Expression and function of glial cell line derived neurotrophic factor family receptor alpha 1 alternatively spliced isoforms

Contents Acnowledgements i Contents ii Summary vii List of tables ix List of figures ix Abbreviations xi Chapter 1: Introduction 1 1.3.4 GDNF family multi-component receptor complex si

EXPRESSION AND FUNCTION OF GLIAL CELL-LINE DERIVED NEUROTROPHIC FACTOR FAMILY RECEPTOR ALPHA 1 ALTERNATIVELY SPLICED

ISOFORMS

PENG ZHONG NI (B Sc., UWA)

A THESIS SUBMITTED FOR THE DEGREE OF

MASTER OF SCIENCE DEPARTMENT OF BIOCHEMISTRY

NATIONAL UNIVERSITY OF SINGAPORE

2003

Acknowledgements

I would like to express my greatest gratitude to my supervisor, Associate Professor Too Heng-Phon for giving me this opportunity to pursue my interest in science and further study His guidance and supports for my research project have been tremendous and invaluable

I would like to thank specially the lab members, Jason, Thai, Nivetha and Lee Foong for their cooperation, assistance and encouragement I appreciate the great help from the staff and friends of the department, and the inspiring, encouraging and friendly environment, which makes my stay memorable and enjoyable

At last, I would like to express my deepest appreciation to my family, my dear husband Jun, my two lovely sons, Tony and Simon for their understanding, inspiring, patience and constant supporting Without them, this project would not be fulfilled

Contents

Acnowledgements i

Contents ii Summary vii List of tables ix List of figures ix Abbreviations xi Chapter 1: Introduction 1

1.3.4 GDNF family multi-component receptor complex signaling 8

1.4 GDNF 9

1.5 GFRα1 11

1.5.2 Expression and functional role of GFRα1a 12

1.6 Alternatively spliced isoforms 13

1.7.1 Quantification of gene expression at the transcription level 14

Chapter 2: Materials and Methods 18

2.1.4 Preparation of competent cells using calcium chloride 20

2.1.7.1 Alkaline lysis method (small scale preparation) 22

2.1.7.2 WizardTM Plus minipreps DNA purification system 22

2.1.7.3 WizardTM Plus midipreps DNA purification system 23

2.1.10 Total RNA extraction from mammalian cells using Quantum Prep AquaPureTM

2.1.13 Polymerase chain reaction (PCR) amplification 27

2.3.2 Cell transfection by FuGENE 6® Transfection Reagent 31

2.3.4 Cell Proliferation measured by Cell Proliferation BiotrakTM ELISA System 32

Chapter 3: Quantitation of GFRα1 Alternatively Spliced Isoforms, GFRα1a and

GFRα1b by Real Time PCR 34

3.1 Introduction 35

3.2.3 Reverse transcription (RT) of total RNA of human tissues and cell samples 38 3.2.4 Sequence dependent real time PCR using specific TaqMan probe for detection 38 3.2.5 Sequence independent real time PCR using SYBR Green I for detection 39

3.3 Results 43 3.3.1 Sequence dependent real time PCR quantitation using specific probes 43

3.3.1.1 Amplification efficiency using the common primers 44

3.3.1.2 Determining the optimum concentration of the specific MGB TaqMan probe

3.3.1.3 Detection with the specific MGB probe using common primers for

amplification is not reliable for the two isoforms 45

3.3.2 Sequence independent real time PCR quantitation using SYBR Green I 47

3.3.2.1 Amplification specificity and efficiency of sequence independent real time

PCR 47 3.3.2.2 Expression levels of GFRα1a, GFRα1b and c-Ret in various human tissues 51

Chapter 4: In Vitro Functional Study of The Two GFRα1 Spliced Receptor Isoforms

54

4.1 Introduction 55

4.2.1 Stably transfected cell lines of the two GFRα1 spliced isoforms 56

4.2.2 Quantitation of expression levels of the two GFRα1 spliced isoforms and c-Ret in

4.2.3 Morphological changes during differentiation of the transfected cell lines 57

4.3 Results 60

4.3.1 Expression levels of GFRα1a, GFRα1b and c-Ret in transfected Neuro2a cells 60

4.3.2 GDNF and NTN induced the differentiation of GFRα1a but not GFRα1b

4.3.3 Cell proliferation profiles of GFRα1a and GFRα1b transfected cells

4.3.4 Kinetic differences in the activation of Erk1/Erk2 signaling pathway between the

Chapter 5: Discussion and Future work 70

References 76 Appendix I: Media and Buffers 84

Appendix II: Bacteria strain and Mammalian cell line 87

Appendix III: Gene Organization and Sequences 89

Summary

Glial cell-line derived neurotrophic factor (GDNF) is a potent neurotrophic factor which shows restorative effects in a wide variety of rodent and primate models of Parkinson’s disease (PD) It promotes the survival of a broad range of central and peripheral neurons and is essential for kidney and enteric nervous system development, as well as regulating the fate of stem cells during spermatogenesis GDNF binds preferentially to GDNF family receptor alpha 1 (GFRα1), which mediates the activation of the proto-oncogene c-Ret receptor protein-tyrosine kinase to form a multi-component system and trigger off downstream signaling events Two alternatively spliced GFRα1 isoforms, GFRα1a and GFRα1b have been previously identified GFRα1a and GFRα1b are highly homologous, with GFRα1a containing an extra 15 nucleotide (exon 5) compared to GFRα1b Currently, the specific physiological functional roles of GFRα1a and GFRα1b are unknown Further more, the distribution and expression levels of these two isoforms have not been reported

To understand the physiological and functional role of GFRα1a and GFRα1b, stably transfected cell lines containing endogenous c-Ret were established to generate cells over-expressing either GFRα1a or GFRα1b Using the cell lines, mechanisms involved in the signaling pathways and functional roles of GFRα1a and GFRα1b in cell morphological differentiation and proliferation were investigated An isoform specific quantitative real-time PCR was used to confirm the existence and to measure the transcriptional expression levels of GFRα1a and GFRα1b in various human tissues

