Sajwan-Khatri Mamta, Balasubramanian Senthilkumaran⁎
Keywords:GFRα-1;GDNF signalling;Brain development;MPTP;Teleosts
ABSTRACT
Glial cell line-derived neurotrophic factor (GDNF)is a potent trophic factor that preferentially binds to GDNF family receptor α-1 (GFRα-1)by regulating dopaminergic (DA-ergic) neuronsin brain. Present study aimed to evaluate the significance of GFRα-1 expression during early brain development in catfish. Initially, the full- length cDNA of GFRα-1 was cloned from adult brain which showed high homology with other vertebrate counterparts. Quantitative PCR analysis of tissue distribution revealed ubiquitous expression of GFRα-1 in the tissues analyzed with high levels in female brain and ovary. Significant high expression was evident in brain at 75 and 100 days post hatch females than the respective age-match males. Expression of GFRα-1 was high in brain during the spawning phase when compared to other reproductive phases. Localization of GFRα-1 revealed its presence in preoptic area-hypothalamus which correlated well with the expression profile in discrete areas of brainin adult catfish. Transient silencing of GFRα-1through siRNA lowered expression levels of GFRα-1, which further down regulated the expression of certain brain-specific genes. Expression of GFRα-1 in brain declined significantly upon treatment with the 1-methyl-1,2,3,6-tetrahydropyridinecausing neurodegeneration which further correlated with catecholamines (CA), L-3,4-dihydroxyphenylalanine, DA and norepinephrine levels. Taken together, GFRα-1 plausibly entrains gonadotropin-releasing hormone and gonadotropin axiseither di- rectly or indirectly, at least by partially targeting CA-ergic activity.
1.Introduction
Glial cell line-derived neurotrophic factor (GDNF) is a potent sur- vival factor for many central and peripheral neurons, including dopa- minergic (DA-ergic) neurons. GDNF signals preferentially through GPI- anchored receptor, GDNF family receptors-α having stronger binding activity over GFRα-1 (Airaksinen et al., 2006; Trupp et al., 1998). GDNF-GFRα-1 complexes recruit the tyrosine kinase transmembrane protein to execute differentiation of DA-ergic neurons in brain (Naughton et al., 2006; Durbec et al., 1996). The expression of GFRα- 1in certain areas of the brain where neurons responsive to GDNF family members reside have been analyzed inmammals (Quartu et al., 2007; Matsuo et al., 2000) and less studied in lower vertebrates including teleosts (Shepherd et al., 2001; Lucini et al., 2010; Lucini et al., 2011). Teleosts serve as an excellent animal model to study neurodegeneration partially due to the abundance of neuronal precursor cells such as glial cells (Zupanc and Clint, 2003). Prominently, glial cells are the most abundant cell type in the teleost brain, which have crucial roles in neuroendocrine systems (Barry et al., 2014; Xing et al., 2014) and also produce neurosteroids (Pellegrini et al., 2007), such as estradiol-17β (E2) and neurotrophic factor, GDNF (Xing et al., 2016). Infact, co-lo- calization of E2 receptors (ER) and DA-neurons reported earlier in tel- eosts indicating a possible interaction of DA-ergic neurons with neu- rosteroids (Dufour et al., 2010). Studies on ER knockout mice revealed a significant loss of DA neuronswhich supports the contention that ER mediated mechanisms involve neuroprotective actions (Wang et al., 2001). E2 also protect the brain against toxicity induced by excitatory neurotransmitters, oxidative stress and neurotoxins (Shahrokhi et al., 2012). Earlier report using Gfra1 knock-out mice showed loss of mo- toneurons in lumbar spinal cord (Moore et al., 1996; Cacalano et al.,1998), which showed the requirement of Gfra1 for survival signalling by GDNF in spinal motoneurons. Furthermore, prominent motoneuron groups that strongly express Gfra1were lost in the Gfra1 mutant, and hence, GFRa1 is critical for the survival of neurons during development.
GFRα-1enhances the survival and differentiation of DA neurons, and decreases apoptosis of damaged DA neurons (Yasuhara et al., 2007) thereby exhibiting neurorestorative and neuroprotective actions (Wissel et al., 2006). It is also well known that the enzyme cyp19a1b, (brain aromatase) catalyzes the conversion of androgens to estrogens (Rasheeda et al., 2010) and found to be expressed in glial cells of adult fish brain (Diotel et al., 2010; Forlano et al., 2001). Previous studies in teleosts revealed a feedback mechanism exerted by gonadal steroids (Habibi et al., 1989; Senthilkumaran and Joy, 1995) by targeting brain through catecholaminergic (CA-ergic) system. In goldfish brain, DA released from DA-ergic neurons affects cyp19a1b expression in glial cells, through DA receptor (Xing et al., 2016) which has an inhibitory role in glial cell proliferation and differentiation through transcription factor-mediated path ways elicited by neurosteroids. It remains to be seen whether GFRα-1 target neurosteroids by regulating DA in brain. Considering this, in the present study an attempt was made to damage DA-ergic neurons using neurotoxin 1-methyl-1,2,3,6-tetra- hydropyridine (MPTP)that is known to cause loss ofDA-ergic neurons in fishes (McKinley et al., 2005) by selectively targeting DA-ergic neurons after systemic administration (Weinreb and Youdim, 2007). This sort of approach will provide a basis to understand the molecular mechanisms of GFRα-1 in DA regulation vis-à-visneurosteroids release at the level of brain. Among monoamines, serotonin (5-HT) and norepinephrine (NE) have a stimulatory influence ongonadotropin-releasing hormone (GnRH) and gonadotropin (GTH) release while DA inhibits the same (Peter et al., 1991; Goos et al., 1999; Senthilkumaran et al., 2001). Considering these findings, the impact of DA on neurosteroids vis-à-vis GFRα-1′s action is an important topic of analysis in teleosts.
