Glial Cell Line-derived Neurotrophic Factor Protein Research Articles

Interactions between neurotrophins, mood, and physical activity under the conditions of sleep deprivation

Sleep deprivation (DS) is the forced elimination of sleep. While brain-derived neurotrophic factor (BDNF) has been extensively studied in the context of in mood changes following DS, the role of other neurotrophins remains elusive. This study explores the impact of DS on BDNF, glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT3), and neurotrophin-4 (NT4) at mRNA and protein level, considering their potential links to mood disturbances. The study involved 81 participants subjected to polysomnography (PSG) and DS. Blood samples, mood assessments, and actigraphy data were collected twice, after PSG and DS. NT mRNA expression and serum protein concentrations of BDNF, GDNF, NT3, and NT4 were measured. Participants were divided into Responders and Non-Responders based on mood improvement after DS. DS reduced BDNF mRNA expression in all participants, with no change in serum BDNF protein. GDNF protein decreased in Non-Responders, while Responders exhibited reduced GDNF mRNA. NT3 protein increased in both groups, while NT3 mRNA decreased in Respondents. NT4 protein rose universally post-DS, but NT4 mRNA remained unchanged. Physical activity (PA) negatively correlated with mRNA expression of BDNF, GDNF, and NT3 post-DS. The study’s short DS duration and exclusion of immature NT forms limit comprehensive insights. GDNF, together with NT3, might play an important role in mood response to DS. PA during DS seems to impair the mRNA expression of NTs in leukocytes. Future studies on the subject of sleep deprivation might consider investigating the relationship between BDNF and NT4 in the context of their apparent redundancy.

Evaluation of the Continuous Positive Airway Pressure Effect on Neurotrophins’ Gene Expression and Protein Levels

Neurotrophins (NT) might be associated with the pathophysiology of obstructive sleep apnea (OSA) due to concurrent intermittent hypoxia and sleep fragmentation. Such a relationship could have implications for the health and overall well-being of patients; however, the literature on this subject is sparse. This study investigated the alterations in the serum protein concentration and the mRNA expression of the brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NTF3), and neurotrophin-4 (NTF4) proteins following a single night of continuous positive airway pressure (CPAP) therapy. This study group consisted of 30 patients with OSA. Venous blood was collected twice after a diagnostic polysomnography (PSG) and PSG with CPAP treatment. Gene expression was assessed with a quantitative real-time polymerase chain reaction. An enzyme-linked immunosorbent assay was used to determine the protein concentrations. After CPAP treatment, BDNF, proBDNF, GDNF, and NTF4 protein levels decreased (p = 0.002, p = 0.003, p = 0.047, and p = 0.009, respectively), while NTF3 increased (p = 0.001). Sleep latency was correlated with ΔPSG + CPAP/PSG gene expression for BDNF (R = 0.387, p = 0.038), NTF3 (R = 0.440, p = 0.019), and NTF4 (R = 0.424, p = 0.025). OSA severity parameters were not associated with protein levels or gene expressions. CPAP therapy could have an impact on the posttranscriptional stages of NT synthesis. The expression of different NTs appears to be connected with sleep architecture but not with OSA severity.

Effects of gestational inflammation on age-related cognitive decline and hippocampal Gdnf-GFRα1 levels in F1 and F2 generations of CD-1 Mice

BackgroundIt has been reported that age-associated cognitive decline (AACD) accelerated by maternal lipopolysaccharide (LPS) insult during late pregnancy can be transmitted to the second generation in a sex-specificity manner. In turn, recent studies indicated that glial cell line‐derived neurotrophic factor (GDNF) and its cognate receptor (GFRα1) are critical for normal cognitive function. Based on this evidence, we aimed to explore whether Gdnf-GFRα1 expression contributes to cognitive decline in the F1 and F2 generations of mouse dams exposed to lipopolysaccharide (LPS) during late gestation, and to evaluate also the potential interference effect of pro-inflammatory cytokines.MethodsDuring gestational days 15–17, pregnant CD-1 mice (8–10 weeks old) received a daily intraperitoneal injection of LPS (50 μg/kg) or saline (control). In utero LPS-exposed F1 generation mice were selectively mated to produce F2 generation mice. In F1 and F2 mice aged 3 and 15 months, the Morris water maze (MWM) was used to evaluated the spatial learning and memory ability, the western blotting and RT-PCR were used for analyses of hippocampal Gdnf and GFRα1 expression, and ELISA was used to analyse IL-1β, IL-6 and TNF-α levels in serum.ResultsMiddle-aged F1 offspring from LPS-treated mothers exhibited longer swimming latency and distance during the learning phase, lower percentage swimming time and distance in targe quadrant during memory phase, and lower hippocampal levels of Gdnf and GFRα1 gene products compared to age-matched controls. Similarly, the middle-aged F2 offspring from the Parents-LPS group had longer swimming latency and distance in the learning phase, and lower percentage swimming time and distance in memory phase than the F2-CON group. Moreover, the 3-month-old Parents-LPS and 15-month-old Parents- and Father-LPS groups had lower GDNF and GFRα1 protein and mRNAs levels compared to the age-matched F2-CON group. Furthermore, hippocampal levels of Gdnf and GFRα1 were correlated with impaired cognitive performance in the Morris water maze after controlling for circulating pro-inflammatory cytokine levels.ConclusionsOur findings indicate that accelerated AACD by maternal LPS exposure can be transmitted across at least two generations through declined Gdnf and GFRα1 expression, mainly via paternal linage.

