Concentrations Of Glial Cell Line-derived Neurotrophic Factor Research Articles

Synergistic effects of glial cell line-derived neurotrophic factor and base-medium on in vitro culture of testicular tissue derived from prepubertal collared peccary.

We evaluated the influence of different media plus various concentrations of Glial cell line-derived neurotrophic factor (GDNF) during the in vitro culture (IVC) of testicular tissues from prepubertal collared peccary. Testes from 5 individuals were collected, fragmented and cultured for 28 days (34°C and 5% CO2). Culture media were Dulbecco's modified essential medium (DMEM) or stem cell serum free media (StemPro-34™ SFM), both supplemented with various concentrations of GDNF (0, 10, or 20 ng/mL). Fragments were cultured on the flat surface of 0.75% agarose gel and were evaluated every 7 days for fragment area, histomorphology, cellular viability, and proliferative activity. Data were expressed as mean ± standard error and analyzed by Kruskal-Wallis's and Tukey test. Fragments area decreased over the 28 days-culture, regardless of the treatment. For morphology, the StemPro-37 SFM medium plus 10 ng/mL GDNF provided higher scores at all time points in comparison to DMEM using any GDNF concentration (p < .05). After 28 days, similar cellular viability (~70%) was observed in all treatments (p > .05). For proliferating cell nuclear antigen assay, only DMEM plus 10 ng/mL GDNF improved (p < .05) cellular proliferation on Days 14 and 28. Looking at argyrophilic nucleolar organizing regions, after 28 days, there were no differences among treatments regarding cell proliferative capacity for both spermatogonia and Sertoli cells (p > .05). In summary, the DMEM and StemPro-34 SFM are adequate medium for IVC of prepubertal peccary testicular tissue. Supplementation with GDNF, especially at a 10 ng/mL concentration, appears to be essential for the maintenance of cell survival and proliferation.

Effect of Regular Aerobic Exercise on Cognitive Function, Depression Level and Regulative Role of Neurotrophic Factor: A Prospective Cohort Study in the Young and the Middle-Aged Sample.

Mild cognitive impairment (MCI) and depressive disorder (DD), which are associated with unhealthy lifestyles, are prevalent worldwide. This study aimed to investigate the effects of regular aerobic exercise on cognitive function, depression, and the regulatory role of neurotrophic growth factors for providing scientific basis in preventing MCI and DD in healthy individuals. Eighty members of the fitness center and 80 community residents were recruited, who were administered by the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) and the Patient Health Questionnaire (PHQ-9). Brain-derived neurotrophic factor (BDNF) and glial cell-line-derived neurotrophic factor (GDNF) in the peripheral blood were detected by enzyme-linked immunosorbent assay (ELISA). The RBANS and other factor scores, except for visuospatial abilities, were higher and PHQ-9 scores were lower in the study group than in the control group. The concentrations of BDNF and GDNF in the study group were higher than those in the control group. RBANS and its factor scores positively and PHQ-9 negatively correlated with BDNF and GDNF levels. Finally, multiple regression analysis showed that BDNF, as a predictor of RBANS, could explain 59.90% of its variance and that GDNF was a predictor of PHQ-9 could explain 12.30% of the variance. Regular aerobic exercise can improve cognitive function and depressive symptoms by increasing the BDNF and GDNF levels.

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.

The significance of glial cell line-derived neurotrophic factor analysis in Progressive Supranuclear Palsy

Progressive Supranuclear Palsy (PSP) is an atypical parkinsonism. Major subtypes of the disease: PSP-Richardson’s Syndrome (PSP-RS) and PSP Parkinsonism Predominant (PSP-P) vary in clinical features, the pathomechanism remains unexplored. The aim of this work is to analyze the relevance of glial cell line-derived neurotrophic factor (GDNF) evaluation in the serum and cerebrospinal fluid (CSF) in PSP subtypes and to verify its significance as a possible factor in the in vivo examination. Authors assessed the concentration of GDNF in the serum and CSF of 12 patients with PSP-RS, 12 with PSP-P and 12 controls. Additionally authors evaluated patients using Unified Parkinson’s Disease Rating Scale—III part (UPDRS-III), Frontal Assessment Battery (FAB) and Magnetic Resonance Imaging (MRI). The evaluation revealed significantly increased concentrations of GDNF in the CSF among PSP-RS patients and substantially increased concentrations of GDNF in the serum in PSP-P. Though the GDNF concentrations differentiated PSP subtypes, no correlations between with clinical factors were observed however certain correlations with atrophic changes in MRI were detected. GDNF is a factor which may impact the pathogenesis of PSP. Possible implementation of GDNF as a therapeutic factor could be a perspective in the search for therapy in this currently incurable disease.

