Glial Cell-derived Neurotropic Factor Research Articles

Unique metabolic phenotype and its transition during maturation of juvenile male germ cells.

Human male reproductive development has a prolonged prepubertal period characterized by juvenile quiescence of germ cells with immature spermatogonial stem cell (SSC) precursors (gonocytes) present in the testis for an extended period of time. The metabolism of gonocytes is not defined. We demonstrate with mitochondrial ultrastructure studies via TEM and IHC and metabolic flux studies with UHPLC-MS that a distinct metabolic transition occurs during the maturation to SSCs. The mitochondrial ultrastructure of prepubertal human spermatogonia is shared with prepubertal pig spermatogonia. The metabolism of early prepubertal porcine spermatogonia (gonocytes) is characterized by the reliance on OXPHOS fuelled by oxidative decarboxylation of pyruvate. Interestingly, at the same time, a high amount of the consumed pyruvate is also reduced and excreted as lactate. With maturation, prepubertal spermatogonia show a metabolic shift with decreased OXHPOS and upregulation of the anaerobic metabolism-associated uncoupling protein 2 (UCP2). This shift is accompanied with stem cell specific promyelocytic leukemia zinc finger protein (PLZF) protein expression and glial cell-derived neurotropic factor (GDNF) pathway activation. Our results demonstrate that gonocytes differently from mature spermatogonia exhibit unique metabolic demands that must be attained to enable their maintenance and growth in vitro.

Quantifying the Risks of Asparagine Deamidation and Aspartate Isomerization in Biopharmaceuticals by Computing Reaction Free-Energy Surfaces.

Early identification of asparagine deamidation and aspartate isomerization degradation sites can facilitate the successful development of biopharmaceuticals. Several knowledge-based models have been proposed to assess these degradation risks. In this study, we propose a physics-based approach to identify the degradation sites on the basis of the free-energy barriers along the prechemical conformational step and the chemical reaction pathway. These contributions are estimated from classical and quantum mechanics/molecular mechanics molecular dynamics simulations. The computed barriers are compared to those for reference reactions in water within GNG and GDG sequence motifs in peptides (which demonstrate the highest degradation rates). Two major factors decreasing the degradation rates relative to the reference reactions are steric hindrance toward accessing reactive conformations and replacement of water by less polar side chains in the solvation shell of transition states. Among the potential degradation sites in the complementarity-determining region of trastuzumab and between two DK sites in glial cell-derived neurotropic factor, this method identified N30T, N55G, D102G, and D95K, respectively, in agreement with experiments. This approach can be incorporated in early computational screening of chemical degradation sites in biopharmaceuticals.

Interaction Between Nonviral Reprogrammed Fibroblast Stem Cells and Trophic Factors for Brain Repair

There are currently no known treatment options that actually halt or permanently reverse the pathology evident in any neurodegenerative condition. Arguably, one of the most promising avenues for creating viable neuronal treatments could involve the combined use of cell replacement and gene therapy. Given the complexity of the neurodegenerative process, it stands to reason that adequate therapy should involve not only the replacement of loss neurons/synapses but also the interruption of multiple pro-death pathways. Thus, we propose the use of stem cells that are tailored to express specific trophic factors, thereby potentially encouraging synergistic effects between the stem cell properties and those of the trophic factors. The trophic factors, brain-derived neurotropic factor (BDNF), glial cell-derived neurotropic factor (GDNF), fibroblast growth factor (FGF) 2, and insulin-like growth factor (IGF) 1, in particular, have demonstrated neuroprotective actions in a number of animal models. Importantly, we use a nonviral approach, thereby minimizing the potential risk for DNA integration and tumor formation. The present study involved the development of a nonviral reprogramming system to transform adult mature mouse fibroblasts into progressive stages of cell development. We also tailored these stem cells to individually express each of the trophic factors, including BDNF, GDNF, FGF2, and IGF1. Significantly, central infusion of BDNF-expressing stem cells prevented the in vivo loss of neurons associated with infusion of the endotoxin, lipopolysaccharide (LPS). This is particularly important in light of the role of inflammatory processes that are posited to play in virtually all neurodegenerative states. Hence, the present results support the utility of using combined gene and cell-targeting approaches for neuronal pathology.