The Polyunsaturated Fatty Acid EPA, but Not DHA, Enhances Neurotrophic Factor Expression through Epigenetic Mechanisms and Protects against Parkinsonian Neuronal Cell Death.

ω-3 Polyunsaturated fatty acids (PUFAs) have been found to exert many actions, including neuroprotective effects. In this regard, the exact molecular mechanisms are not well understood. Parkinson's disease (PD) is the second most common age-related neurodegenerative disease. Emerging evidence supports the hypothesis that PD is the result of complex interactions between genetic abnormalities, environmental toxins, mitochondrial dysfunction, and other cellular processes, such as DNA methylation. In this context, BDNF (brain-derived neurotrophic factor) and GDNF (glial cell line-derived neurotrophic factor) have a pivotal role because they are both involved in neuron differentiation, survival, and synaptogenesis. In this study, we aimed to elucidate the potential role of two PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and their effects on BDNF and GDNF expression in the SH-SY5Y cell line. Cell viability was determined using the MTT assay, and flow cytometry analysis was used to verify the level of apoptosis. Transmission electron microscopy was performed to observe the cell ultrastructure and mitochondria morphology. BDNF and GDNF protein levels and mRNA were assayed by Western blotting and RT-PCR, respectively. Finally, methylated and hydroxymethylated DNA immunoprecipitation were performed in the BDNF and GDNF promoter regions. EPA, but not DHA, is able (i) to reduce the neurotoxic effect of neurotoxin 6-hydroxydopamine (6-OHDA) in vitro, (ii) to re-establish mitochondrial function, and (iii) to increase BNDF and GDNF expression via epigenetic mechanisms.

Overexpression of microRNA-211 in Functional Dyspepsia via Downregulation of the Glial Cell Line-Derived Neurotrophic Factor (GDNF) by Increasing Phosphorylation of p38 MAPK Pathway.

Overexpression of miRNA-211 suppresses the differentiation of bone marrow stem cells into intestinal ganglion cells via downregulation of GDNF, a regulator of intestine barrier function. The study aimed to investigate the interaction between miR-211 and GDNF on intestinal epithelial cells. The expression levels of miR-211 and GDNF in duodenal biopsy specimens from FD patients and healthy controls were compared. Enteric glia cell (EGCs) cell line transfected with miR-211 mimics and inhibitors were used to clarify the expression levels of GDNF were analyzed by qRT-PCR and ELISA. Intestine epithelial cell (IECs) cell line cultured in medium from ECGs in different transfection conditions were used in wound healing assay, cell proliferation assay, and western blotting for evaluation of p38 MAPK phosphorylation level. MiR-211 expression was significantly upregulated in the duodenal tissue of patients with FD compared to healthy subjects, whereas GDNF expression was significantly downregulated (both p < 0.05). Transfection with miR-211 mimics significantly decreased GDNF mRNA expression and protein secretion (p < 0.001). An inhibited intestinal epithelial cell wound healing (p < 0.05) and increased expression levels of phosphorylated p38 MAPK (p < 0.05) were found in IECs cultured with medium from EGCs transfected with miR-211 mimics. MiR-211 may downregulates GDNF mRNA and protein expression via activation of the pp38 MAPK signaling pathway. Targeting miR-211 or the MAPK pathway may be a potential intervention for FD.

Glial cell line-derived neurotrophic factor contributes to alcoholic-induced liver injury by regulating the NF-κB pathway.

Alcoholic liver disease (ALD) is associated with high morbidity and mortality worldwide. The pathogenesis of ALD is not completely understood. Although accumulating evidence suggests an important role of glial cell line-derived neurotrophic factor (GDNF) in several diseases, there are no data concerning its role in ALD. This study compared patients with ALD with control subjects and used a mouse model and a cell culture model to investigate the function of GDNF in ALD and its mechanism of action in hepatocyte injury. Serum levels of GDNF were measured in 25 patients with ALD and 25 healthy control subjects. A 4-week Lieber-DeCarli ethanol (EtOH) liquid diet combined with the Gao-Binge model was used in the mouse study. Mouse primary hepatocytes and Huh-7 cells were used for cell experiments. The parameters of liver injury, inflammatory cytokines, and lipid metabolism were measured. Patients with alcoholic hepatitis had higher serum GDNF than control subjects. Expression of GDNF mRNA and protein was markedly increased in mice in the chronic-plus-binge ALD mouse model. The level of GDNF mRNA was upregulated in primary hepatic stellate cells isolated from ethanol-fed mouse liver. Ethanol induced GDNF expression in LX2 cells. The levels of inflammatory cytokines (tumor necrosis factor α, interleukin 1β, and monocyte chemotactic protein 1) were significantly increased after GDNF stimulation in primary hepatocytes and Huh-7 cells. After GDNF stimulation, levels of both p-AKT and p-NF-κB were significantly increased in primary hepatocytes and Huh-7 cells. The NF-κB activity induced by GDNF was significantly decreased by an NF-κB inhibitor, which limited hepatocyte injury and inflammation. The concentration of GDNF is increased in the circulation of ALD patients. GDNF promotes alcohol-induced liver injury and inflammation via the activation of NF-κB, which mediates hepatocyte injury and inflammatory cytokine expression. Based on these findings, GDNF is a potential therapeutic target for preventing or ameliorating liver injury in ALD.