Глиальный нейротрофический фактор у пациентов с возрастной катарактой

Purpose. To study the content of glial cell line-derived neurotrophic factor (GDNF) in the aqueous humor (AH), lacrimal fluid (LF), and blood serum (BS) in patients with age-related cataract. Material and methods. The content of GDNF in the LF, AH and BS was studied in 47 patients (47 eyes) with age-related cataract. Collection of stimulated LF was performed with a pipette on the day preceding surgery; the AH and BS were sampled during the phacoemulsification of a cataract. The concentration of GDNF was measured using an enzyme immunoassay. Results. The concentration of GDNF in the AH was 88.9±46.9 (2.0–212.5) pg/ml, in the BS – 201.1±44.6 (103.2–287.2) pg/ml, in the LF – 343.6±133.7 (172.8– 683.0) pg/ml. The levels of GDNF in the studied biological fluids did not show any significant correlations with each other. Conclusion. In patients with age-related cataract, the level of GDNF in the AH was relatively low: the concentration of GDNF in the AH was more than 2 times lower than in the BS and almost 4 times lower than in the LF. No significant correlations were found between the concentrations of GDNF in LF, AH and BS. Key words: glial cell line-derived neurotrophic factor, lacrimal fluid, aqueous humor, blood serum, age-related cataract

GDNF Promotes Astrocyte Abnormal Proliferation and Migration Through the GFRα1/RET/MAPK/pCREB/LOXL2 Signaling Axis.

Glial cell-line derived neurotrophic factor (GDNF) is a powerful astroglioma (AG) proliferation and migration factor that is highly expressed in AG cells derived from astrocytes. However, it is still unclear whether high levels of GDNF promote AG occurrence or if they are secondary to AG formation. We previously reported that high concentrations of GDNF (200 and 500ng/mL) can inhibit DNA damage-induced rat primary astrocytes (RA) apoptosis, suggesting that high concentrations of GDNF may be involved in the malignant transformation of astrocytes to AG cells. Here we show that 200ng/mL GDNF significantly increased the proliferation and migration ability of RA cells and human primary astrocytes (HA). This treatment also induced RA cells to highly express Pgf, Itgb2, Ibsp, Loxl2, Lif, Cxcl10, Serpine1, and other genes that enhance AG proliferation and migration. LOXL2 is an important AG occurrence and development promotion factor and was highly expressed in AG tissues and cells. High concentrations of GDNF promote LOXL2 expression and secretion in RA cells through GDNF family receptor alpha-1(GFRα1)/rearranged during transfection proto-oncogene (RET)/mitogen-activated protein kinase (MAPK)/phosphorylated cyclic AMP response element binding protein (pCREB) signaling. GDNF-induced LOXL2 significantly promotes RA and HA cell proliferation and migration, and increases the expression of Ccl2, Gbp5, MMP11, TNN, and other genes that regulate the extracellular microenvironment in RA cells. Our results demonstrate that high concentrations of GDNF activate LOXL2 expression and secretion via the GFRα1/RET/MAPK/pCREB signal axis, which leads to remodeling of the astrocyte extracellular microenvironment through molecules such as Ccl2, Gbp5, MMP11, TNN. This ultimately results in abnormal astrocyte proliferation and migration. Collectively, these findings suggest that high GDNF concentrations may promote the malignant transformation of astrocytes to AG cells.

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.

Neurotrophic Factors in Human Milk in Early Lactation and the Effect of Holder and Microwave Pasteurization on Their Concentrations.