The air- breathing catfish, Clarias gariepinusfollows a seasonal reproductive cycle with recrudescence where the brain under goes physiological cyclicity in accordance to gonadal changes and returns to spawning stage as season approaches. This unique characteristic feature facilitates present work to comprehend at the transcript levels of various genes that play crucial roles in neuroendocrine control of reproduction and neural plasticity focusing on the interaction of GFRα-1 on DA-ergic system. Secondly, teleost (catfish) will be an ideal experimental model as distinct changes of mono aminergic system drive GnRH-GTH axis to entrain reproductive cycle.Main objective of the present study is to investigate the involvement of GFRα-1 in the neuroendocrine control of reproduction through DA- ergic system. Hence to implicate GFRα-1 as an intermediate between GDNF-DA-ergic system, GFRα-1 was cloned and the ontogenic expres- sion was analyzed in the brain of catfish, C. gariepinus. The impact of transient silencing of GFRα-1, in vivo through siRNA complexed with polyethylenimine (PEI) in brain was analysed in catfish to understand its functional significance by analysing various brain-specific genes. Further, to examine the neurotoxin effects of MPTP, catecholamines (CA) levels were measured in brain to understand the interaction of GFRα-1 and DA-ergic activity.
2.Materials and methods
2.1.Animal and sampling
The air-breathing catfish, C. gariepinus, bred and reared as per the method described earlier (Raghuveer et al., 2011), were used for the present study. Soon after hatching, the fingerlings were retained in plastic tubs with continuous aeration under ambient photothermal conditions. Live tube worms(Tubifex tubifex) were fed for catfish hatchlings ad libitum till adulthood. Annual reproductive cycle of catfish is divided into preparatory, pre-spawning, spawning and post- spawning/regressed phases. Commercial pelleted fish feed, was given ad libitum to adult catfish (∼ 1-year-old) and reared in the outdoor tanks in ambient photothermal conditions. Fish sampling was done by fol- lowing the general guidelines of the Institutional Animal Ethics Com- mittee, University of Hyderabad(Reg./No./151/1999 dt. 22.07.1999). Animals were briefly anesthetized with 100 mg/L of ethyl 3-amino- benzoate methane sulfonate (MS-222; Sigma; St. Louis, MO, USA) in mild ice-cold water and, samples were dissected out on ice and stored briefly at −80 °C until analysis for various parameters.
2.2. Cloning of GFRα-1 from catfish brain
Degenerate primers were designed for GFRα-1 by using the nu- cleotide information available in NCBI database. Total RNA was pre- pared using TRI reagent® by following the manufacturer’s protocol (Sigma). The quality and quantity of total RNA was analyzed using Nanodrop spectrophotometer (ND-2000, Nano Drop Technologies and Wilmington, DE, USA). About 500 ng of total RNA was used for reverse transcription by following the protocol of verso® cDNA synthesis kit (Thermo Scientific Inc., Waltham, MA, USA). PCR amplification was accomplished with Taq 2X master mix (New England Biolabs Inc., Ipswich, MA) using degenerate primers as per subsequent conditions: initial step of 94 °C (2 min), 94 °C (1 min), 53 °C (1 min), 72 °C (1 min), for 35 cycles and final extension at 72 °C (10 min). The PCR amplicon was gel purified and ligated in pGEM®-T easy vector (Promega, Madison, WI, USA) and sequenced bidirectionally. Gene specific pri- mers were then designed for 5′ and 3′ rapid amplification cDNA ends (RACE) using SMARTer™ RACE cDNA amplification kit (Clontech, Mountain View, CA, USA). The amplicons were cloned into pGEM®-T vector and nucleotide information was obtained through bidirectional DNA sequencing. Both partial and RACE derived nucleotide sequences were aligned through Lasergene software (DNASTAR, Madison, WI, USA) to deduce the full-length cDNA.
2.3.Sequence and phylogenetic analysis
The deduced amino acid (aa) sequences of cloned GFRα-1 of catfish and other vertebrates from GenBank were used for the Clustal Omega alignment and the phylogenetic tree was constructed using neighbor- joining method. The GenBank accession numbers of GFRα-1 sequences used are as follows. Ictalurus punctatus (XM_017482772), Danio rerio_a (NM_131730), D.rerio_b (NM_131731.1), Oreochromis mossambicus (KR779759.1),Takifugu rubripes (XM_003961583.2), Labeo rohita (HM130051.2),Salmo salar (LOC106577126),Bubalus bubalis (XM_006071676), Oryzias latipes (XM_004080268.2), Mus musculus (NM_010279), C. gariepinus (KY553234). Clustal Omega (https://www. ebi.ac.uk/Tools/msa/clustalo/) multiple alignment tool was used for the construction of phylogenetic tree by neighbor-joining method and expressed using Jalview 2.8 and TreeView 1.6.6 software.