The signaling pathway studies showed that cells expressing GFRα1a or GFRα1b when stimulated by specific GDNF family ligands (GFL), GDNF and Neurturin (NTN), resulted

in rapid activation of MAPK (Erk1/2) and significantly different morphological changes

A modurate but significant difference in the kinetics of the phosphorylation of Erk1/2 was detected when GFRα1a and GFRα1b transfected cells were exposed to NTN No significant changes in the proliferation profile in the transfected cells were observed compared to parental cells containing the vector only Real time PCR revealed that both GFRα1a and GFRα1b isoforms existed in all the human tissues examined, and the transcriptional expression levels of the two isoforms were similar in human fetal and adult brain GFRα1b levels were found to be much higher than GFRα1a in peripheral tissues

In conclusion, the two spliced isoforms, GFRα1a and GFRα1b are differentially expressed

in the human tissues examined A close investigation reveals that the both isoforms, despite having only 5 amino acid sequence difference, show remarkable differences in their signaling and capabilities in inducing morphological differentiation

List of Tables

Table 3.1 Sequences and concentrations of the primers used in real time

List of Figures

Fig.1.1 GDNF family ligands and their preferential receptors signal through

Fig.1.2 NCAM as an alternative receptor for GDNF signaling receptor

Fig.3.1 Exon organization of human GFRα1a and GFRα1b spliced isoforms

and locations of the primers and the specific MGB TaqMan probe 41 Fig.3.2 Exon organization of c-Ret isoforms, Ret 51, Ret 43 and Ret 9, the common

primers Ret-NF and Ret-NR were used for the amplification and

Fig.3.3 Amplification of five log dilutions of standards plasmids

Fig 3.4 Agarose gel electrophoresis of amplified products by real time PCR 45 Fig.3.5 Optimization of the specific probe concentration for the sequence

Fig.3.6 Real time PCR detection using GFRα1a specific MBG probe 47 Fig.3.7 Amplification and detection of GFRα1a and GFRα1b using specific exon

Fig.3.8 Amplification of GAPDH and c-Ret standards by real time PCR 50

Fig.3.10 GFRα1 isoforms and c-Ret expression levels in various human tissues 53 Fig.4.1 Expression levels of GFRα1a, GFRα1b and c-Ret in the transfected cell

Fig.4.2 Expressions of c-Ret in the three transfected cell lines detected by Western

blotting 62

Fig.4.4 Percentages of differentiated cells (N-GFRα1a, N-GFRα1b and

Fig.4.5 Proliferation study of the transfected cells expressing GFRα1a or GFRα1b

Fig.4.6 Proliferation study by measuring BrdU incorporation 66

Fig.4.7 Activation of Erk1/2 in the transfected cells stimulated by GDNF or NTN

Fig.4.8 Phosphorylation of Erk1/Erk2 stimulated by GDNF and NTN in Neuro 2a

EDTA ethylene diamine tetra-acetate

GDNF Glial-cell line derived neurotrophic factor

IPTG Isopropyl thio-β-D-galactoside

MAPK mitogen activated protein kinases

MOPS 3-[N-morpholino] propane-sulphonic acid

NTN Neurturin

PAGE poly-acrylamide gel electrophoresis

Chapter 1

Introduction

1.1 Introduction

Neurotrophic factors play crucial roles in the development, growth and maintenance of the developing and adult nervous systems They are secreted polypeptides, regulating many aspects of neuronal and glial structures and functions via paracrine and autocrine mechanisms (Yuen and Mobley 1996)

Since the discovery of the first neurotrophic factor, Nerve growth factor (NGF) (Bradshaw

et al 1993), many other neurotrophic factors have been since identified and characterized based on either their structural or functional similarities These neurotrophic factors include neurotrophins, neurokines and the glial cell line-derived neurotrophic (GDNF) family of ligands All these neurotrophic factors signal via specific multi-component receptor complexes (Siegel and Chauhan 2000)

In the vertebrate nervous system, ample evidence suggest that neurotrophic factors play critical roles in the survival and phenotypic differentiation of developing neurons and the maintenance and protection of mature and injured neurons (Ebadi et al 1997; Connor and Dragunow 1998) Alteration in neurotrophic factor levels due either to age, genetic background or other factors might contribute to neuronal degenerations characteristic of Alzheimer’s disease (AD), Parkinson’s disease (PD) and other neurodegenerative diseases (Miyazaki et al 2003) Some recent therapeutic strategies have included the use of neurotrophic factors in preventing, reducing and rescuing the neuronal loss and atrophy that occur in neurodegenerative disorders such as AD and PD (Martin et al 1995; Martin

et al 1996; Bjorklund et al 1997; Kordower et al 2000; Tuszynski et al 2002)

1.2 GDNF in Parkinson’s disease (PD)

PD is a common progressive neurodegenerative disease which affects almost 1 % of the worldwide population over 65 (Giasson and Lee 2000) It is caused by degeneration of neurons in a region of the brain that controls movement PD is characterized by selective degeneration of substantia nigra dopaminergic neurons and was first described by a London physician James Parkinson in 1817 This neuronal degeneration creates a shortage

of the brain signaling chemical, dopamine, which in turn leads to impaired motor function

PD is characterized clinically by rigidity, tremor and bradykinesia, which result from the progressive death of dopaminergic neurons in substantia nigra region in the brain The defining feature of classical PD is the accumulation of Lewy bodies revealed in microscopic observation of brain sections, which contain ubiquitin and α-synuclein (Baba

et al 1998) Although the symptoms and pathology of PD have been well characterized, the underlying mechanisms and the cause of the disease still remained unknown