The objective of this study was to determine the level of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) in human milk in the first 2 weeks of breast-feeding and compare the effects of Holder pasteurization (HoP, 62.5 °C, 30 minutes) and microwave pasteurization (MP) at constant temperature (62.5 °C) on the concentraion of both neurotrophic factors (NFs). Concentration of NFs in human milk was determined using a commercially available ELISA assay. The average concentration of BDNF and GDNF in milk was 11 ± 6 ng/mL and 336 ± 238 pg/mL, respectively. There was a positive correlation between the concentrations of BDNF and GDNF in human milk and day of lactation (r = 0.441, P < 0.05 and r = 0.482, P < 0.05, respectively). In addition, there was a significant correlation between the levels of BDNF and GDNF (r = 0.366, P < 0.05). HoP and MP for 10 minutes affected both NF levels similarly, causing degradation of BDNF by about 24% and 17%, and GDNF by 47% and 45%, respectively. Use of MP for 5 minutes resulted in preservation of nearly 91% BDNF and 79% GDNF in human milk. In the pasteurization processes carried out, results showed that GDNF is more susceptible to degradation under the influence of high temperature. To the authors' best knowledge, this is the first study evaluating the effects of HoP and MP at constant temperature on the concentration of NFs in human milk. It was found that the MP for 5 minutes is the optimal method.

High levels of NGF during anxiety-like behavior in a murine model of brain ischemic stroke

PurposeNeuropsychiatric disorders following stroke, including depression and anxiety, are often associated with long-term disability. Neurotrophic factors play an important role in the pathophysiology of many neurodegenerative and neuropsychiatric disorders. In the current study, we investigated a potential participation of neurotrophic factors in stroke-associated behavioral and pathological changes 14 days after ischemia. MethodsTransient global cerebral ischemia was induced by bilateral occlusion of common carotid arteries (BCCAo) in C57BL/6 mice. Neurological evaluation was performed daily up to 14 days after induction. The Open Field (OF) and Elevated Plus Maze (EPM) tests were performed. After behavioral tests, brains from sham and ischemic mice were removed and processed to evaluate histopathology and immunomarcation for cleaved caspase-3 as well as the neurotrophic factors brain-derived neurotrophic factor (BDNF), glial cell line derived neurotrophic factor (GDNF), and neural growth factor (NGF) by ELISA. ResultsIschemic animals presented anxiety-like behavior and histopathological alterations mainly characterized by formation of small necrotic cavities surrounded by penumbra zone and neuropil vacuolation in the frontal cortex, ischemic neurons in the hippocampus and gliosis in the midbrain. Some immunopositive neurons for cleaved caspase-3 were observed in the penumbra by immunohistochemical analysis. Higher levels of NGF were found in the brain of BCCAo mice compared with sham animals. Similar concentrations of BDNF and GDNF were detected in both groups. ConclusionOur results suggested the participation of NGF in anxiety-like behavior at 14 days after induction of experimental cerebral ischemia and reperfusion.

Do serum GDNF levels correlate with severity of Alzheimer's disease?

A growing body of evidence that glial cell line-derived neurotrophic factor (GDNF) levels are probably involved in pathogenesis and disease course of Alzheimer's disease (AD) suggested that its blood levels could potentially be used as a biomarker of AD. The aim of this study was to compare serum GDNF levels in patients with AD and age-matched controls. Serum concentrations of GDNF were compared in 25 AD patients and 25 healthy volunteers using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Severity of the disease in AD patients was assessed using Functional Assessment Staging (FAST). Cognitive assessment of the patients was done using the Mini-Mental State Examination (MMSE). Mean GDNF levels were found to be 2.45 ± 0.93ng/ml in AD patients and 4.61 ± 3.39ng/ml in age-matched controls. There was a statistically significant difference in GDNF serum levels in patients with AD compared to age-matched controls (p = 0.001). Moreover, GDNF serum levels were significantly correlated with disease severity (p< 0.001) and cognitive impairment (p< 0.001). This study showed that serum levels of GDNF are significantly decreased in AD patients in comparison with age-matched controls, thus suggesting a potential role of GDNF as a disease biomarker. However, a comprehensive study of changes in serum levels of multiple neurotrophic factors reflective of different neurobiological pathways in large-scale population studies is recommended.

Acute normobaric hypoxia does not affect the simultaneous exercise-induced increase in circulating BDNF and GDNF in young healthy men: A feasibility study.