2.4.Quantitative real-time PCR (qRT-PCR)
Expression analysis for all the genes were determined through qPCR using SYBR green detection method. Total RNA isolation and first strand cDNA synthesis for all the samples were done as described above. The purity of RNA was checked using a Nano Drop spectro- photometer (ND-2000, NanoDrop Technologies and Wilmington, DE, USA) and the integrity of RNA was confirmed by running the sample in a formaldehyde agarose gel. Random hexamers were utilized for the reverse transcription using verso® reverse transcriptase (Thermo Scientific Inc., Waltham, MA, USA) with 1 μg of brain total RNA iso- lated from brain using TRI-reagent®(Sigma) as per the manufacturer’s protocol followed by DNase I treatment to eliminate the genomic DNA. In addition, one of the qRT-PCR primers chosen were at exon–exon junction. The reaction was done in triplicate using qPCR primers in MicroAmp® 96-Well plates with SYBR green master mix in a 7500-fast thermal cycler (Applied Biosystems, Foster City, CA, USA) as per the manufacturer’s universal thermal cycling conditions. After performing melting-curve analysis to check the amplicon specificity,cycle threshold (Ct) value was calculated from the exponential phase of PCR amplification and the gene expression was normalized against the ex- pression of 18S rRNA (as reference gene) to generate a ΔCt value (Ct of target gene – Ct of reference gene). Relative expressions of the genes were calculated using 2 −ΔCtmethod.
2.5.Tissue distribution and diferential expression of GFRα-1 in adult catfish brain
qRT-PCR was performed to analyze the tissue distribution of GFRα-1 in adult male and female catfishusing the GFRα-1 specific primers (listed in Table 1). Total RNA was extracted from different tissues (gill, spleen, heart, muscle, kidney, liver, ovary, testis and brain of male and female) of adult catfish using the method described above. Catfish at different age groups [0, 10, 20, 30, 40, 50, 75, 100, 150, 200, and 250 days post hatch (dph)] were collected (5 fish brains were pooled till 100 dph and taken as one biological sample, n = 5 at 0 dph head was taken) for ontogeny analysis (n = 5). Morphological distinction of the gonad occurs in catfish around 50 dph and hence sexing of brain was done from that stage for ontogeny studies. All the samples were stored briefly at −80 °C before being used for total RNA preparation and cDNA synthesis as described earlier.
2.6.Reproductive phase expression
Since catfishis an annual breeder, brain from different reproductive phases were dissected as described earlier and stored to analyze the expression pattern of GFRα-1. Total RNA was prepared from each sample (n = 5) followed by cDNA synthesis was done as explained before. Further qPCR was performed in triplicate and the relative ex-
pression was determined.
2.7.Expression of GFRα-1 in diferent regions of the adult female brain
GFRα-1 mRNA levels were analyzed in different regions of the adult female brain such as telencephalon + olfactory bulb (TEL + OB), pi- tuitary (PT), preoptic area-hypothalamus (POA-HYP), thalamus (TH) and medulla oblongata (MO), after dissecting those out as per Raghuveer et al. (2011).
2.8. In situ hybridization (ISH)
Localization of the transcripts of GFRα-1 in adult catfish brain was done by using ISH, as per the method described earlier (Rajakumar and Senthilkumaran, 2014). Fixation and sectioning of adult brain was done as explained before (Sudhakumari et al., 2017). Based on the sequence information of pGEM-T easy- GFRα-1 cDNA either T7 or SP6 RNA polymerase was used for sense and antisense ‘cRNA’ probe preparation using digoxigenin (DIG; Roche Diagnostics GmbH, Mannheim, Ger- many), 2 μg of digested plasmid was used as a templateand RNAase inhibitor (20U) were added then reaction were incubated at 37 °C for 2 h in a thermocycle (ABI Prism 2720 thermocycler) based on in vitro transcription. The sections were treated and fixed using phosphate buffered saline (PBS) with Tween 20 in diethyl pyrocarbonate treated water (0.1% v/v), proteinase K and paraformaldehyde (PFA). About 1 μl of purified sense and antisense cRNA probes were diluted in 200 μl of hybridization buffer and heat denatured at 80 °C for 5 min. The mixture was then added onto the sections and kept overnight at 50 °C in a sterile RNase free incubator. Slides were washed with wash and blocking buffer solution (Roche). Anti-DIG-ALP antibody fab fragments (Roche Cat. No. 1109324910) were added onto the sections and kept at 4 °C overnight. Slides were washed with DIG washing buffer (Roche) and incubated further with detection buffer (Roche). The sections were color developed using BCIP-NBT (Roche) as substrates while nuclear red was used as acounter stain. After color development, the slides were washed and dehydrated using a graded ethanol series then mounted using DPX mountant. Finally, photomicrographs were taken in CX41 Olympus microscope (Olympus corporation, Tokyo, Japan) with Q capture pro 6 software-controlled micropublisher 3.3 RTV-CCD camera (Quantitative Imaging corporation, BC, Canada).