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.

TNF-α inhibits GDNF levels in Sertoli cells, through a NF-κB-dependent, HES1-dependent mechanism.

Glial cell line-derived neurotrophic factor (GDNF) is a soluble molecule crucial for the regulation of the spermatogonial stem cells (SSC) of the testis. The effects of GDNF on target cells have been extensively described, but mechanisms underlying GDNF regulation are currently under investigation. In the nervous system, GDNF expression is regulated by pro-inflammatory cytokines including lipopolysaccharide (LPS), interleukin 1 beta (IL-1β), and tumor necrosis factor alpha (TNF-α) but the effect of these cytokines on GDNF expression in the testis is unclear. The aim of the present study was to investigate the impact of TNF-α on GDNF expression levels using primary murine Sertoli cells as experimental model. The expression of TNF-α-regulated genes including Gdnf in different culture conditions was determined by real-time PCR. GDNF protein levels were determined by ELISA. The activation of the NF-κb pathway and HES1 levels were assessed by Western Blot analysis and immunofluorescence. HES1 expression was downregulated by RNAi. In primary Sertoli cells, TNF-α downregulates GDNF levels through a nuclear factor-κB (NF-κB)-dependent mechanism. Mechanistically, TNF-α induces the transcriptional repressor HES1 by a NF-Κb-dependent mechanism, which in turn downregulates GDNF. Under physiological conditions, TNF-α is secreted by germ cells suggesting that this cytokine plays a role in the paracrine control of SSC niche by modulating GDNF levels. HES1, a well-known target of the Notch pathway, is implicated in the regulation of GDNF expression. In Sertoli cells, TNF-α and Notch signaling may converge at molecular level, to regulate the expression of HES1 and HES1- target genes, including GDNF. Because of the importance of GDNF for spermatogonial stem cell self-renewal and proliferation, this data may give important insights on how cytokine signals in the testis modulate the expression of niche-derived factors.

The Effects of Deep Brain Stimulation of the Subthalamic Nucleus on Vascular Endothelial Growth Factor, Brain-Derived Neurotrophic Factor, and Glial Cell Line-Derived Neurotrophic Factor in a Rat Model of Parkinson’s Disease

Objective: Deep brain stimulation (DBS) of the subthalamic nucleus (STN) has evolved as a powerful therapeutic alternative for the treatment of Parkinson’s disease (PD). Despite its clinical efficacy, the mechanisms of action have remained poorly understood. In addition to the immediate symptomatic effects, long-term neuroprotective effects have been suggested. Those may be mediated through neurotrophic factors (NFs) like vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), and glial cell line-derived neurotrophic factor (GDNF). Here, the impact of DBS on the expression of NFs was analysed in a rat model of PD. Methods: Unilateral 6-hydroxydopamine (6-OHDA) lesioned rats received DBS in the STN using an implantable microstimulation system, sham DBS in the STN, or no electrode placement. Continuous unilateral STN-DBS (current intensity 50 µA, frequency 130 Hz, and pulse width 52 µs) was conducted for 14 days. Rats were then sacrificed and brains shock frozen. Striata and motor cortices were dissected with a cryostat. Levels of VEGF, BDNF, and GDNF were analysed, both by quantitative PCR and colorimetric ELISA. Results: PCR revealed a significant upregulation of only BDNF mRNA in the ipsilateral striata of the DBS group, when compared to the sham-stimulated group. There was no significant increase in VEGF mRNA or GDNF mRNA. ELISA analysis showed augmentations of BDNF, VEGF, as well as GDNF protein in the ipsilateral striata after DBS compared to sham stimulation. In the motor cortex, significant increases after DBS were observed for BDNF only, not for the other 2 NFs. Conclusions: The upregulation of trophic factors induced by STN-DBS may participate in its long-term therapeutic efficacy and potentially neuroprotective effects.