Physical exercise has a neuromodulatory effect on the central nervous system (CNS) partially by modifying expression of neuropeptides produced and secreted by neurons and glial cells, among which the best examined are brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). Because both neurotrophins can cross the brain-blood barrier (BBB), their blood levels indirectly reflect their production in the CNS. Moreover, both neuropeptides are involved in modulation of dopaminergic and serotoninergic system function. Because limited information is available on the effects of exercise to volition exhaustion and acute hypoxia on CNS, BDNF and GDNF formation, the aims of the present study were to verify whether 1) acute exercise to exhaustion in addition to neurons also activates glial cells and 2) additional exposure to acute normobaric moderate hypoxia affects their function. In this feasibility study we measured blood concentrations of BDNF, GDNF, and neuropeptides considered as biomarkers of brain damage (bFGF, NGF, S100B, GFAP) in seven sedentary healthy young men who performed a graded exercise test to volitional exhaustion on a cycle ergometer under normoxic (N) and hypoxic conditions: 2,000 m (H2; FiO2 = 16.6%) and 3,000 m altitude (H3; FiO2 = 14.7%). In all conditions serum concentrations of both BDNF and GDNF increased immediately after cessation of exercise (p<0.01). There was no effect of condition or interaction (condition x time of measurement) and exercise on any of the brain damage biomarkers: bFGF, NGF, S100B, GFAP. Moreover, in N (0<0.01) and H3 (p<0.05) exercise caused elevated serum 5-HT concentration. The results suggest that a graded effort to volitional exhaustion in normoxia, as well as hypoxia, simultaneously activates both neurons and astrocytes. Considering that s100B, GFAP, bFGF, and NGF (produced mainly by astrocytes) are markers of brain damage, it can be assumed that a maximum effort in both conditions is safe for the CNS.

Glial cell line-derived neurotrophic factor as a treatment after spinal cord injury.

Glial cell line-derived neurotrophic factor as a treatment after spinal cord injury.

Bioactive Components in Human Milk Along the First Month of Life: Effects of Iodine Supplementation during Pregnancy

Background/Aims: Human milk is considered the most suitable food for infants. The potential benefits of breastfeeding can be explained by the presence of different growth and neurotrophic factors in human milk. This study was designed to detect some biomarkers in human milk, which could be involved in the infant neurodevelopment and in the regulation of the maturation of neonatal intestine (brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), glial fibrillary acidic protein (GFAP), fibroblast growth factor 21 (FGF21), lysophosphatidic acid (LPA) and autotaxin (ATX)), and compare them on the basis of the consumption of iodine supplements or multivitamins. Methods: A prospective study included 37 healthy breastfeeding mothers, divided into 3 different groups: (1) 10 mothers who did not take supplements, (2) 17 mothers who took potassium iodine (KI) 200 µg/day and (3) 10 mothers who took a multivitamin supplement. Results: The concentrations of BDNF, GDNF, GFAP, FGF21, LPA and ATX in human milk were not significantly different in women who took a multivitamin or KI supplement compared with those who did not take any supplement. Conclusions: The presence of neurotrophic factors in human milk is neither modified by the consumption of supplements nor by their type.

230 EFFECT OF DIFFERENT CONCENTRATIONS OF GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR ON EXPRESSION OF SELF-RENEWAL RELATED GENES IN GOAT (CAPRA HIRCUS) SPERMATOGONIAL STEM CELLS

Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor-β superfamily produced by Sertoli cells, is essential for the self-renewal of spermatogonial stem cells in vivo. The present study evaluated the effects of different concentrations of GDNF (human recombinant expressed in Escherichia coli) on expression of some self-renewal related genes in spermatogonial stem cells (SSC). The SSC were isolated from prepubertal goat testes (3–6 months of age) by using double enzymatic digestion method and filtration through 80- and 60-µm nylon mesh filters. Further enrichment was achieved by differential plating on Datura stramonium agglutinin (DSA) lectin-coated dishes and Percoll density gradient centrifugation. The enriched cells were cultured on goat Sertoli cell feeder layer in DMEM + 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Primary SSC colonies were formed within 7 to 10 days. These colonies were characterised through alkaline phosphatase and immunofluorescence staining on Day 10 for different SSC-specific protein markers. Colonies were found to be positive for DBA, THY1, PLZF, UCHL1, OCT-4, SOX2, and NANOG, and negative for c-Kit expression. These colonies were cultured for 15 days without or with supplementation of GDNF forming following groups: (1) without GDNF (control), (2) 10 ng mL–1 GDNF, (3) 20 ng mL–1 GDNF, and (4) 40 ng mL–1 GDNF. RNA was isolated from 100 colonies from 3 different trials on Day 15 of culture, and relative expression of different self-renewal related genes was determined by qRT-PCR. Relative mRNA abundance of PLZF was higher (P &lt; 0.05) following supplementation with 40 ng mL–1 GDNF than in other groups (i.e. control, 10 and 20 ng mL–1 GDNF). Expression of BCL6B and ID4 was found to be significantly higher (P &lt; 0.05) after supplementation of GDNF at all concentrations compared with the control group. Expression of UCHL1 was higher with addition of 20 and 40 ng mL–1 GDNF (P &lt; 0.05), whereas expression of THY1 was higher with supplementation of 10 ng mL–1 GDNF (P &lt; 0.05). In conclusion, GDNF was found to benefit expression of goat SSC candidate genes at a concentration of 40 ng mL–1.