2.9. Western blot analysis
Western blot analysis was performed to detect GFRα-1 protein using GFRα-1/GFRα polyclonal antibody (Life Span Biosciences, Cat. No. LS- C294196/70558; Seattle, WA, USA) raised against the conserved N-terminal regions of human GFRα-1 which showed 85% homology with the conserved domain region of catfish GFRα-1. Tissue homogenate prepared from adult catfish brain and muscles (negative control) by homogenization with ice cold 1 M Tris, pH 7.4, 150 mM NaCl, 1 mM DTT, and protease inhibitor cocktail. The mixture was centrifuged to 10,000 × g at 4 °C to isolate supernatant and later quantification of protein was determined using Bradford method. About 50 μg of tissue homogenate protein was run onto a 12% SDS-polyacrylamide gel electrophoresis. Separated proteins were then transferred onto a ni- trocellulose membrane (Pall Life sciences, Port Washington, NY, USA) and the bands were visualized by ponceau S staining. Further, the membrane was blocked with 5% skimmed milk in Tris-buffered saline (TBS) for 1 h at room temperature (RT). After several washes with TBS containing 0.1% Tween 20, membrane was incubated with the re- spective antibody (1: 1000) dilution for overnight at 4 °C. After washing steps, secondary antibody of goat anti-rabbit IgG coated with HRP was added (1: 5000; Merk Bangalore Genei, Bengaluru, India) and the bands were developed by treating with BCIP-NBT (Roche, Roche Diagnostics GmbH, Mannheim, Germany) as substrates.
2.10.Immunohistochemistry (IHC)
Adult catfish brain was dissected to localizeGFRα-1 protein as per the procedure described (Rajakumar and Senthilkumaran, 2014).In brief, the tissues were dissected and fixed in 4% PFA in PBS for over- night at 4 °C. It was then washed with PBS several times before being embedded using cryomedium. Brain embedded incryoblock was cut at 7 μm thickness onto Poly-L-Lysine coated glass slide using cryostat (Leica CM1850, Leica Microsystems). The sections were hydrated using PBS and later blocked with 10% normal goat serum (Merck Bangalore Genei) for 1 h. Polyclonal antibody of GFRα-1 (1:1000) and pre-ad- sorbed antibody with excess GFRα-1 antigen (for negative control) was added on the sections and kept overnight at 4 °C. Then, the sections were washed thrice with PBS and incubated with HRP-conjugated secondary antibody (1: 5000; Merck Bangalore Genei) for 1 h at RT. Sections were later treated with ABC reagent(avidin-biotinylated horseradish peroxidase complex) supplied in VECTASTAIN® Elite ABC kit (Vector Laboratories, Burlingame, CA, USA) for 30 min. The slides were washed with PBS and developed using commercially supplied 3′, 3′-diaminobenzidine as chromogen and H2O2 (Vector Laboratories) as a substrate for horseradish peroxidase. The color development was stopped by washing the sections with PBS and counterstained with hematoxylin (Qualigens fine chemicals, Mumbai, India), followed by dehydration using a graded ethanol series and finally mounted using DPX mountant. All the photomicrographs were taken using CX41Olympus microscope (Olympus corporation, Tokyo, Japan) fitted with Q capture pro 6 software-controlled micropublisher.
2.11. GFRα-1-siRNA PEI mediated transfection, in vivo
Small interfering RNAs (siRNAs) was commercially synthesized using ∼500 bp length of GFRα-1 gene cloned from catfish brain. Experiment was carried out with custom made GFRα-1 siRNA ( 5′-UUGGUACAGAUUAUCACUGUUGUAGCU-3′) designed (Sigma) from catfish GFRα-1nucleotide sequence. About 2 μg of siRNA was taken and combined with branched PEI (MW 25 kDa, Sigma) dissolved in sterile HEPES-NaCl pH 7.4, constructed on the protocol explained earlier (Höbel and Aigner, 2010, 2013). The mixture was kept for 20 min at RT to form siRNA-PEI complex. Fish were anesthetized with 100 mg/L of MS 222 (Sigma) in mild ice-cold water following the guidelines of in- stitutional animal ethical committee. The mixture was injected (n = 5) directly into the brain through pineal window followed by topical ap- plication of denture adhesive powder was applied gently to cure the injected area. Scrambled (control) siRNA (MISSION siRNA Fluorescent Universal Negative Control #1, Cat. No. SIC007 conjugated with 6-FAM, Sigma) was used for control groups. All the injected catfishes were maintained with continuous aeration and replenishment of fresh water daily for 3 days. Administration of siRNA-PEI complex has been done and validated earlier for catfish (Sudhakumari et al., 2017).The samples were immediately frozen using liquid nitrogen and stored briefly at −80 °C for further analysis.Expression analysis was performed with gene specific primers (see Table 1) of several genes such as tryptophan hydroxylase (tph), tyrosine hydroxylase (th), catfish gonadotropin-releasing hormone (cfGnRH), 3β-hydroxysteroid dehydrogenase (hsd3b), 11β-hydroxylases (h11b), aromatase (cyp19a1b)and β-actin,and relative expression was calcu- lated using 2−ΔCtmethod. Relative expression of GFRα-1transcripts was calculated in PEI alone and control scrambled siRNA treated catfish brain to check the off-target effects, if any.