A regulatable adenovector system for GDNF and GFP delivery in the rat hippocampus

Spatial memory performance declines in both normal aging and Alzheimer's disease. This cognitive deficit is related to hippocampus dysfunction. Gene therapy using neurotrophic factors like Glial cell line-derived neurotrophic factor (GDNF) emerges as a promising approach to ameliorate age-related cognitive deficits. We constructed a two vector regulatable system (2VRS) which consists of a recombinant adenoviral vector (RAd) harboring a Tet-Off bidirectional promoter flanked by GDNF and Green Fluorescent Protein (GFP) genes. A second adenovector, RAd-tTA, constitutively expresses the regulatory protein tTA. When cells are cotransduced by the 2VRS, tTA activates the bidirectional promoter and both transgenes are expressed. In the presence of the antibiotic doxycycline (DOX) transgene expression is silenced. We tested the 2VRS in CHO-K1 cells where we observed a dose-dependent GFP expression that was completely inhibited by DOX (1 mg/ml). The 2VRS injected in the hippocampal CA1 region transduced both neurons and astrocytes and was efficiently inhibited by DOX added to the drinking water. In order to assess GDNF biological activity we injected 2VRS and its Control (CTRL) vector in the hypothalamus and monitored body weight for one month. The results showed that GDNF retards weight recovery 6 days more than CTRL. In conclusion, our 2VRS demonstrated optimal GFP expression and showed a bioactive effect of transgenic GDNF in the brain.

Decreased abundance of GDNF mRNA transcript in the immature Sertoli cells of cattle in response to protein kinase inhibitor staurosporine

Sertoli cells (SC) have important functions in spermatogenesis by regulating development of spermatogenic cells. Glial cell line-derived neurotrophic factor (GDNF) are produced by SC. Although the effects of GDNF on spermatogenesis have been well studied, the understanding of how GDNF is synthesized is still limited, especially in food animal producing species. Because protein kinase (PK) has varied functions in multiple cellular processes and the PK pathway modulates SC functions, the objective of the present study was to determine whether PK modulates the abundance of GDNF protein in SC of cattle. To conduct this study, immature SC were enriched from cryopreserved testicular tissues of 1-day-old bulls. These cells had a marked proliferation capacity. Results from immunostaining analysis indicated that there was a sustained abundance of SC mRNA marker protein transcripts and marker proteins: androgen bind protein (ABP), GATA4 and VIMENTIN. There was subsequent characterization of SC treated with the PK inhibitor staurosporine for 0, 1 or 2 h. Results from real-time-PCR and Western blot analyses indicated the treatment (2 h) resulted in a decrease in Gdnf mRNA transcript and GDNF protein. Additionally, the staurosporine treatment resulted in an increase in the abundance of anti-apoptosis Bcl2 and decrease in pro-apoptosis Bax mRNA transcripts. Furthermore, results of the TUNEL assay indicated there was a decrease in apoptosis in the staurosprine-treated SC. Collectively, results indicate the PK signaling is involved in regulation of GDNF protein abundance in the immature SC and the survival of these cells in cattle.

FTY720-Mitoxy reduces synucleinopathy and neuroinflammation, restores behavior and mitochondria function, and increases GDNF expression in Multiple System Atrophy mouse models

Multiple system atrophy (MSA) is a fatal disorder with no effective treatment. MSA pathology is characterized by α-synuclein (aSyn) accumulation in oligodendrocytes, the myelinating glial cells of the central nervous system (CNS). aSyn accumulation in oligodendrocytes forms the pathognomonic glial cytoplasmic inclusions (GCIs) of MSA. MSA aSyn pathology is also associated with motor and autonomic dysfunction, including an impaired ability to sweat. MSA patients have abnormal CNS expression of glial-cell-line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF). Our prior studies using the parent compound FTY720, a food and drug administration (FDA) approved immunosuppressive for multiple sclerosis, reveal that FTY720 protects parkinsonian mice by increasing BDNF. Our FTY720-derivative, FTY720-Mitoxy, is known to increase expression of oligodendrocyte BDNF, GDNF, and nerve growth factor (NGF) but does not reduce levels of circulating lymphocytes as it is not phosphorylated so cannot modulate sphingosine 1 phosphate receptors (S1PRs). To preclinically assess FTY720-Mitoxy for MSA, we used mice expressing human aSyn in oligodendrocytes under a 2,’ 3′-cyclic nucleotide 3′-phosphodiesterase (CNP) promoter. CNP-aSyn transgenic (Tg) mice develop motor dysfunction between 7 and 9 mo, and progressive GCI pathology. Using liquid chromatography-mass spectrometry (LC-MS/MS) and enzymatic assays, we confirmed that FTY720-Mitoxy was stable and active. Vehicle or FTY720-Mitoxy (1.1 mg/kg/day) was delivered to wild type (WT) or Tg littermates from 8.5–11.5 mo by osmotic pump. We behaviorally assessed their movement by rotarod and sweat production by starch‑iodine test. Postmortem tissues were evaluated by qPCR for BDNF, GDNF, NGF and GDNF-receptor RET mRNA and for aSyn, BDNF, GDNF, and Iba1 protein by immunoblot. MicroRNAs (miRNAs) were also assessed by qPCR. FTY720-Mitoxy normalized movement, sweat function and soleus muscle mass in 11.5 mo Tg MSA mice. FTY720-Mitoxy also increased levels of brain GDNF and reduced brain miR-96-5p, a miRNA that acts to decrease GDNF expression. Moreover, FTY720-Mitoxy blocked aSyn pathology measured by sequential protein extraction and immunoblot, and microglial activation assessed by immunohistochemistry and immunoblot. In the 3-nitropropionic acid (3NP) toxin model of MSA, FTY720-Mitoxy protected movement and mitochondria in WT and CNP-aSyn Tg littermates. Our data confirm potent in vivo protection by FTY720-Mitoxy, supporting its further evaluation as a potential therapy for MSA and related synucleinopathies.