Lentiviral Vector-Mediated Gradients of GDNF in the Injured Peripheral Nerve: Effects on Nerve Coil Formation, Schwann Cell Maturation and Myelination

Although the peripheral nerve is capable of regeneration, only a small minority of patients regain normal function after surgical reconstruction of a major peripheral nerve lesion, resulting in a severe and lasting negative impact on the quality of life. Glial cell-line derived neurotrophic factor (GDNF) has potent survival- and outgrowth-promoting effects on motoneurons, but locally elevated levels of GDNF cause trapping of regenerating axons and the formation of nerve coils. This phenomenon has been called the “candy store” effect. In this study we created gradients of GDNF in the sciatic nerve after a ventral root avulsion. This approach also allowed us to study the effect of increasing concentrations of GDNF on Schwann cell proliferation and morphology in the injured peripheral nerve. We demonstrate that lentiviral vectors can be used to create a 4 cm long GDNF gradient in the intact and lesioned rat sciatic nerve. Nerve coils were formed throughout the gradient and the number and size of the nerve coils increased with increasing GDNF levels in the nerve. In the nerve coils, Schwann cell density is increased, their morphology is disrupted and myelination of axons is severely impaired. The total number of regenerated and surviving motoneurons is not enhanced after the distal application of a GDNF gradient, but increased sprouting does result in higher number of motor axon in the distal segment of the sciatic nerve. These results show that lentiviral vector mediated overexpression of GDNF exerts multiple effects on both Schwann cells and axons and that nerve coil formation already occurs at relatively low concentrations of exogenous GDNF. Controlled expression of GDNF, by using a viral vector with regulatable GDNF expression, may be required to avoid motor axon trapping and to prevent the effects on Schwann cell proliferation and myelination.

Effects of glial cell line-derived neurotrophic factor, fibroblast growth factor 2 and epidermal growth factor on proliferation and the expression of some genes in buffalo (Bubalus bubalis) spermatogonial cells

The present study evaluated the effects of glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor (FGF) 2 and epidermal growth factor (EGF) on proliferation and the expression of some genes in spermatogonial cells. Spermatogonial cells were isolated from prepubertal buffalo testes and enriched by double enzyme treatment, filtration through 80- and 60-μm nylon mesh filters, differential plating on lectin-coated dishes and Percoll density gradient centrifugation. Cells were then cultured on a buffalo Sertoli cell feeder layer and formed colonies within 15-18 days. The colonies were found to predominantly contain undifferentiated Type A spermatogonia because they bound Dolichos biflorus agglutinin and did not express c-kit. The colonies expressed alkaline phosphatase, NANOG, octamer-binding transcription factor (OCT)-4 and tumour rejection antigen (TRA)-1-60. Cells were subcultured for 15 days, with or without growth factor supplementation. After 15 days, colony area and the relative mRNA abundance of PLZF were higher (P<0.05) following supplementation with 40 ng mL⁻¹ GDNF + 10 ng mL⁻¹ EGF + 10 ng mL⁻¹ FGF2 than with the same concentrations of GDNF alone or GDNF plus either EGF or FGF2. Expression of TAF4B was higher (P<0.05) in the presence of FGF2, whereas the expression of THY1 was not affected by growth factor supplementation. In the Sertoli cell feeder layer, EGF and FGF2 decreased (P<0.05), whereas GDNF increased (P<0.05), the relative mRNA abundance of ETV5 compared with control. In conclusion, an in vitro culture system that incorporates various growth factors was developed for the short-term culture of buffalo spermatogonia.