2.12.MPTP treatment, in vivo
MPTP purchased from Hychem laboratories (Catalogue No. 28289- 54-5) was used for understanding its effect on DA-ergic system vis-à-vis on GFRα-1. Dose-response study was performed to understand the ef- fect of neurotoxin MPTP, in vivo. The prespawning fish (n = 5) were injected intraperitoneally (IP) with five different concentrations of the MPTP 5, 10, 50, 100 and 150 μgonce and then maintained for 5 days, however, the control group received saline injection(vehicle). All the injected fishes were maintainedin different tanks each holding 50 l capacity of fresh water. This experimentwas done to understand the significance of GFRα-1 considering the changes in DA-ergic system.
2.13.Quantification CA levels in the brain after the exposure of neurotoxin,
MPTP using high performance liquid chromatography-electrochemical detection (HPLC-ECD)
Brain CA levels were measured by HPLC-ECD as described pre- viously (Nagao and Tanimura, 1988). Brain tissues were dissected after MPTP exposure and quickly frozen on dry ice and weighed (n = 5). The brain tissues were homogenized by ultrasonic disintegration (Bronson Sonifer 450, Danbury, CT, USA) 0.1 M perchloric acid (PCA) containing 0.1 mM EDTA, after centrifugation at 10,000g for 20 min at 4 °C, the clear supernatants were filtered through a 0.45 μm filter (Millex-HV, Millipore Corporation, Billerica, MA, USA). 20 μl of filtered sample was injected onto the C-18 reversed-phase column. The mobile phase con- tained 20% (vol/vol), methanol, 0.06 M sodium acetate/0.02 M citric acid buffer, 0.035% (wt/vol.) heptasulfonic acid and 0.1 mM EDTA, pH 3.92. The column temperature (17–30 °C) and flow rate (1 ml/min) was adjusted by HPLC-ECD (Waters, Milford, MA, USA) system (Murai et al., 1988). The working standard solutions were prepared in 0.1 M PCA containing 0.1 mM EDTA and stored at −80 °C. The 20 μl injection contained 20 pmol L-3,4-dihydroxyphenylalanine (L-DOPA), 30 pmol DA and 50 pmol NE. Elution peaks were recognized by associating the retention time of each peak in the sample solution to the peak in the standard solution (Murai et al., 1988). The levels of CA were expressed as nanograms per gram of wet tissue weight.
2.14. Statistical analysis
The data of qPCR analysis were expressed as mean ± standard error of mean (SEM). All the data passed homogeneity and normality testswere evaluated by one-way analysis of variance (ANOVA) followed by Student-Newman–Keuls’(SNK) post hoc test, Student’s t-test and a probability of P < 0.05 was considered statistically significant. All the statistical analysis was performed using Sigma Plot 11.0 software (Systat Software Inc., Chicago, IL, USA).
Fig. 1. Multiple sequence alignment of catfish GFRα-1 with deduced amino acid sequences of GFRα-1 of other vertebrates. Protein-BLAST of catfish GFRα-1 showed high homology with other sequences in GFRα-1 domain regions. GenBankaccession numbers used for different GFRα- were followed Ictalurus punctatus (XM_ 017482772), C. gariepinus (KY553234), Danio rerio_a (NM_131730), D. rerio_b (NM_131731.1), Oreochromis mossambicus (KR779759.1), Takifugu rubripes (XM_003961583.2), Labeo rohita (HM130051.2), Salmo salar (LOC106577126), Bubalus bubalis (XM_006071676), Oryzias latipes (XM_004080268.2), Mus musculus (NM_010279).
3.Results
3.1. Molecular cloning of GFRα-1
Partial cDNA of 244 bp was cloned from adult catfish brain by RT- PCR. cDNA fragments of 5′ and 3′ ends of GFRα-1was obtained from
brain by following RACE strategies. The nucleotide sequences of partial and RACE cDNAs were aligned using Lasergene software to generate complete cDNA fragment 1401 bp of GFRα-1. The open reading frame (ORF) of GFRα-1 encodes a putative protein of about 467 aa. All the nucleotide sequence information of the genes was submitted to NCBI GenBank and the primers used for cloning were specified in Table 1, the nucleotide sequence of GFRα-1 of catfish was submitted to GenBank having accession no. KY553234. Multiple sequence alignment (Fig. 1) was performed using Clustal Omega by aligning the catfish GFRα-1 deduced aa sequence with its vertebrate counter parts to check. Homology of the deduced aa from catfish GFRα-1 ORF was compared with its counterparts from other vertebrates. To further clarify its classification, phylogenetic analysis was performed (Fig. 2) using neighbor joining method software which revealed that the catfish, C. gariepinus GFRα-1 showed more homology with GFRα-1 of I. punctatus and GFRα-1b of D. rerioand formed a separate clade.
3.2.Tissue distribution and brain ontogeny expression analysisby qRT-PCR
The expression of GFRα-1 were ubiquitous in the tissues (n = 5) analyzed, however, significant (P < 0.05) high expression was evident in female brain (Fig. 3A). Moderate transcript levels were detected in male brain and ovary. Low level of expression was seen in testis, gills, spleen, heart and intestine (Fig. 3A). In ontogeny analysis, expression of GFRα-1was significantly (P <0.05) high in the developing female brain, at 75 and 100 dph while there were no significant differences in other stages analyzed (Fig. 3B).
3.3.Expression of GFRα-1 during reproductive phases
Phase-wise expression analysis of GFRα-1 showed elevated levels in the spawning phase when compared to other reproductive phases (Fig. 4).