APEX-1은 GDNF/GFRα1 시그널을 통해 세포증식을 조절한다

APEX-1 (인간 apyrimidinic/apurinic 효소)은 염기성 사이트 및 DNA단일 가닥 결손으로 손상된 DNA을 복구 할 수 있는 다기능 단백질이다. 또한 APEX-1은 많은 전사 인자들의 redox-modifying factor (산화 환원 수정 요소)로서의 역할을 한다고 알려져 있다. 이런 APEX-1의 전사 타겟을 동정하는 것은 APEX-1의 다양한 세포 내 작용 메커니즘을 이해하는데 필수적이다. 따라서 이 논문에서는 먼저 Expression array analysis를 통해 glial cell-derived neurotropic factor receptor <TEX>${\alpha}1$</TEX> (<TEX>$GFR{\alpha}1$</TEX>)을 동정하였다. <TEX>$GFR{\alpha}1$</TEX>은 glial cell-derived neurotropic factor (GDNF) family 수용체이며 APEX-1에 의해 발현이 증가된다. APEX-1이 과발현된 세포에서 GDNF처리에 의해 GDNF/<TEX>$GFR{\alpha}1$</TEX> 시그널 타겟인 c-Src가 Tyr418잔기에서 인산화 됨을 관찰하였다. 또한 APEX-1이 과발현된 세포에 GDNF처리하면, 세포증식이 증가함을 보았다. 반면, APEX-1 발현을 siRNA을 이용하여 감소시키면 <TEX>$GFR{\alpha}1$</TEX> 발현과 GDNF에 의한 c-Src 인산화 및 세포증식이 감소함을 확인하였다. 이상의 결과는 APEX-1은 GDNF/<TEX>$GFR{\alpha}1$</TEX> 시그널을 통해 세포 생존과 증식을 조절함을 증명하였다. 따라서 본 연구를 통해 APEX-1의 세포 증식을 조절하는 새로운 기전을 규명하였다. Human apurinic/apyrimidinic endonuclease (APEX-1) is a multifunctional protein that is capable of repairing abasic sites and single-strand breaks in damaged DNA. In addition, it serves as a redox-modifying factor for a number of transcription factors. Identifying the transcriptional targets of APEX-1 is essential for understanding how it affects various cellular outcomes. Expression array analysis was used to identify glial cell-derived neurotropic factor receptor <TEX>${\alpha}1$</TEX> (<TEX>$GFR{\alpha}1$</TEX>), which is an encoding receptor for the glial cell-derived neurotropic factor (GDNF) family, the expression of which is induced by APEX-1. A target of GDNF/<TEX>$GFR{\alpha}$</TEX> signaling, c-Src (Tyr418) was strongly phosphorylated by GNDF in the APEX-1 expressing cells. Moreover, GDNF initiated cell proliferation, measured by counting the number of cells, in the APEX-1 expressing cells. Importantly, the down-regulation of APEX-1 by siRNA caused a marked reduction in the <TEX>$GFR{\alpha}1$</TEX> expression level, and it reduced the ability of GDNF to phosphorylate c-Src (Tyr418) and stimulate cell proliferation. These results demonstrate an association between APEX-1 and GDNF/<TEX>$GFR{\alpha}$</TEX> signaling and suggest a potential molecular mechanism for the involvement of APEX-1 in cell survival and proliferation.

In vitro neuraotropic growth of cholangiocarcinoma: an experimental study

SummaryObjectivePerineural invasion of cholangiocarcinoma happens in the early stage of the disease but is often not recognized until its later stages. Research about the behaviour and mechanism of perineural invasion by cholangiocarcinoma is urgently needed for a useful new model. The aim of this work is to establish a novel model to address the problem.DesignNeural cells and cholangiocarcinoma cells were co-cultured to mimic the neurotropic invasion of cholangiocarcinoma.SettingHuman embryonic stem cells were induced to form neural cells by glial cell-derived neurotropic factor and retinoic acid; neural cells and cholangiocarcinoma cells were co-cultured in Transwell chamber.ParticipantsHuman embryonic stem cells and cholangiocarcinoma cells were applied.Main outcome measuresPaired t-test was used to compare the counts of penetrating cholangiocarcinoma cells in co-culture and control group.ResultsFormation of neurospheres and neural-like cells were observed following induction at 24 and 48 h, respectively; synapses were viewed to protrude from neural-like cell bodies after incubation for 96 h. Forty-eight hours after incubation, immunocytochemical staining of the cells showed that synaptophysin and glial fibrillary acidic protein were expressed in the neuron-like cells and gliocytes-like cells, respectively. The cholangiocarcinoma cells that had penetrated through the Matrigel/polyethylene terephthalate membrane from the upper chamber to the lower chamber of the Transwell in the co-culture group were significantly more numerous than those in the control group (68 ± 8.3/field versus 46 ± 5.7/field, P < 0.05).ConclusionThe novel model is a valuable tool to study the perineural invasion of cholangiocarcinoma.