A number of studies have shown the potential of GDNF as a therapeutic agent for neurodegenerative diseases like PD Injection of GDNF into the substantia nigra or striatum potently protected dopaminergic system and significantly restored the dopamine levels and dopaminergic fiber densities after the administration of MPTP,(1-methyl-4 phenyl-1,2,3,6 - tetrahyropyridine), a neurotoxin known to cause Parkinsonian-like symptoms, in experimental animals (Opacka-Juffry et al 1995; Tomac et al 1995; Shults

et al 1996; Gash et al 1998) GDNF has also been shown to promote the differentiation

and survival of mesencephalic substantia nigral dopaminergic neurons both in vitro and in

vivo and enhances their dopamine up-take (Hudson and A.C Granholm 1993; Lile et al

1993; Stromberg et al 1993; Bowenkamp et al 1995; Gash et al 1995; Krieglstein et al

1995; Heller et al 1996; Connor et al 2001) In various experimental PD models, the level of GDNF in the substantial nigra was found to decrease prior to the onset of symptoms, which may be indicative of early symptom resulting from selective neuronal degeneration in this region (Meyer et al 1999; Oo et al 2003) These studies are suggestive of a potential therapeutic role of GDNF in the treatment of PD (Lile et al 1993; Schaar et al 1993; Schmidt-Kastner et al 1994; Gash et al 1995; Opacka-Juffry et al 1995; Martin et al 1996; Shults et al 1996) Further studies have demonstrated a neurochemical and behavioral improvement in unilateral dopamine-lesioned animal models after intranigral administration of GDNF This suggests that GDNF maintains dopaminergic neuronal phenotype survival after a nigrostriatal lesioning in the rat brain This is consistent with a possible physiological role of GDNF in regulating the event The

most recent in vivo study involving direct administration of GDNF into striatal target

demonstrated a role for this neurotrophic factor in preventing the early phase of neuronal death by suppressing apoptosis in dopaminergic neurons (Oo et al 2003) The recent success of the clinic phase 1 trial of GDNF for the treatment for Parkinson patients has lend further evidence that GDNF is a promising therapeutic agent (Gill et al 2003)

Despite the many studies of the physiological significance of GDNF, the biochemical and molecular mechanisms underlying its neuroprotective effects are only beginning to be unraveled

1.3 GDNF family

1.3.1 GDNF family ligands (GFLs)

The GDNF family of growth factors consists of four members, glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN), persephin (PSP) and artemin (ART) They are structurally related homologous proteins and belong to a subfamily of growth factors within the transforming growth factor-β (TGF-β) superfamily GDNF was first isolated from B49 rat glial cells, and was shown to have a potent neurotrophic effects in cultured rat embryonic dopaminergic neurons (Lin et al 1993) NTN was purified as a survival factor for sympathetic neurons (Kotzbauer et al 1996) ART and PSP were then identified based on their sequence homology to GDNF and NTN (Baloh et al 1998; Milbrandt et al 1998) NTN and ART have many neurotrophic effects that are similar to GDNF In contrast, PSP is expressed at low level in most tissues and supports CNS dopamine and motor neurons survival, but not peripheral neurons (Milbrandt et al 1998) All the four members exert their neurotrophic effects through a multi-component receptor signaling system formed by the interactions between the Ret tyrosine kinase (Takahashi

et al 1985) and a cysteine-rich GPI-anchored specific binding subunit called GDNF family receptor alpha (GFRα) (Fig.1.1) Recently, this multi-component signaling complex is shown to include the neural cell adhesion molecule (NCAM) (Fig.1.2)(Paratcha et al 2003)

GDNF NTN ART PSP

RET GFRα1 GFRα2 GFRα3 GFRα4

Fig.1.2 NCAM as an alternative receptor for GDNF signaling receptor complex

GDNF signals through the interacting with GFRα1and NCAM complex, which leads to activation of Fyn tyrosine kinase It is currently unknown if other GDNF family of ligands signal in the same manner as GDNF or cross-talk exist between the different receptors and ligands PM – plasma membrane

1.3.2 GDNF family receptors

Until recently, GFLs were thought to signal through a receptor complex comprising of a high-affinity binding receptor (GFRα1 - GFRα4) and a common component, Ret All the four members of GFLs have their own preferred receptors (Fig.1.1) GDNF signals preferentially via GFRα1 (Jing et al 1996; Treanor et al 1996), NTN via GFRα2 (Baloh

et al 1997; Jing et al 1997; Klein et al 1997), while ART signals through GFRα3 (Jing et

al 1997; Baloh et al 1998), and GFRα4 (Thompson et al 1998) is the receptor for PSP NTN can also stimulate GFRα1 almost equally as well as GDNF in cell lines (Baloh et al 1997; Creedon et al 1997; Jing et al 1997), whereas ART and GDNF only interact weakly to GFRα1 and GFRα2, respectively (Baloh et al 1997; Jing et al 1997; Baloh et al 1998) High concentrations of NTN can also mediate responses through the activation of GFRα4 (Enokido et al 1998)

GFRα receptors are plasma membrane proteins attached to the membrane by a glycosyl phosphatidylinositol (GPI) anchor The amino acid sequences of GFRα1 and GFRα2 have revealed internal homologies within a conserved cysteine-rich domain and suggests a common domain structure shared by the two receptors (Suvanto et al 1997) A similar domain is also present in GFRα3 and GFRα4

1.3.3 c-Ret as co-receptor for GDNF family

As the specific GFRα receptor of GDNF is a GPI linked membrane surface protein, it was thought that the GFRα may require the interaction with another transmembrane spanning receptor for intracellular signaling c-Ret was subsequently identified as the second signaling so-receptor GDNF as c-Ret was shown to co-immunoprecipitate with

GDNF/GFRα and is functionally involved in mediating the biological responses of GDNF (Durbec et al 1996; Trupp et al 1996)