GDNFOS1 knockdown decreases the invasion and viability of glioblastoma cells.

Glioblastoma multiforme is the most aggressive primary brain cancer in adults. Therefore, it is important to investigate the mechanisms associated with cell viability and invasion ability of the cells in glioblastoma multiforme. The opposite strand of the glial cell line-derived neurotrophic factor (GDNF) gene is used to transcribe the cis-antisense GDNF opposite strand (GDNFOS) gene, which belongs to the long noncoding RNAs. The current study assessed the effects of GDNFOS1 overexpression and interference on GDNF expression, cell viability and invasion ability in U87 and U251 MG glioblastoma cells. Overexpression and interference were performed using constructed lentiviral vectors, including long non-coding RNA GDNFOS1 overexpression vector, pL-short hairpin RNA (shRNA)-GDNFOS1-9, pL-shRNA-GDNFOS1-49, pL-shRNA-GDNFOS1-248, pL-shRNA-GDNFOS1-9+49, pL-shRNA-GDNFOS1-9+248 and pL-shRNA-GDNFOS1-49+248. Reverse transcription-quantitative PCR was used to determine the efficiency of interference and overexpression of GDNFOS1 in U87 and U251 MG cells. GDNF protein expression in U87 and U251 MG cells was detected using western blot analysis. In addition, cell viability was detected using a cell counting kit-8 assay at 24, 48 and 72 h after GDNFOS1 overexpression or interference. A transwell invasion assay was used to detect invasion ability. Different shRNA sequences were tested and the results revealed that a combination (pL-shRNA-GDNFOS1-49+248) was most effective in the knock-down GDNFOS1. Compared with the control group, GDNF expression in U87 MG cells was significantly increased in the GDNFOS1 overexpression group and decreased in the shRNA-GDNFOS1-248 group. U87 MG cell viability was significantly increased in the GDNFOS1 overexpression group at 24, 48 and 72 h compared with the negative control group. The viability of U87 MG cells was decreased in the GDNFOS1 interference group at 72 h when compared with the control group. The relative invasive ability was significantly increased in the GDNFOS1 overexpression group when compared with the negative control group. The invasive ability was significantly decreased in the GDNFOS1 interference group when compared with the negative control group. Similar results were exhibited by the U251 MG cells. Overall, GDNF expression, cell viability and invasion ability of glioblastoma cells significantly increased with GDNFOS1 overexpression and decreased with GDNFOS1 interference.

Effects of Transplanted Umbilical Cord Blood Mononuclear Cells Overexpressing GDNF on Spatial Memory and Hippocampal Synaptic Proteins in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive incurable neurodegenerative disorder. Glial cell line-derived neurotrophic factor (GDNF) is a prominent regulator of brain tissue and has an impressive potential for use in AD therapy. While its metabolism is still not fully understood, delivering neuropeptides such as GDNF via umbilical cord blood mononuclear cells (UCBMCs) to the sites of neurodegeneration is a promising approach in the development of innovative therapeutic avenues. UCBMCs were transduced with adenoviral vectors expressing GDNF and injected into AD transgenic mice. Various parameters including homing and survival of transplanted cells, expression of GDNF and synaptic proteins, as well as spatial memory were evaluated. UCBMCs were observed in the hippocampus and cortex several weeks after transplantation, and their long-term presence was associated with improved spatial memory. Post-synaptic density protein 95 (PSD-95) and synaptophysin levels in the hippocampus were also effectively restored following the procedure in AD mice. Our data indicate that gene-cell therapy with GDNF-overexpressing UCBMCs may produce long-lasting neuroprotection and stimulation of synaptogenesis. Such adenoviral constructs could potentially possess a high therapeutic potential for the treatment of AD.

GDNF-ADSCs-APG embedding enhances sciatic nerve regeneration after electrical injury in a rat model.