67 SUPPLEMENTATION WITH EPIDERMAL GROWTH FACTOR AND GLIAL CELL-DERIVED NEUROTROPIC FACTOR DURING PORCINE OOCYTE MATURATION STIMULATES BLASTOCYST HATCHING RATE AND QUALITY

The growth factors glial cell-derived neurotropic factor (GDNF) and epidermal growth factor (EGF) have previously been shown to elicit a functional effect on oocyte competence and embryo development; however, their effects on embryo quality remain elusive. The objective of the present study was to investigate whether supplementation with EGF, GDNF, or a combination of the 2 growth factors during in vitro porcine oocyte maturation has a beneficial effect on the rate of embryonic development, cell number and relative expression of genes related to apoptosis (anti-apoptotic: BCLX; pro-apoptotic: BAK, CASPASE), cellular stress (HSP70), mitochondrial function (TFAM) and pluripotency (TERT). Cumulus-oocyte complexes (COC) aspirated from slaughterhouse ovaries were matured for 42 h in maturation medium supplemented with either 50 μg mL–1 of EGF (n = 3398), 50 μg mL–1 of GDNF (n = 1870), or a combination of both growth factors [EGF (50 μg mL–1) + GDNF (50 μg mL–1); EGF+GDNF-group; n = 1993). Subsequently, the oocytes were inseminated, the cleavage rate scored and the resultant embryos cultured in vitro for 7 days. The experiments were replicated a total of 7 times and zona-enclosed blastocysts were collected for Hoechst staining (cell counting) and for gene expression analyses. For gene expression analyses, total RNA was isolated from pools of embryos (n = 10), reverse transcribed into cDNA and subjected to comparative real-time PCR using the delta-delta Ct method (2–ΔΔCT) and statistical method with a significance level of P &lt; 0.05. The blastocyst rate was significantly lowest in the GDNF group (P &lt; 0.05), whereas the hatched blastocyst rate (P &lt; 0.05) was highest in the EGF+GDNF-group (Table 1). The GDNF-group displayed 2-fold up-regulation of BAK compared with the EGF group (P &lt; 0.05), 1.75-fold up-regulation of TFAM compared with the EGF+GDNF group (P &lt; 0.05) and 2.4-fold up-regulation of CASPASE compared with the EGF+GDNF group (P &lt; 0.05). In contrast, an 11-fold decrease of TERT compared with the EGF+GDNF and EGF group was observed (P &lt; 0.05). The EGF+GDNF-group displayed 1.16 and 1.56-fold up-regulation of BCLX compared with the EGF and GDNF (P &lt; 0.05) groups, respectively and a 1.1-fold up-regulation of TERT compared with the EGF group; in contrast, there was a 1.35 and 1.25 fold down-regulation of CASPASE and TFAM compared with the EGF group, respectively. The EGF-group displayed the lowest (P &lt; 0.05) expression level of BAK. In conclusion, compared among treatments, supplementation with a combination of EGF and GDNF during porcine in vitro oocyte maturation resulted in an increased rate of hatched blastocysts and increased expression of anti-apoptotic (BCLX) and pluripotency related (TERT) genes, whereas it resulted in decreased expression of the pro-apoptotic gene (CASPASE). The present study may help to improve the culture conditions, which are important for optimizing blastocyst rates and quality. Table 1.Effect of using growth factor in in vitro maturation on the cleavage rate and blastocyst yield (mean ± standard error of the means) in vitro