Bone mesenchymal stromal cells stimulate neurite outgrowth of spinal neurons by secreting neurotrophic factors

It has been demonstrated that bone mesenchymal stromal cells (BMSCs) stimulate neurite outgrowth from dorsal root ganglion (DRG) neurons. The present in vitro study tested the hypothesis that BMSCs stimulate the neurite outgrowth from spinal neurons by secreting neurotrophic factors. Spinal neurons were cocultured with BMSCs, fibroblasts and control medium in a non-contact system. Neurite outgrowth of spinal neurons cocultured with BMSCs was significantly greater than the neurite outgrowth observed in neurons cultured with control medium or with fibroblasts. In addition, BMSC-conditioned medium increased the length of neurites from spinal neurons compared to those of neurons cultured in the control medium or in the fibroblasts-conditioned medium. BMSCs expressed brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). The concentrations of BDNF and GDNF in BMSC-conditioned medium were 132±12 and 70±6 pg ml−1, respectively. The addition of anti-BDNF and anti-GDNF antibodies to BMSC-conditioned medium partially blocked the neurite-promoting effect of the BMSC-conditioned medium. In conclusion, our results demonstrate that BMSCs promote neurite outgrowth in spinal neurons by secreting soluble factors. The neurite-promoting effect of BMSCs is partially mediated by BDNF and GDNF.

Glial cell line-derived neurotrophic factor influences proliferation of osteoblastic cells

Little is known about the role of neurotrophic growth factors in bone metabolism. This study investigated the short-term effects of glial cell line-derived neurotrophic factor (GDNF) on calvarial-derived MC3T3-E1 osteoblasts. MC3T3-E1 expressed GDNF as well as its canonical receptors, GFRα1 and RET. Addition of recombinant GDNF to cultures in serum-containing medium modestly inhibited cell growth at high concentrations; however, under serum-free culture conditions GDNF dose-dependently increased cell proliferation. GDNF effects on cell growth were inversely correlated with its effect on alkaline phosphatase (AlP) activity showing a significant dose-dependent inhibition of relative AlP activity with increasing concentrations of GDNF in serum-free culture medium. Live/dead and lactate dehydrogenase assays demonstrated that GDNF did not significantly affect cell death or survival under serum-containing and serum-free conditions. The effect of GDNF on cell growth was abolished in the presence of inhibitors to GFRα1 and RET indicating that GDNF stimulated calvarial osteoblasts via its canonical receptors. Finally, this study found that GDNF synergistically increased tumor necrosis factor-α (TNF-α)-stimulated MC3T3-E1 cell growth suggesting that GDNF interacted with TNF-α-induced signaling in osteoblastic cells. In conclusion, this study provides evidence for a direct, receptor-mediated effect of GDNF on osteoblasts highlighting a novel role for GDNF in bone physiology.

S100B Protein, Brain-Derived Neurotrophic Factor, and Glial Cell Line-Derived Neurotrophic Factor in Human Milk

BackgroundHuman milk contains a wide variety of nutrients that contribute to the fulfillment of its functions, which include the regulation of newborn development. However, few studies have investigated the concentrations of S100B protein, brain-derived neurotrophic factor (BDNF), and glial cell line-derived neurotrophic factor (GDNF) in human milk. The associations of the concentrations of S100B protein, BDNF, and GDNF with maternal factors are not well explored.Methodology/Principal FindingsTo investigate the concentrations of S100B protein, BDNF, and GDNF in human milk and characterize the maternal factors associated with their levels in human milk, human milk samples were collected at days 3, 10, 30, and 90 after parturition. Levels of S100B protein, BDNF, and GDNF, and their mRNAs in the samples were detected. Then, these concentrations were compared with lactation and other maternal factors. S100B protein levels in human milk samples collected at 3, 10, 30, and 90 d after parturition were 1249.79±398.10, 1345.05±539.16, 1481.83±573.30, and 1414.39±621.31 ng/L, respectively. On the other hand, the BDNF concentrations in human milk samples were 10.99±4.55, 13.01±5.88, 13.35±6.43, and 2.83±5.47 µg/L, while those of GDNF were 10.90±1.65, 11.38±1., 11.29±3.10, and 11.40±2.21 g/L for the same time periods. Maternal post-pregnancy body mass index was positively associated with S100B levels in human milk (r = 0.335, P = 0.030<0.05). In addition, there was a significant correlation between the levels of S100B protein and BDNF (z = 2.09, P = 0.037<0.05). Delivery modes were negatively associated with the concentration of GDNF in human milk.ConclusionsS100B protein, BDNF, and GDNF are present in all samples of human milk, and they may be responsible for the long term effects of breast feeding.