The pluripotency of adipose-derived stem cells (ADSCs) makes them appropriate for tissue repair and wound healing. Owing to the repair properties of autologous platelet-rich gel (APG), which is based on easily accessible blood platelets, its clinical use has been increasingly recognized by physicians. The aim of this study was to investigate the effect of combined treatment with ADSCs and APG on sciatic nerve regeneration after electrical injury. To facilitate the differentiation of ADSCs, glial cell line-derived neurotrophic factor (GDNF) was overexpressed in ADSCs by lentivirus transfection. GDNF-ADSCs were mingled with APG gradient concentrations, and in vitro, cell proliferation and differentiation were examined with 5-ethynyl-2'-deoxyuridine staining and immunofluorescence. A rat model was established by exposing the sciatic nerve to an electrical current of 220 V for 3 seconds. Rat hind-limb motor function and sciatic nerve regeneration were subsequently evaluated. Rat ADSCs were characterized by high expression of CD90 and CD105, with scant expression of CD34 and CD45. We found that GDNF protein expression in ADSCs was elevated after Lenti-GDNF transfection. In GDNF-ADSCs-APG cultures, GDNF was increasingly produced while tissue growth factor-β was reduced as incubation time was increased. ADSC proliferation was augmented and neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) expression were upregulated in GDNF-ADSCs-APG. In addition, limb motor function and nerve axon growth were improved after GDNF-ADSCs-APG treatment. In conclusion, our study demonstrates the combined effect of ADSCs and APG in peripheral nerve regeneration and may lead to treatments that benefit patients with electrical injuries.

The Effects of Aging and Exercise on Glial Cell Line‐Derived Neurotrophic Factor Content in Rat Hearts

Heart function is regulated by the sensory nervous system and the two branches of the autonomic nervous system, the sympathetic and parasympathetic nervous systems. Neural development, plasticity, and survival of these branches of the nervous system are controlled by neurotrophic factors such as the glial cell line‐derived neurotrophic factor (GDNF). Studies in our laboratory have shown that aging can shift the innervation pattern in mesenteric vessels from a balanced sensory/sympathetic pattern to a predominately sympathetic pattern and this is associated with an increase in blood pressure. However, the same studies suggest that exercise is associated with an increase in GDNF protein content in mesenteric vessels, a restored balance of innervation, and a lowering of blood pressure. Little is known about how aging and exercise impacts neurotrophic factor expression in cardiac muscle. Therefore, the purpose of this study was to measure GDNF protein content in the heart of rats at different ages and to investigate the effects of exercise on GDNF protein levels. Control groups consisted of 4‐week‐old, 14‐week‐old, 6‐month‐old, 12‐month‐old, and 18‐month‐old sedentary rats (5 or 6 animals per group). In the exercise study, 4‐week‐old, 6‐month‐old, and 12‐month‐old rats were placed in cages with access to voluntary running wheels and were allowed to exercise for 10 weeks (4‐week‐old), or 6 months (6‐ and 12‐month‐old). Sedentary and exercised animals were euthanized and cardiac tissues were removed. Each heart was divided into three parts: right atria (RA), left atria (LA), and ventricles (VT). Tissues were processed and neurotrophic factor protein content was measured using enzyme‐linked immunosorbent assay. GDNF protein content in right atria (0.95 ± 0.14pg of GDNF/mg of tissue), left atria (0.91 ± 0.29pg of GDNF/mg of tissue), and ventricles (0.54 ± 0.35pg of GDNF/mg of tissue) of 4‐week‐old animals was significantly higher than that in heart chambers of older animals (6‐month‐old sedentary RA: 0.17 ± 0.02pg, LA: 0.14 ± 0.07pg, VT: 0.03 ± 0.02pg of GDNF/mg of tissue; 12–month‐old sedentary RA: 0.02 ± 0.01pg, LA: 0.018 ± 0.012pg of GDNF/mg of tissue, VT: Non‐detectable; 18‐month‐old sedentary RA: 0.000172 ± 0.000170pg, LA: 0.00128 ± 0.00236pg of GDNF/mg of tissue, VT: Non‐detectable). The results show that exercise significantly increased GDNF protein content in all heart chambers, in all age‐matched groups (14‐week‐old sedentary: RA: 1.20 ± 0.17pg, LA: 1.30 ± 0.14pg, VT: 1.27 ± 0.10pg of GDNF/mg of tissue and 14‐week‐old exercised: RA: 2.97 ± 0.27pg, LA: 2.06 ± 0.23pg, VT: 2.57 ± 0.32pg of GDNF/mg of tissue; 12‐month‐old exercised RA: 0.25 ± 0.07pg, LA: 0.11 ± 0.03pg, VT: 0.02 ± 0.01pg of GDNF/mg of tissue; 18‐month‐old exercised RA: 0.18 ± 0.21pg, LA: 0.04 ± 0.03pg, VT: 0.006 ± 0.006pg of GDNF/mg of tissue). These results suggest that GDNF protein content decreases with aging in all heart chambers and that exercise increases GDNF protein content in all heart chambers. The observed lowering of GDNF content with age may contribute to altered neural function observed in aged individuals, while the increased GDNF expression observed following exercise could help explain beneficial effects of exercise on cardiac innervation.Support or Funding InformationNIH 1 R15 AG022908‐01A2/NSF 0552517This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of long‐term exercise on GDNF expression and innervation in rat skeletal muscle

