMotor symptoms of Parkinson's disease (PD) are caused by degeneration and progressive loss of nigrostriatal dopamine neurons. Currently, no cure for this disease is available. Existing drugs alleviate PD symptoms but fail to halt neurodegeneration. Glial cell line–derived neurotrophic factor (GDNF) is able to protect and repair dopamine neurons in vitro and in animal models of PD, but the clinical use of GDNF is complicated by its pharmacokinetic properties. The present study aimed to evaluate the neuronal effects of a blood‐brain‐barrier penetrating small molecule GDNF receptor Rearranged in Transfection agonist, BT13, in the dopamine system.MethodsWe characterized the ability of BT13 to activate RET in immortalized cells, to support the survival of cultured dopamine neurons, to protect cultured dopamine neurons against neurotoxin‐induced cell death, to activate intracellular signaling pathways both in vitro and in vivo , and to regulate dopamine release in the mouse striatum as well as BT13's distribution in the brain.ResultsBT13 potently activates RET and downstream signaling cascades such as Extracellular Signal Regulated Kinase and AKT in immortalized cells. It supports the survival of cultured dopamine neurons from wild‐type but not from RET‐knockout mice. BT13 protects cultured dopamine neurons from 6‐Hydroxydopamine (6‐OHDA) and 1‐methyl‐4‐phenylpyridinium (MPP+)–induced cell death only if they express RET. In addition, BT13 is absorbed in the brain, activates intracellular signaling cascades in dopamine neurons both in vitro and in vivo, and also stimulates the release of dopamine in the mouse striatum.ConclusionThe GDNF receptor RET agonist BT13 demonstrates the potential for further development of novel disease‐modifying treatments against PD. © 2019 The Authors. Movement Disorders published by Wiley Periodicals LLC. on behalf of International Parkinson and Movement Disorder Society.