3.4.Expression of GFRα-1 mRNA in diferent regions of adult female brain
Expression pattern ofGFRα-1 was quantified in five different regions of adult female brain (TEL +OB, POA + HYP, PT, TH and MO), wherein GFRα-1 mRNA level was higher in POA + HYP when compared to other regions of brain (Fig. 5).
3.5. ISH of GFRα-1 mRNA in catfish brain
Localization of GFRα-1 selleck products mRNA in catfish brain revealed positive signals in the POA-HYP(Fig. 6A–C), while the sense probe showed no signal confirming the specificity (Fig. 6D) of the reaction.
3.6. Immunolocalizationand western blot analysis of GFRα-1 in catfish brain
Immunoreactivity of GFRα-1 was observed in POA-HYP of adult brain (Fig. 7A–C) confirming the antibody specificity. Negative control (pre-adsorbed antibody) of GFRα-1 counter stained with haematoxylin displayed no immunoreactivity in the catfish brain (Fig. 7D). Further, validation of catfish GFRα-1 antibody was tested using western blot- ting. A sharp band of GFRα-1, ∼50 kDa, was observed in the brain- protein gel-segregation which is equivalent to the size of deduced cat- fish GFRα-1peptide. Upon transient silencing of GFRα-1using siRNA in brain, the levels GFRα-1 decreased considerably (Fig. 8C). Both these results validate the specificity of antibody used in IHC.
Fig. 2. Phylogenetic tree showing the evolutionary status of catfish GFRα-1. The phylogenetic analysis was performed using the neighbor-joining method and a bootstrap analysis with 1000 replicates was used to assess the strength of the nodes in the tree. The phylogenetic tree was generated using the TreeView software.
3.7. In vivo transfection of GFRα-1-siRNA in catfish brain
To check the efficiency of GFRα-1-siRNA-PEI complex administra- tion, control groups PEI and controlscrambled siRNA (n = 5) were in- jected separately (Fig. 8A). After the treatment, GFRα-1 mRNA was quantified and compared with respective control groups. PEI alone and control scrambled siRNA injected fish did not show any significant differences in the expression of GFRα-1. These results specify that in- jection of PEI and control scrambled siRNA independently (n = 5) did not affect the expression of genes analyzed including the levels of GFRα- 1(Fig. 8A). But, GFRα-1-siRNA-PEI complex injected brain showed significant (P < 0.05) decrease in Antiviral medication the expression of GFRα-1 when compared to all the other groups (Fig. 8A) confirming that GFRα-1- siRNA-PEI complex transiently silenced the expression of GFRα-1 (P < 0.05). Further,expression analysis on certain brain-specific genes such as tph, th, cfGnRH, hsd3b, hb11 and cyp19a1b (Fig. 8B) revealed significant (P < 0.05) reduction when compared to the control groups and unaffected β-actin gene indicating the impact of GFRα-1 transient gene silencing.
Fig. 4. qRT-PCR analysis of GFRα-1 expression during reproductive phases in adult female brain. Means with different alphabets differs significantly while means with similar alphabet did not have any significance.The relative ex- pression was normalized with 18S rRNA and the values were calculated using2 −ΔCtmethod. Data (n = 5) were expressed as mean ± SEM (* P < 0.05; one –way ANOVA followed by SNK test).
Fig. 3. Tissue distribution (A) and ontogeny (B)of GFRα-1expression in the catfish brain. The relative expression was normalized with 18S rRNA and the values were calculated using 2 −ΔCtmethod. (A) Data (n = 5) were expressed as mean ± SEM (*P < 0.05; one-way ANOVA followed by SNK test). Means with different al- phabets differs significantly while means with similar alphabets did not have any significance. (B) Data (n = 5) were expressed as mean ± SEM (*P < 0.05 Student’s t-test). Abbreviation: M-male; F-female; dph- day post hatch.
Fig. 5. qRT-PCR analysis of GFRα-1 expression patterns in different regions of the adult female brain. The relative expression was normalized with 18S rRNA and the values were calculated using 2 −ΔCtmethod. Data (n = 5) were ex- pressed as mean ± SEM (*P < 0.05; one-way ANOVA followed by SNK test). Means with different alphabets differs significantly while means with similar alphabets did not have any significance. Abbreviation: TEL + OB- tele- ncephalon + olfactory bulb, PT- pituitary, POA-HYP- preoptic area-hypotha- lamus, TH-thalamus and MO- medulla oblongata.
3.8. Efect of MPTP on catfish brain, in vivo
MPTPtreatment, in vivo in catfish brain showed dose related de- crease in the expression of GFRα-1 (P < 0.05) when compared to control (Fig. 9).
3.9.Changes in the level of CA in catfish brain after the exposure of MPTP
The levels of CA were significantly (P < 0.05) lower in the MPTP- treated brain when compared with the control group (Fig. 10). The levels of L-DOPA were significantly reduced (P < 0.05) at higher concentration of MPTP treatment when compared to control brain. However, no significant difference was observed at lower concentration of MPTP (Fig. 10A). Similarly, the levels of DA and NE were sig- nificantly decreased (P < 0.05) in the brain at two different con- centrations as compared to the control groups (Fig. 10B, C).