395 OVINE SPERMATOGONIA ISOLATION AND IN VITRO GROWTH ASSAY

Spermatogonia, as adult stem cells from the male reproductive system, are attracting strong interest from those studying male reproductive gamete preservation and developing new approaches in transgenic animals. Protein gene product 9.5 (PGP9.5) is a marker of sheep spermatogonia, which has been validated by J. R. Rodriguez-Sosa et al. (2006).The objective of this study was to develop an in vitro culture system for spermatogonial growth. Twenty 2- to 3-month-old rams were randomly selected at a local slaughter house for testicular tissue collection. Two-step enzymatic digestion methods were used for spermatogonia isolation from seminiferous tubules. In brief, mechanically isolated seminiferous tubules from testicular tissue were incubated in 1:1 1 mg mL-1 collagenase and hyaluronidase with 5 μg mL-1 DNase I for 20 min at 37°C. Most of the surrounding interstitial cells will fall off from seminiferous tubules by slightly pipetting. Seminiferous tubules were from cell suspensions after natural sedimentation in PBS and then were digested in 0.25% trypsin + 0.04% EDTAfor 5 to 7 min at 37°C to disassociate tubules into single cells. Data were analyzed with ANOVA procedures. Means of 5 specimens were presented. Of the total isolated cells, 19.7 ± 5.3% were identified as PGP9.5+cells, and 23.8 ± 3.6% were identified as c-kit+ cells. C-kit, the transmembrane tyrosine kinase receptor for stem cell factor, has been identified, which is expressed and functional in differentiating A1-A4 spermatogonia but not in spermatogonial stem cells (OhtaH2000). For in vitro culture of spermatogonia, DMEM supplemented with 1X ITS (insulin, transferrin, selenium), 100 μM β-mercaptoethano, 6 mM L-Glutamine, and 1X nonessential amino acids were used as basic culture medium. We have found that in primary spermatogonia culture, cells cultured together with testis somatic cells (sertoli cells) in basic medium supplied with 2.5% fetal bovine serum (FBS) were much more efficient than culturing with a supplement of 10% FBS. In primary culture, growing round-shaped cell colonies were visible from Day 5 in basic culture medium. In subculture, colonies were enzymically digested into smaller pieces from Day 8 to 10 and then placed onto mouse embryonic fibroblasts feeder layer in 2.5% FBS basic medium with the addition of 100 ng mL-1 glial cell-derived neurotropic factor, 10 ng mL-1 leukemia inhibitory factor, and 10 ng mL-1 basic fibroblast growth factor. PGP9.5+ spermatogonia cell colonies maintained their normal round shape until 4 to 5 passages. However, in subsequent passages, the colonies became flattened and cells gradually lost their interconnection and the growth pattern present in the early passages. Also in subsequent passages, cells began expressing more C-kit than PGP9.5. In conclusion, PGP9.5+ spermatogonia were successfully isolated from 2- to 3-month-old ram testis, and PGP 9.5 cell colonies were maintained and proliferated in the in vitro culture system up to 2 months.

Ape1/Ref-1 Induces Glial Cell-Derived Neurotropic Factor (GDNF) Responsiveness by Upregulating GDNF Receptor α1 Expression

Apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1) dysregulation has been identified in several human tumors and in patients with a variety of neurodegenerative diseases. However, the function of Ape1/Ref-1 is unclear. We show here that Ape1/Ref-1 increases the expression of glial cell-derived neurotropic factor (GDNF) receptor alpha1 (GFRalpha1), a key receptor for GDNF. Expression of Ape1/Ref-1 led to an increase in the GDNF responsiveness in human fibroblast. Ape1/Ref-1 induced GFRalpha1 transcription through enhanced binding of NF-kappaB complexes to the GFRalpha1 promoter. GFRalpha1 levels correlate proportionally with Ape1/Ref-1 in cancer cells. The knockdown of endogenous Ape1/Ref-1 in pancreatic cancer cells markedly suppressed GFRalpha1 expression and invasion in response to GNDF, while overexpression of GFRalpha1 restored invasion. In neuronal cells, the Ape1/Ref-1-mediated increase in GDNF responsiveness not only stimulated neurite outgrowth but also protected the cells from beta-amyloid peptide and oxidative stress. Our results show that Ape1/Ref-1 is a novel physiological regulator of GDNF responsiveness, and they also suggest that Ape1/Ref-1-induced GFRalpha1 expression may play important roles in pancreatic cancer progression and neuronal cell survival.

Association study for genes at chromosome 5p13‐q11 in attention deficit hyperactivity disorder

Linkage of attention deficit hyperactivity disorder (ADHD) to the short arm-centromeric region of chromosome 5 has been reported in multiple studies. The overlapping region (5p13-q11) contains a number of strong candidate genes for ADHD, based on their role in brain function or neurodevelopment. The aim of this study was to investigate some of the top candidates among these genes in relation to ADHD in a sample of 245 nuclear families from the Toronto area. We investigated the genes for the glial cell-derived neurotropic factor (GDNF), the fibroblast growth factor 10 (FGF10), islet-1 (ISL1), the hyperpolarized potassium channel (HCN1) and the integrin alpha 1 (ITGA1). In addition to these genes, we assessed the 3'region of the SLC1A3 gene, a glutamate transporter implicated in ADHD by a previous association study. A total of 36 polymorphisms were selected across the six genes. We performed family-based association and haplotype analyses. ADHD is a dimensional disorder, with symptoms of inattention and hyperactivity-impulsivity therefore, we also conducted quantitative analysis in relation to symptom scores for both dimensions. Single marker and haplotype analyses yielded little evidence of association for any of the genes tested in this study. Moreover, we were unable to replicate the positive association findings reported for SLC1A3. Our results suggest that these six genes are unlikely to be susceptibility genes in the chromosome 5p13-q11 region and other genes should now be considered for priority study.