In human, activating germline Ret mutations are associated with inherited cancer syndrome known as multiple endocrine neoplasia type 2A and 2B (MEN2A, MEN2B), familial medullary thyroid carcinoma (FMTC) and papillary thyroid carcinomas (PTC) (Santoro et al 1995; Eng et al 1996; Edery et al 1997) On the other hands, the lose of function of Ret causes Hirschsprung’s disease, a congenital disorder of the enteric nervous system

Ret was originally identified and described as a novel oncogene in 1985 (Takahashi et al 1985) The Ret gene encodes a single-pass transmembrane receptor tyrosine kinase with four cadherine-like repeats in its extracellular domain and an intracellular tyrosine kinase domain The intracellular domain of Ret consists of 14 tyrosine residues (a short isoform

of Ret lacks 2 tyrosine residues at the c-terminus) Phosphorylated tyrosine residues were identified as docking sites adaptor proteins such as Grb10, Grb7, Shc and Src (Pandey et

al 1995; Asai et al 1996; Pandey et al 1996; Airaksinen et al 1999; Encinas et al 2004) The Ret gene was mapped to human chromosome 10q11.2 (Ishizaka et al 1989), with a total of 21 exons and multiple alternative spliced isoforms (Myers et al 1995)

1.3.4 GDNF family multi-component receptor complex signaling

GFLs bind to specific GFRα receptors, which leads to the activation of Ret by tyrosine autophosphorylation In the original model, a GDNF molecule as a homodimer first binds

to the two monomeric GFRα1, and the GDNF-GFRα1 complex then interacts with Ret, promoting Ret dimerization and autophosphorylation (Jing et al 1996) Ret has been

shown to activate several pathways when the tyrosine autophosphorylation occurs This includes the Ras-MAPK (Santoro et al 1994; Worby et al 1996), phosphatidylinositol 3-kinaes (PI3K) (van Weering and Bos 1997), Jun N-terminal kinase (JNK) (Chiariello et al 1998; Xing et al 1998) and PLCγ (Borrello et al 1996) dependent pathways

The stoichiometry and kinetics of the multicomponent ligand-receptor complex are not well understood GPI linked membrane receptors were thought to cluster into lipid rafts to form a signaling patch, which is essential for the recruitment of Ret to the lipid raft after GDNF stimulation, resulting in Ret/Src association (Tansey et al 2000) It is assumed that the other GFL members bind to their specific receptor and activate Ret in a similar manner GFRα receptors utilize transmembrane Ret as a signaling co-receptor upon the stimulations of the GDNF family ligands However, in the absence of Ret, GDNF family ligands can still signal in some cells which expresses GFRα receptor, indicating the involvement of a possible new member of co-receptor (Poteryaev et al 1999; Trupp et al 1999) The most recent report of the neural cell adhesion molecule, NCAM as an alternative signaling receptor for GDNF family ligands proves the existence of a Ret independent signaling pathway NCAM can associates with GFRα1 and promotes high-affinity binding of GDNF and NCAM, resulting in rapid activation of cytoplasmic protein tyrosine kinases Fyn and FAK in cells lacking Ret (Paratcha et al 2003)

1.4 GDNF

1.4.1 Discovery and identification of GDNF

GDNF is the first member of the GDNF family discovered It was initially isolated and cloned as a factor produced by B49 rat glial cells with potent neurotrophic effects in cultured embryonic rat midbrain dopaminergic neurons (Lin et al 1993; Lin et al 1994) GDNF is a glycosylated, disulfide-bonded homodimer with a molecular weight of approximately 33-45 kDa Analyses of the sequence of GDNF isolated from B49 rat glial cells suggests that GDNF is secreted as a mature protein of 134 amino acids (Lin et al 1993) Its amino acids sequence revealed that it is closely related to transforming growth factor beta (TGF-β) superfamily, containing seven cysteine residuals, but shared less than 20% of overall sequence homology as compared to TGF- β (Milbrandt et al 1998) The potential receptor binding surfaces have been identified based on the crystal structure of GDNF (Eigenbrot and Gerber 1997) In human, the gene coding for GDNF is located on chromosome 5p12-p13.1 (Schindelhauer et al 1995)

1.4.2 Neurotrophic effects of GDNF

GDNF is a potent neurotrophic factor which promotes the survival of a wide variety of neurons, including dopaminergic, motor, noradrenergic, enteric, parasympathetic, sympathetic and sensory neurons (Henderson et al 1994; Airaksinen et al 1999; Manie et

al 2001; Airaksinen and Saarma 2002) It is also essential for ureteric branching in kidney formation (Towers et al 1998; Sariola and Saarma 1999) and regulates the fate of stem cells during spermatogenesis (Meng et al 2000) GDNF is now known to promote the

survival neuronal populations in both the central and peripheral nervous systems (Baloh et

al 2000; Takahashi 2001) and has important functions both in neuronal and non-neuronal

tissues (Sariola 2001) In vitro, GDNF supports the survival of embryonic, dopaminergic

neurons (Lin et al 1993), increases potently the survival of embryonic rat motor neurons

in culture by increasing cell number, neurite outgrowth, choline acetyltransferase activity (Henderson et al 1994; Zurn et al 1996) and promotes regeneration and protection of

injured neurons (Bennett et al 1998) In vivo, GDNF almost completely rescues motor

neurons in rats from lesion-induced cell death (Yan et al 1995; Rosenblad et al 1999) In gene knockout model of GDNF-/-mouse, a subset of spinal motor neurons as well as neurons in superior cervical ganglion, dorsal root ganglion and NPG (Baloh et al 2000) were missing, indicating that those neurons require GDNF for survival

GDNF exerts its neurotrophic effect via a multi-component signaling system In vivo,