4.Discussion
In the current study, full-length cDNA of neurotrophic family receptor,GFRα-1was cloned from catfish brain using degenerate primers and RACE. Tissue distribution and ontogeny expression analysis re- vealed high expression of GFRα-1 in the developing and female adult brain, indicating a plausible role for GFRα-1 in the brain development of catfish. Expression pattern of GFRα-1 was in accordance with the transcript changes of th and DA levels in the female brain reported earlier (Mamta et al., 2014). Clustal Omega alignment showed its homology with the conserved signature GDNF superfamily domain of other vertebrates including teleosts. Transcript variants GFRα-1a, GFRα- 1b and GFRα-2 have been reported in other teleostsean member, D. rerio by using RACE (Shepherd et al., 2004). However, our cloning strategies such as degenerate RT-PCR, RACE and genomic PCR with the primers designed onto two neighboring highly conserved exons flanking a variable intron employed in the present study resulted in a single transcript of GFRα-1 in catfish. The phylogenetic analysis of GFRα-1 revealed that GFRα-1 of C. gariepinus showed high homology with GFRα-1 of I. punctatus and GFRα-1b of D. rerio forming a separate clade (Fig. 2). Expression analysis revealed higher GFRα-1 mRNA levels in the female brain and ovary with moderate levels in several other tissues and male brain. GDNF receptor complex expressed very early in the development of enteric nervous system which has been shown to pro- mote survival, proliferation, differentiation in D. rerio (Lucini et al., 2010). In mammals, GFRα-1 mRNA was predominantly expressed in the developing and adult DA-ergic neurons (Nosrat et al., 1997) to assume a role for GFRα-1 in brain development perhaps by targeting DA-ergic activity. In catfish, ontogeny analysis of GFRα-1 in brain suggested that this gene might be essential for early stage of brain development vis-à- vis gonadal development. As per our earlier studies, Tph and Th vis-à- vis 5-HT and CA might have an important role in gonadal differentia- tion either directly or indirectly (Raghuveer et al., 2011; Mamta et al., 2014). In addition, higher expression of GFRα-1 in spawning phases of catfish suggested that gene might have role in GnRH-GTH axis. How- ever, the levels of GTHsshowed seasonal variation warranting their regulation (Joy et al., 2000; Acharjee et al., 2015) through GnRH cy- clicity (Senthilkumaran et al., 1999).
Fig. 6. In situ hybridization of GFRα-1 in POA- HYP regions of adult catfish brain. Antisense probe in POA-HYP (A,B,C) and Sense probe in POA-HYP (D), Scale bar indicates: A and D:50 μm;B: 25 μm; and C: 10 μm. Note: No signal was detected in sense probe as against the antisense probe. Arrows indicates presence of transcript localization.
Fig. 7. Immunohistochemistry of GFRα-1 protein in POA-HYP regions of adult catfish brain. GFRα- 1 immunoreactivity was observed in POA-HYP (A–C) while preabsorption antibody treatment showed no immunoreactivity (D). Black arrow showing immunoreactive cells in POA- HYP re- gions. Scale bar indicates:A:50μm;B: 25μm;C:10 μm;D:120μm.Abbreviation: TEL +OB- telencephalon+olfactory bulb, PT- pituitary, POA-HYP- preoptic area-hypothalamus, TH- thalamus and MO- medulla oblongata.
Fig. 8. In vivo PEI mediated GFRα-1-siRNA transfection in adult catfish. The transcript of GFRα-1 were quantified and compared (A) between PEI alone, scrambled siRNA (control) and GFRα-1-siRNA (treated) group. Data (n = 5) were expressed. as mean ± SEM (*P < 0.05; one-way ANOVA followed by SNK test). Expressions of certain brain-specific genes (B) wereobserved after transfection with GFRα-1-siRNA.Data (n = 5) were expressed as mean ± SEM (*P < 0.05 Student’s t-test). Validation of western blot (C) showed a sharp band ∼50 kDa size while upon siRNA treatment the intensity of band is diminished.
Fig. 9. qPCR analysis showed expression of GFRα-1 between control and MPTP treated group, in vivo.The relative expression was normalized with 18S rRNA and the values were calculated using comparative 2 −ΔCtmethod. Data (n = 5) were expressed as mean ± SEM (*P < 0.05; one-way ANOVA followed by SNK test).Means with different alphabets differs significantly while means with similar alphabets did not have any significance. Abbreviation: cont-control.GFRα-1 might regulate DA to control GnRH-GTH axis on gonads (Peter et al., 1991) through medicare current beneficiaries survey DA-ergic activity. Over expression of GFRα-1 may alsofavour DA inhibitory tone either directly or indirectly to regulate GnRH-GTH axis which is well demonstrated in teleosts (Dufour et al., 2010; Peter et al., 1991; Goos et al., 1999; Senthilkumaran and Joy, 1996). Localization of GFRα-1 mRNA showed its predominant expression in POA-HYP. By and large, these results creditably support vital sig- nificance for GFRα-1 in brain. Earlier study reported intense GFRα-1 protein and high mRNA expression in different regions of brain and its rolein the central nervous system in adult D. rerio (Lucini et al., 2010). Interesting part of thepresent study is to analyze the regulatory role of GFRα-1 and its function by GFRα-1 siRNA transient silencingin vivo, through brain targeted injection wherein delivery of GFRα-1-siRNA-PEI complex decreased the expression of GFRα-1in catfish brain. After si- lencingGFRα-1, protein levels and the expression of various brain-spe- cific genes i.e. tph, th, cfGnRH, hsd3b,h11b, cyp19a1b were down- regulated and incidentally these genes are implicated to “brain sex differentiation” in catfish (Senthilkumaran et al., 2015). The outcome of the study demonstrated that the silencing of GFRα-1 act as foremost target to regulate the expression of genes at the level of brain and for the first time that signalling through a neurotrophic factor receptor absolutely required for the brain has been shown. However, it is im- portant to knowhow GFRα-1 regulate brain related genes as the present study demonstrated an effect, yet,exact mode of action is not clear. Considering these reports low expression of th and tph in brain after GFRα-1 silencing indicate a probable effect on CA-ergic and 5-HT-ergic neurons, respectively. This contention needs to be validated further.