Commentary (Research Highlights)

Putting a Finger on Neurotrophic Protein Therapy in Parkinson's Disease Parkinson's disease (PD) was first described in the essay entitled “An Essay of the Shaking Palsy” by James Parkinson in 1817. PD is the most common neurodegenerative movement disorder, whose neuropathological hallmarks are characterized by progressive and profound loss of neuromelanin-containing dopaminergic neurons in the substantia nigra pars compacta with the presence of eosinophilic intracytoplasmic, proteinaceous inclusions termed Lewy bodies and dystrophic Lewy neurites in surviving neurons. Although neuronal cell loss in the substantia nigra pars compacta is pronounced, there is widespread neurodegeneration in the CNS with the pars compacta being involved in midstages of the disease. Clinical manifestations of this complex disease include motor impairments involving resting tremor, bradykinesia, postural instability, gait difficulty and rigidity, along with non-motoric symptoms like autonomic, cognitive, and psychiatric problems. As with many multifactorial diseases, the incidence of PD increases with age. Because of an aging population, improved diagnosis, and prolonged survival, especially in developing countries, the number of patients suffering from PD is predicted to double by the year 2030. The power of neurotrophic factors to regulate neuronal cell survival in the developing nervous system and to promote also survival after injury or protect neurons in toxin-mediated disease models in animals has encouraged the idea that such proteins could be harnessed for the treatment of neurodegenerative disease. In the case of dopaminergic neurons, glial cell line-derived neurotrophic factor (GDNF), a member of the basic fibroblast growth factor superfamily, given intracerebrally has neuroprotective and neurorestorative effects in neurotoxin-induced rodent and non-human primate models of PD. In spite of extensive positive preclinical data and encouraging results from phase I clinical trials, results from phase II studies have been disappointing. Indeed, neurotrophic factor treatment of CNS diseases presents an especially complex problem, since these polypeptides have poor pharmacokinetics and bioavailability and are not able to cross the blood-brain barrier. At the same time, excessive levels of a neurotrophic protein may provoke deleterious side-effects, as evidenced by toxicity (including multifocal cerebellar Purkinje cell loss) observed in rhesus monkeys that received intraputamenal infusion of high concentrations of GDNF. A novel approach to this problem has recently been reported by Laganiere and colleagues, who explored the possibility of engineering zinc finger protein transcription factors (ZFP TFs) that activate the expression of the endogenous GDNF gene. Transcription factors based on Cys2-His2 ZFPs can be engineered to specifically target virtually any gene. By targeting the endogenous gene and exploiting the native promoter, which imposes a physiological upper limit on the level of gene expression from each allele, a ZPF can produce sufficient, but not supraphysiological levels of the therapeutic protein needed to achieve both long-term efficacy and safety. Laganiere et al. designed a human GDNF ZFP TF (hGDNF-ZFP) that recognized a site that is fully conserved between human and rhesus macaque. Having confirmed that hGDNF-ZFP activated rhesus GDNF in rhesus macaque cell lines, the authors then designed and assembled a panel of ZFP activators specific to the rat GDNF promoter. Using an adeno-associated viral vector serotype 2 as the delivery vehicle, the engineered transcription factor that gave the highest levels of rat GDNF activation was tested in the rat 6-hydroxydopamine model of PD. Vector was infused bilaterally into the striatum by convection-enhanced delivery. The modest (∼60%) overall increase in striatal GDNF levels provoked by the ZFP was sufficient to provide marked improvement in all behavioral tests performed. Moreover, functional neuroprotection by the GDNF activator was supported by immunohistochemical demonstration of preservation of tyrosine hydroxylase-positive nigrostriatal neurons. While activation of endogenous GDNF was adequate to protect again neurotoxin lesion in rat, demonstration of efficacy and safety in a nonhuman primate model of PD will be a prerequisite before advancing the ZFP-based approach to the clinic. The dual species-specific GDNF activator should facilitate such studies. In addition, convection-enhanced delivery is expected to provide a more complete coverage of the putamen, unlike previously used infusion protocols. Engineered ZFP activators that drive specific activation of endogenous GDNF combined with highly efficient convection-enhanced delivery may well offer hope to realize the therapeutic potential of GDNF.