GDNF is thought to be released in limited amounts by distinct target tissues in which it binds to its specific receptors and activate Ret Using PCR techniques, the regional distribution and cellular localization of GDNF has been examined in the human and mouse CNS (Schaar et al 1993; Stromberg et al 1993; Schaar et al 1994; Springer et al 1994) In human CNS, GDNF transcripts have been identified in the striatum, caudate, hippocampus, cortex and spinal cord Similarly, in the rat CNS, GDNF is expressed in all major regions such as the striatum, hippocampus, cortex, cerebellum and the spinal cord

1.5 GFRα1

1.5.1 GFRα1 and its spliced isoforms

GFRα1 is an essential component of the multi-component receptor complex in GDNF signaling It also responds to the stimulation of NTN and mediates its signaling transduction GFRα1 was discovered by expression cloning and screening of GDNF binding protein From the studies, a cDNA clone of GFRα1 from mouse retinal culture was identified to encode a polypeptide of 486 amino acids (Jing et al 1996; Treanor et al 1996) GFRα1 is a GPI (glycosyl phosphatidylinositol) linked cell surface protein, binds GDNF with high affinity and mediates the association of GDNF with Ret GFRα1 is rich

in cysteine (31 of 468 amino acids) and has a N-terminal hydrophobic domain with the characteristics of a secretory signal peptide and a C-terminal hydrophobic region of about

20 amino acids in length The human GFRα1 was isolated by cross-species hybridization screening using rat cDNA and a cDNA library prepared from adult human substantial nigra The human GFRα1 is a polypeptide of 465 amino acids and is 93% identical to mouse GFRα1 The human GFRα1 gene locus spans a genomic region of around 70 kb and is located at chromosome 10q25 The gene is encoded by 11 exons, which includes an untranslated exon upstream of the first methionine and 10 additional exons (Eng et al 1998)

Two alternative spliced transcripts of GFRα1, GFRα1a (Jing et al 1996; Treanor et al 1996) and GFRα1b (Jing et al 1996; Treanor et al 1996; Dey et al 1998) were identified Both isoforms are highly homologous except that GFRα1b lacks exon 5 (5 amino acids) compared to GFRα1a Currently, the functional and physiological roles of the two spliced

isoforms are unknown and remain to be elucidated Furthermore, the tissue distribution and expression levels of these two isoforms have not been reported

1.5.2 Expression and functional role of GFRα1a

Since GFRα1 was originally discovered and described as GPI-linked protein required by GDNF for its physiological responses (Jing et al 1996; Treanor et al 1996), the expression and functional role of GFRα1 as receptor for neurotrophic factor GDNF have

been widely reported In vivo, GFRα1 is coexpressed with Ret in the developing kidney

and enteric system GFRα1 deficient mice have deficits in the enteric nervous system and kidneys (Cacalano et al 1998; Enomoto et al 1998) GFRα1 is widely expressed in a number of GDNF-responsive neuronal populations, including midbrain dopaminergic and spinal cord motor neurons (Garces et al 2000; Marco et al 2002) It has also been detected in peripheral ganglia, including the trigeminal, nodose, superior cervical and dorsal root ganglia (Trupp et al 1997; Cacalano et al 1998; Widenfalk et al 1999) In human, the abnormal expression of GFRα1 has been associated with Hirschsprung’s disease and sporadic modular thyroid carcinoma (Gimm et al 2001; Lui et al 2002) Expression of GFRα1 has been detected in human developing and adult spinal cord, dorsal root ganglia and testis during fetal development and in adult men (Davidoff et al 2001), suggesting a critical role for GDNF and GFRα1during the differentiation of testicular structure A recent report of soluble and bound forms of GFRα1 eliciting different GDNF-independent neurite growth responses indicates that GFRα1 may signal without GDNF and plays additional physiological roles other than being the receptor for GDNF (Mikaels-Edman et al 2003)

1.6 Alternatively spliced isoforms

Alternative splicing is a mechanism, by which more than one transcript (mRNA) can be expressed form a single gene locus (Gilbert 1978) This increases the information contained within one gene and allows complexity in the regulation of its expressions It was estimated that 5 % of genes in higher eukaryotes have alternative spliced forms (Sharp 1994) The completed human genome revealed a number of human expressed mRNA that is much higher than the number of genes (30,000 – 40,000 human genes), suggesting a major role for alternative splicing in the production of genetic complexity (Modrek and Lee 2002)

Various alternative spliced isoforms of different growth factor receptors have been reported, examples of these are FGFR1, TrkA, TrkB, TrkC (Barbacid 1994; Wang et al 1995; Tam et al 1997),VEGF receptors (Giasson and Lee 2000), GFRα2 (Wong and Too 1998) and GFRα4 (Lindahl et al 2000) Spliced variants of Ret have been demonstrated

to have distinct biochemical and physiological functions (Giasson and Lee 2000) The alternative spliced forms of the receptors showed differences in specificities and affinities

to the cognate ligands as well as their signaling pathways Characterization of NGF (Selby

et al 1987) in rat, mouse and human have shown that several different transcripts are produced from as many as four exons found upstream of the coding region In the case of the BDNF gene, alternative splicing give rise to 6 different transcripts (Nakayama et al 1994) The tissue specific expressions of the isoform provide evidence that alternative splicing of the BDNF gene produced proteins of different biological significance Hence, the existence of spliced isoforms and their differential distributions may influence the physiological responses profoundly

1.7 Quantification of gene expression

1.7.1 Quantification of gene expression at transcription level

Determination of the expressions of the alternatively spliced variants of a gene is increasingly important as many cellular decision involving survival, growth and differentiation are reflected in the quantitative differential expressions of these variants(Zamorano et al 1996) Four methods are commonly used for the quantification of

transcription These include Northern blotting, RNA in situ hybridization (Parker and