Our findings showed possible effects of GFRα-1 transient knock- down on CA-ergic neurons and reduction of Th levels, the rate limiting enzyme of CA biosynthesis. In view of this, it is important to analyze the relationship between GFRα-1 and DA-ergic activity. Towards this, pre- sent study was extended to analyze the impact of neurotoxin, MPTP on DA levels,in vivo. MPTP treatment reduced GFRα-1 expression and CA levelsafter neurotoxin post injection in catfish brain and causes cellular toxicity to target DA-ergic system. Based on this, we showed that catfish DA-ergic neurons are susceptible to neurotoxicity after MPTP
Fig. 10. Quantitative estimation of catecholamines (CA), such as L-DOPA, DA and NEafter the exposure of MPTP with two different concentrations 10 μg and 150 μg. L-DOPA (A), DA (B) and NE levels(C)with respective controls using HPLC-ECD.Data (n = 5) were expressed as mean ± SEM (*P < 0.05; Student’s t-test). Abbreviation: cont-control, L-DOPA- L-3,4-dihydroxyphenylalanine, DA-dopamine and NE- norepinephrine treatment. This contention is supported by the reports that identified significant glial cell activation associated with the loss of DA-ergic neurons in MPTP-induced Parkinson disease models (Yokoyama et al., 2010) and causing damage to DA neurons in teleosts (Xing et al., 2016). However, the exact role of activated glial cells in DA neurodegeneration and regeneration remains to be seen. In goldfish, DA neurodegeneration starts after neurotoxin post injection in the HYP (Popesku et al., 2012), though recovery of DA and NE levels was observed after MPTP ex- posure. In the present study, the effect of MPTP was observed 5 days after an IP injection,however it was not extended to analyze any re- trieval of GFRα-1 and CA levels in the catfish brain. Certainly, in goldfish (Popesku et al., 2012) 6 days after MPTP exposure, DA levels were shown to be decreased by about 70% in the HYP and Tel. Unlike mammals, DA neurons in teleosts might regenerate following injection with MPTP (Poli et al., 1992; Pollard et al., 1992).
In addition, high expression of cyp19a1b implicating endogenous E2 having an important role in glial cells reaction in response to the neurotoxin MPTP (Xing et al., 2016) and the production of neurotrophic factors and genes in- volved in DA neuron development under the modulation of E2 espe- cially after damage. The involvement of GFRα-1, based on its impact on CA levels needs to be considered as the inhibitory tone of DA seems important for GnRH-GTH release during recrudescence (Peter et al., 1991; Senthilkumaran and Joy, 1995).The GDNF acts solely via GFRα-1 receptor to regulate DA-ergic neuron. GFRα-1 siRNA silencing led to reduction th expression wherein both DA and NE might have been subdued due to fact that Th being rate limiting enzyme of CA biosynthesis. Hence, low level of NE might have reduced GnRH rather than reduction in DA. During recrudescence, re- duction in NE levels might influence GnRH-GTH axis as NE plays a crucial role in gonadal recrudescence in seasonally reproducing teleosts (Senthilkumaran and Joy, 1995; Goos et al., 1999). MPTPis a well- known blocker of DA-ergic neuron and hence, reduction in GFRα-1 expression wasseen, which is in accordance with the report of Popesku et al. (2012). Further, reduction in DA andNE levels confirms that MPTP did have an impact on CA biosynthesis. Hence, association of GFRα-1and CA in particular DA is evident. In addition, GFRα-1 ex- pression in accordance to CA levels may have role for early brain de- velopment, plausibly through DA-ergic activity, either directly or in- directly.
5.Conclusion
In conclusion, abundance of GFRα-1 mRNA and protein expression in the brain seems to suggest asignificant role for this correlate perhaps through GDNF signalling in the brain of catfish, C. gariepinus. In addi- tion, tissue distribution and ontogeny analysis showed high expression in developing (female) brain. Localization of GFRα-1 protein by IHC and mRNA by ISH in the POA-HYP of brain indicates plausible sig- nificance in relation to GnRH-GTH axis in catfish. Transient silencing of GFRα-1-siRNA and MPTP neurotoxin treatment indicate an important role for GFRα-1 in coordination with DA-ergic activity. Decreased ex- pression of certain brain-specific genes after GFRα-1-siRNA transient silencing revealed the regulatory influence of GFRα-1 on brain more importantly on GnRH-GTH, either indirectly or directly.