Sarcopenia is a major cause of frailty and loss of independence in aging individuals. Results of recent studies suggest that changes in neural tissues may contribute to development of sarcopenia. Exercise provides neuroprotection by promoting neurogenesis, decreasing apoptosis, and modulating inflammation, however the mechanisms by which exercise generates these effects are not well understood. Neurotrophic factors are powerful regulators of neuronal maintenance and synaptic strength. Glial cell line‐derived neurotrophic factor (GDNF) is a neurotrophic factor that has been shown to be a potent survival factor for somatic motor neurons that innervate skeletal muscle, but as age continues, GDNF levels in muscle tend to decrease. This study seeks to expand our understanding of the regulatory processes controlling GDNF expression, which will aid in identifying how these processes become disturbed with aging, injury or in neurodegenerative diseases. Our hypothesis is that long‐term exercise will increase GDNF expression, and support neuromuscular junction (NMJ) structures. For these studies Sprague‐Dawley rats (6‐months of age) were exercised, in the form of voluntary running, for 6 months. Controls consisted of an age‐matched sedentary group maintained in cages without access to running wheels. After 6 months of exercise hind‐limb muscles were collected and processed for measurement of GDNF protein content via enzyme‐linked immunosorbent assay. The length and area of motor end plates were quantified via staining with a‐bungarotoxin. Immunohistochemical analysis was performed to detect co‐localization of GDNF and choline acetyltransferase (ChAT), using antibodies against GDNF and ChAT. There was a significant (p ≤ 0.05) increase in length of stained end plates in muscles from exercised rats (36.6 ± 3.5μm) when compared to that in the age‐matched sedentary group (27.6 ± 1.15μm). There was a significant (p ≤ 0.05) increase in area of stained end plates in muscles from exercised rats (616.3 ± 84.9μm2) when compared to that in the age‐matched sedentary group (430.3 ± 30.8μm2). We also observed a trend towards an increase GDNF protein content in muscle from exercised rats compared to tissues from sedentary control rats; however, the effect was not significant. Although, co‐localization between GDNF and ChAT appeared more apparent in tissues from exercised rats. These findings suggest that increased physical activity enhances structural neuroplasticity in NMJ elements and may possibly lead to enhanced neuromuscular function. Understanding the activity dependent regulation of neurotrophic factor expression and neural plasticity in the neuromuscular system may help in identifying novel targets for pharmacological development.Support or Funding InformationThis work was supported by a grant from the Faculty Research and Creative Activities Award, Western Michigan University, NIH grant 1 R15 AG022908‐01A2, NSF grant DBI 0552517.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Alterations of DNA Methylation at GDNF Gene Promoter in the Ventral Tegmental Area of Adult Depression-Like Rats Induced by Maternal Deprivation

Objective: To study the expression and DNA methylation of the Glial cell line-derived neurotrophic factor (GDNF) gene in the development of depression-like behaviors in rats experiencing maternal deprivation stress in early life.Methods: Newborn SD rats were randomly assigned to a normal control group (NOR) or maternal deprivation group (MD). An open field test (OPT), sucrose preference test (SPT), and a forced swimming test (FST) were used to evaluate rats' behaviors. Protein, mRNA, and methylation levels were measured by ELISA/Western blot, real-time PCR, and BiSulfte Amplicon sequencing PCR, respectively.Results: MD rats had significantly shorter total distance and more fecal pellets in OPT, a lower sucrose preference rate in SPT, and a longer immobility time in FST than NOR rats. Compared with NOR rats, MD rats showed a significantly higher plasma corticosterone (CORT) level. The levels of plasma dopamine (DA) and the GDNF were significantly lower in the MD rats than in NOR rats. In the ventral tegmental area (VTA) tissues, MD rats had a significantly higher level of methylation at the GDNF gene promoter than NOR rats. The expression of the GDNF mRNA and protein were significantly lower in MD rats than in NOR rats. The total distance was significantly correlated with plasma DA and GDNF, the DNA methylation level at the GDNF promoter and the GDNF mRNA level in the VTA. Fecal pellets showed a significant correlation with plasma CORT. The sucrose preference rate was significantly correlated with plasma DA, the DNA methylation level at the GDNF promoter and the GDNF mRNA level in the VTA. Immobility time showed a significant correlation with the plasma DA, the plasma GDNF and the GDNF mRNA level in the VTA.Conclusion: up-regulation of DNA methylation at the GDNF gene promotor and the subsequent down-regulation of the GDNF gene expression in the VTA, may be involved in the development of depression-like behaviors in rats experiencing MD in early life.