Barnes 1999), RNAse protection assays (Hod 1992) and reverse transcription polymerase chain reaction methods (RT-PCR) (Weis et al 1992) Northern blotting is the only method that provides information about the mRNA size, alternative spliced transcripts and the integrity of the sample The RNase protection method is most useful for mapping the

initiation and termination sites and intron/exon boundaries of transcripts RNA In situ

hybridization allows the localization of transcripts to specific cellular location within a tissue These three methods have a low sensitivity compared to RT-PCR (Melton et al

1984) RT-PCR is an in vitro method that involves enzymatical amplification of target

sequence of mRNA (Rappolee et al 1988) All these methods have their own limitations Northern blotting is time-consuming and requires relatively large amounts of RNA RNase protection analysis is more sensitive than Northern blotting, but it is not sensitive enough

to detect low abundance transcripts To quantify gene expressions using RT-PCR, the reaction must be terminated and quantified in the early exponential phase, which is often not easily controlled (Vandenbroucke et al 2001) The development of real time PCR techniques has overcome this limitation and it is now widely used for the quantitation of gene expression

1.7.2 Absolute quantification using real time PCR

The use of real time PCR was first reported in 1992, where the process of amplification was monitored by ethidium bromide (EtBr) (Higuchi et al 1992; Zamorano et al 1996) EtBr is no longer favored for monitoring PCR progress in real time as it fluoresces in the presence of both double- and single-stranded DNA SYBR green I is now the fluorescent dye of choice as it binds preferentially to double-stranded DNA, thus replacing EtBr for real time PCR detection

In real time PCR, the accumulation of products generated during each cycle of the reaction process can be reliably correlated with the amount of starting template Here, the product

is detected at each cycle by measuring the fluorescence emitted As the amount of product

is directly proportional to the intensity of fluorescence emitted, it is possible to measure the progress of amplification by real time monitoring of fluorescence Thus, by measuring

a defined intensity of fluorescence (Ct, threshold cycle), it is then possible to compare the progress of PCR of two separate reactions (Gibson et al 1996) The Ct values are determined at the early stages of amplification or even at the exponential phase of PCR This is the most accurate moment of the reaction for the detection as the reagents for amplification are not limited during the exponential phase Real time PCR offers a wide dynamic range of quantification (> 107 fold) and is ideally suited for high-throughput analyses

1.8 Aim of this study

Identification of GFRα1b, a novel spliced receptor isoform of GFRα1(Dey et al 1998), has prompted our investigation into the distribution and function of this isoform Both isoforms share high sequence homologies The objective of this study was firstly, to investigate expression levels of the two isoforms in various human tissues This will enable a better understanding of their potential functions in the tissues The expression levels of the two isoforms and their signaling partner, Ret, were measured in various human tissues including adult and fetal brain by real time PCR Secondly, Western blot analyses were carried out to measure the activations of MAPK/Erk1/2, JNK, p38, PI3K/AKT in GFRα1a and GFRα1b expressing cells treated with GDNF and NTN to determine the signaling pathways used by these two isoforms Finally, the functional roles

of the two spliced isoforms in cell morphological differentiation and proliferation were investigated using Neuro 2a cells transfected with GFRα1a and GFRα1b

Chapter 2

Materials and Methods

2.1 Molecular techniques

2.1.1 Agarose gel electrophoresis of DNA

A 1 % (w/v) agarose gel was prepared by melting 0.3 g of powdered agarose (SeaKem LE, BioWhittaker Molecular Application, USA) in 30 ml of 1 x TAE buffer (Appendix I) containing 0.25 µg/ml of ethidium bromide (Sigma-Aldrich, USA) The molten agarose was poured into a gel casting tray and allowed to set at room temperature The DNA samples premixed with appropriate amount of 6x loading dye (0.25 % bromophenol blue, 0.25 % xylene cyanol FF and 30 % glycerol) were loaded into the gel and electrophoresed

in 1x TAE buffer at 80 to 110 volts The migration of bromophenol blue dye in the samples was monitored till it reached the desired distance The gel was then viewed under

UV illumination and recorded by ChemiDocTM system (ChemiDocTM MZL, Bio-Rad Laboratories, Italy)

2.1.2 DNA recovery from agarose gel

The desired DNA fragment was gel purified using the Qiagen DNA purification system (Qiagen GmbH, Germany) according to the manufacturer’s instruction After electrophoresis, the agarose gel slice containing the desired DNA fragment was excised under visualization with UV illumination (300 nm) and weighed Three gel volume of QG buffer was added and incubated with shaking to completely dissolve the gel slice One gel volume of isopropanol was added and mixed The mixture was then added to the column where DNA was bound by hydrophobic interaction with the silica in the column in high

salt buffer The column was then washed to remove impurities and the DNA eluted with

50 µl of TE buffer (Appendix I)

2.1.3 Ligation of DNA fragment with vector

DNA fragments isolated from agarose gel were ligated to linearised vector using an insert

to vector ratio of 5 to 1 Ligation was carried out by mixing appropriate amounts of insert DNA and vector in T4 DNA ligase buffer (30 mM Tris-HCl, pH 7.8, 10 mM MgCl2, 10

mM DTT and 1mM ATP) and 2 - 3 U T4 DNA ligase (Promega, USA) in a 20 µl reaction and incubated at 4ºC overnight Half of the reaction was analyzed on an agarose gel to check the extent of the ligation

2.1.4 Preparation of competent cells using calcium chloride

One milliliter of overnight E.coli (Appendix II) culture was used to seed a 50 ml of 2 x PY

media (Appendix I) and grown in an incubator with vigorous shaking (200 rpm) to an

OD590 of 0.5 at 37°C Cells were harvested by first chilling on ice for 30 min and then centrifuged in a cold rotor at 3,500 X g for 10 min at 4°C The supernatant was removed and the pellet was resuspended gently in 50 ml of cold CaCl2 solution (Appendix I) and kept on ice for 20 min before they were pelleted as before Competent cells were finally resuspended in 2.5 ml of cold CaCl2 solution and stored as 100 µl aliquots at -80°C

2.1.5 DNA transformation of E.coli cells by heat shock

The competent E coli cells prepared by the calcium chloride method (Section 2.1.4) were

thawed on ice and mixed with DNA The mixture was allowed to stand on ice for 30 min, after which the competent cell was heat shocked at 42oC in a water-bath for 90 sec Immediately after the heat shock, the DNA-competent cell mixture were chilled on ice for another 10 min One milliliters of pre-warmed 2 x PY media (Appendix I) was then added

to the heat-shocked cell and incubated at 37oC for an hour At the end of the incubation,

200 µl of the heat-shock cells were plated onto the 2 x PY agar plate (Appendix I) and selection was carried out with the appropriate antibiotics (Appendix I)

2.1.6 Selection of recombinant DNA

With vectors exploiting blue/white color selections, the bacteria culture was plated on 2 x

PY agar plate (Appendix I) with 100 µg/ml ampicillin (Sigma-Aldrich, USA) 80 µg/ml gal (Promega, USA) and 1 mM IPTG (Promega, USA) The antibiotic selection differentiates between the transformed bacteria and wild type bacteria, while the X-gal/IPTG selects for transformants with an insert (white colony) and those transformants harboring self-ligated vector without insert (blue colony)

X-2.1.7 Isolation of plasmid DNA

2.1.7.1 Alkaline lysis method (small scale preparation)

Plasmid preparation from bacterial culture (1-3 ml) was carried out using conventional plasmid preparation by alkaline lysis (Birnboim and Doly 1979) Two milliliters of overnight bacterial culture was collected by centrifugation at 13,000 X g for half a minute and the pellet was subsequently suspended in 100 µl of TE buffer Cells were then thoroughly lysed in 200 µl of lysis buffer (0.5M NaOH and 4 % SDS) One hundred fifty microliters of 3 M sodium acetate, pH 4.5, was added and gently mixed to prevent shearing of genomic DNA The precipitate was collected by centrifugation at 12,000 X g for 15 min The supernatant was subsequently extracted with phenol/chloroform (1:1; v/v) Plasmid DNA was then precipitated by adding 2.5 volume of 100 % ethanol and centrifuged at 13,000 X g for 20 min The pellet was then washed with equal volume of ethanol (70 %) and air-dried at room temperature The pellet was resuspended in 50 µl of

TE buffer (Appendix I) containing a RNase cocktail (5 U, Ambion)

WizardTM Plus minipreps DNA purification system (Promega, USA) provides a simple and reliable way for the rapid isolation of plasmid of up to 20 kbp One to 5 ml overnight bacterial culture was centrifuged at 13,000 X g for 2 min The supernatant was removed and the cell pellet was resuspended in 300 µl of cell resuspension solution (50 mM Tri-HCl, pH 7.5, 10 mM EDTA and 100 µg/ml RNase A) Three hundred microliter of cell lysis solution (0.2 M NaOH, 1 %SDS) was then added and mixed gently After that 300 µl

of neutralization solution (1.32 M potassium acetate, pH 4.8) was added and mixed by inverting the tube several times The mixture was then centrifuged at 13,000 X g for 10 min The supernatant was transferred into a fresh tube and 1 ml of resuspended resin was added The mixture was transferred to a minicolumn and washed with 2 ml column wash solution (80 mM potassium acetate, 8.3 mM Tris-HCl pH 7.5, 40 µM EDTA and 55 % ethanol) The minicolumn was then transferred to a centrifuge tube and centrifuge at 13,000 X g for 3 min to get rid of any residual column wash solution and the DNA was eluted in 50 µl TE buffer (10 mM Tris-HCl pH 7.5 and 1 mM EDTA)

Overnight bacterial culture (10-100 ml) was centrifuged at 13,000 X g for 5 min The supernatant was removed and the cell pellet was completely resuspended in 3 ml of cell resuspension solution (50 mM Tri-HCl, pH 7.5, 10 mM EDTA and 100 µg/ml RNase A) Three milliliters of cell lysis solution (0.2M NaOH, 1 %SDS) was then added and mixed gently After that 3 ml of neutralization solution (1.32 M potassium acetate, pH 4.8) was added and mixed by inverting the tube several times The mixture was then centrifuged at 13,000 X g for 15 min The supernatant was transferred into a fresh tube and 10 ml of resuspended resin was added The mixture was transferred into a column and then washed with 15 ml of column wash solution (80 mM potassium acetate, 8.3 mM Tris-HCl pH 7.5,

40 µM EDTA and 55 % ethanol) twice and centrifuge at 13,000 X g for 3 min to get rid of any residual column wash solution The plasmids DNA was eluted either in 300 µl of TE buffer (10 mM Tri-HCl pH 7.5 and 1 mM EDTA) or double distilled water

2.1.7.4 NucleoBond ® Plasmids purification system

Plasmids for transfection were prepared using NucleoBond® Plasmids purification system (Clontech laboratories, USA) NucleoBond® AX (Clontech laboratories, USA) is a silica based anion exchange resin The purified nucleic acids products are suitable for use in the most demanding molecular biological application, including transfection The following protocol is used to purify plasmids ranging from 3-10 kb

Fifty milliliters of overnight bacterial culture were centrifuged at 6,000 X g for 15 min at 4°C and the supernatant was discarded The pellet was resuspended in 0.4 ml of Buffer S1 + RNase A (50 mM Tris-HCl, 10 mM EDTA, 100 mg/ml RNase A) Buffer S2 (4 ml) (200 mM NaOH, 1 % SDS) was then added and mixed by gentle inversion Buffer S3 (0.3ml) (2.8 M KAc, pH 5.1) was then added and incubated on ice for 5 min The mixture was centrifuged at 10,000 X g for 20 min at 4°C The cleared lysate was loaded into a NucleoBond cartridge The cartridge was washed with 1 ml of Buffer N3 (100 mM Tris,