Expression of BDNF and GDNF in brain of attention deficit hyperactivity disorder rats

Objective To observe the changes of protein and mRNA expression levels of brain derived neurotrophic factor(BDNF) and glial cell line derived neurotrophic factor(GDNF) in the prefrontal cortex, hippocampus and striatum of the attention deficit hyperactivity disorder(ADHD) model animal, and to explore the relationship between BDNF, GDNF and ADHD. Methods Junior spontsneously hyoertensive rat(SHR) rats were used as ADHD model group and Wistar Kyoto rats(WKY) as control group.The protein and mRNA expression levels of BDNF and GDNF in prefrontal cortex, hippocampus and striatum were detected by Western blot and RT-PCR methods. Results The expression of BDNF and GDNF protein was (0.561±0.095) and (0.507±0.155) in the prefrontal cortex, (0.606±0.054) and (0.457±0.198) in hippocampus and (0.558±0.191) and (0.554±0.267) in striatum of SHR rats.The expression of BDNF and GDNF protein of WKY rats were (0.855±0.211) and (0.891±0.244) in the prefrontal cortex, (0.827±0.163) and (0.898±0.207) in hippocampus and (0.998±0.220) and (0.955±0.146) in striatum.The expression of BDNF and GDNF mRNA was (0.636±0.141) and (0.544±0.161) in the prefrontal cortex, (0.996±0.196) and (0.563±0.302) in hippocampus and (0.664±0.145) and (0.689±0.049) in striatum of SHR rats.The expression of BDNF and GDNF mRNA of WKY rats were (0.861±0.232) and (0.840±0.124) in prefrontal cortex, (1.372±0.423) and (1.180±0.441) in hippocampus, and (1.107±0.185) and (0.950±0.247) in striatum.The protein and mRNA expression levels of BDNF and GDNF in prefrontal cortex, hippocampus and striatum of ADHD rats were significantly lower than those in the control group (all P<0.05). Conclusion The low expression of BDNF and GDNF in the prefrontal cortex, hippocampus and striatum of SHR rats may be related to the pathophysiology of ADHD. Key words: Attention deficit hyperactivity disorder; Spontsneously hyoertensive rat; Brain derived neurotrophic factor; Glial cell line derived neurotrophic factor

Effect of Voluntary Exercise on Neurotrophic Factor Protein Content in Cardiac Muscle of Young Rats

The sensory nervous system and the two branches of the autonomic nervous system, the sympathetic and parasympathetic nervous systems, work complementarily to regulate function of the heart. The literature shows that neurotrophic factors such as nerve growth factor (NGF) and glial cell line‐derived neurotrophic factor (GDNF) are responsible for controlling development, plasticity, and survival of sensory, sympathetic and parasympathetic innervation in cardiovascular tissues. Preliminary studies in our laboratory have shown that as we age, the innervation pattern in mesenteric vessels may shift from a balanced sensory/sympathetic to primarily sympathetic, with a concomitant increase in blood pressure. Little is known about how neurotrophic factor expression is regulated in cardiac muscle of adult rats and how changes in expression will impact the pattern of innervation in the heart. Therefore, the purpose of this study is to measure neurotrophic factor content and patterns of innervation in the hearts of sedentary and exercised rats. Sedentary animals (n=6) were kept in individual cages, while the exercised animals (n=6) were kept in individual cages with access to running wheels. Animal weight was measured throughout the study. Following 10 weeks of exercise animals were euthanized and cardiac tissues were removed. Each heart was divided into three parts: right atria, left atria, and ventricles. Tissues were processed and neurotrophic factor protein content was measured using enzyme‐linked immunosorbent assay. Our results show that body weight was lower in the exercised group compared to the sedentary group (P&lt;0.05). GDNF protein content in right atria (2.97 ± 0.27 pg of GDNF/mg of tissue), left atria (2.06 ± 0.23 pg of GDNF/mg of tissue), and ventricles (2.58 ± 0.31 pg of GDNF/mg of tissue) of exercised animals were significantly higher than in right atria (1.20 ± 0.16 pg of GDNF/mg of tissue), left atria (1.30 ± 0.13 pg of GDNF/mg of tissue), and ventricles (1.27 ± 0.10 pg of GDNF/mg of tissue) of sedentary animals (P&lt;0.001). NGF protein content in right atria (0.029 ± 0.019 pg of NGF/mg of tissue), left atria (0.112 ± 0.100 pg of NGF/mg of tissue), and ventricles (0.043 ± 0.030 pg of NGF/mg of tissue) of exercised animals were significantly lower than in right atria (0.037 ± 0.016 pg of NGF/mg of tissue), left atria (0.66 ± 0.45 pg of NGF/mg of tissue), and ventricles (0.67 ± 0.45 pg of NGF/mg of tissue) of sedentary animals (P&lt;0.05). Previous studies have shown that exercise may restore the balance in autonomic neural drive to the heart. Our results suggest that exercise increases GDNF and reduces NGF protein content in the heart. This could provide a potential mechanism by which the innervation balance is restored in the heart, preventing cardiovascular diseases and improving quality of life.Support or Funding InformationNIH grant 1 R15 AG022908‐01A2, Western Michigan University, Conselho Nacional de Desenvolvimento Científico e TecnológicoThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.