15 % ethanol, 1.15 M KCl, adjusted to pH 6.3 with H3PO4) for three times by centrifugation The plasmid DNA was eluted with 0.8 ml of Buffer N5 (100 mM Tris, 15

% ethanol, 1 M KCl, adjusted to pH 8.5 with H3PO4) 0.6 ml of isopropanol was added to the eluate and the mixture was centrifuged at 10,000 X g for 30 min at 4°C The pellet was washed once with 70 % ethanol, air dried and re-dissolved in 50 µl of double distilled water

2.1.8 Restriction enzyme digestion of plasmid DNA

Plasmid DNA was digested using the recommended buffers according to the manufactory’s instructions (Promega USA, or Amersham USA) One to four enzyme units

(U) were used per µg of DNA Restriction digestions were incubated at 37ºC for about 1

hr (depending on the restriction enzyme) and the reaction was terminated by heating at 70

ºC for 20 min The digests were analyzed by agarose gel electrophoresis (Section 2.1.1)

2.1.9 DNA sequencing

DNA for sequencing was prepared using the Big-Dye sequencing kit (PE Applied Biosystems), following the manufacturer’s instructions Sequencing reaction was carried out either with 0.5 µg of circular plasmid DNA or 0.2 µg of PCR products and 10 pmole

of primer with 8 µl of the reaction pre-mix (Big-Dye) in a total of 20 µl reaction in a thermal cycler (PT-100, MJ Research Inc., Watertown, MA, USA) using the following parameters, 95oC for 15 sec, 50oC for 30 sec and 68oC for 4 min for 25 cycles To recover the sequencing reaction product, the sample (20 µl) was precipitated with 2 µl of 3 M sodium acetate (pH 5.0) (Sigma-Aldrich), and 44 µl of 100 % ethanol (Merck, Germany) and the pellet was collected by centrifugation at 13,000 X g for 20 min The supernatant was aspirated, while the pellet was washed with 250 µl of 70 % ethanol, air dried and sequenced (NUMI NUS, Singapore) The sequence data was analyzed by using DNASTAR (DNASTAR Inc, Madison, WI, USA) and DNAMAN (Version5.2.9, Lynnon Biosoft, USA) softwares

2.1.10 Total RNA extraction from mammalian cells using Quantum Prep

AquaPureTM RNA isolation system

Total RNA was extracted from cultured cells using AquaPureTM RNA isolation kits Rad, USA) Cells were grown in 75 cm2 tissue culture flask untill confluent Growth medium was removed and 1.5 ml of RNA lysis solution (Citric acid 2.5-5 %, EDTA 2.5-5

(Bio-%, SDS 2.5-5 %) was added to the cells and the lysate was transferred to a tube Five hundred microliter of protein-DNA precipitation solution (NaCl 20-35 %, citric acid 2.5-5

%) was then added and mixed gently The mixture was placed on ice for 5 min and then centrifuged for 3 min at 13,000 X g The supernatant containing RNA was transferred to a fresh tube and 1.5 ml of 100 % isopropanol was added The mixture was then centrifuged

at 13,000 X g for 10 min The pellet was washed with 70 % ethanol; air dried and resuspended with 50 µl of RNA hydration solution The concentrations and purity of the RNA were determined by spectral wavelength scan from 220 to 320 nm and calculated based on 1OD260 is equal to 40 µg/ml of RNA The integrities of the total RNA were validated by denaturing agarose gel electrophoresis (Section 2.1.11)

2.1.11 Agarose gel electrophoresis of RNA

Denaturing agarose-formaldehyde gel was used for RNA electrophoretic analysis A 1% (w/v) agarose (SeaKem LE, BioWhittaker Molecular Application, USA) formaldehyde gel was prepared in 1 x MOPS buffer (Appendix I) containing 1.5 % formaldehyde (v/v, Sigma Chemical Co Germany) RNA loading sample was prepared by mixing the total RNA (2-10 µl, up to 20 µg) with 25 µl loading buffer (Appendix I) containing 0.03 mg/ml

of ethidium bromide (Sigma Chemical Company, USA) and electrophoresis in 1 x MOPS buffer The gel was viewed and recorded by ChemiDocTM system (ChemiDocTM MZL, Bio-Rad Laboratories, Italy)

2.1.12 Reverse transcription (RT) of RNA

Ten microgram of total RNA was heat denatured with 0.5 µg of random hexamer (Promega, USA) at 65oC for 5 min and snap chilled on ice for 2 min Reverse transcription was carried out in 5 mM DTT (Invitrogen Corporation, USA), 40 unit of RNase inhibitor (Promega, USA), 10 mM of dNTP (Promega, USA) and 400 U of SuperscriptTM II reverse transcriptase (Invitrogen Corporation, USA) in a final volume of 40 µl The reaction was incubated at 42oC for 60 min and terminated by heating at 70oC for 5 min

2.1.13 Polymerase chain reaction (PCR) amplification

A typical PCR amplification was performed in a 100 µl reaction volume containing 10-20

ng of DNA template, 0.1-0.5 µM of each primer, 2mM MgCl2, 0.2 mM dNTP, 1 x Taq polymerase buffer and 2.5 units of Taq polymerase (Promega, USA) PCR reaction was carried out after an initial denaturation for 5 min at 95oC followed by 30 cycles of 30sec denaturation at 95oC, 1 min at 55°C, and 2 min extension at 72oC with one final cycle for

10 min at 72°C in a thermal cycler (PTC-100 TM Programmable thermal Controller, MJ Research Inc., USA) The amplified product was analyzed by agarose gel electrophoresis (Section 2.1.1)