The role of neurotrophin-3 in primary sensory neurons

Neurotrophin-3 (NT-3) is a member of the nerve growth factor family of neurotrophins. These molecules regulate aspects of sensory neuron survival, proliferation, phenotype, regeneration, and nociception. NT-3 presumably acts upon binding to its preferred receptors, trkC and p75, but much of the literature concerning its role involves embryological studies; little is known regarding its role in the adult, aside from its part in proprioception. Here, the effect of a 7 day intrathecal infusion of NT-3 on the phenotype of intact or axotomized adult rat dorsal root ganglion (DRG) neurons was examined. Serial sections were processed for in situ hybridization and computer-assisted image analysis was undertaken to characterize the NT-3 responsive subpopulation and to compare relative levels of mRNA for neurotrophin receptors, neuropeptides, cytoskeletal elements, injury- and regeneration-associated molecules, and other markers in individual neurons. 125I-NGF was utilized to determine the influence of NT-3 on high-affinity NGF binding site densities. Data show that ~ 40% of neurons coexpress trkC and p75 mRNAs; some of these cells also exhibit transcripts for trkA, NFM, Ta1 a-tubulin, a-CGRP, SP, galanin, NPY, GAP-43, cjun, and SNAP-25 - establishing the presence of potentially functionally significant micropopulations within the trkC-positive subset. Following injury, levels of many biochemical markers are altered in a positive or negative fashion. In all cases described here, if the marker colocalizes with trkC, post-trauma treatment with NT-3 allows for a return towards normal message levels, suggesting a role for NT-3 in the maintenance of normal adult phenotype in these cells. In the intact state, NT-3 effects a reduction in trkA, high-affinity NGF binding sites, and SP levels, within non-trkC neurons. The importance of these markers in nociception suggests a role for NT-3 in analgesia. But, in addition, NT3 also reduces SNAP-25 mRNA levels in otherwise normal trkC-expressing cells, which might negatively alter their functioning. Together, these data indicate that multiple subsets of mature DRG neurons are responsive to NT-3, not all of which express trkC, and not all of which respond to the neurotrophin in the same manner: these factors must be taken into account when considering therapeutic applications for NT-3

Do neurotrophins play a role in adult neurogenesis?

Tese de doutoramento em Ciências Biomédicas, (Neurociências Básicas), apresentada à Universidade de Lisboa através da Faculdade de Medicina, 2009O processo de neurogénese continua activo ao longo da vida adulta em mamíferos graças a células estaminais em determinadas zonas do cérebro. Esta capacidade representa um importante potencial para terapias regenerativas. Em roedores, a zona subventricular (SVZ) gera milhares de neuroblastos diáriamente, os quais migram para o bolbo olfactivo (OB) e se diferenciam em interneurónios. Descobertas recentes indicam que a neurotrofina Brain-Derived Neurotrophic Factor (BDNF) estimula a neurogénese adulta na SVZ, mas o mecanismo pelo qual actua é desconhecido. Na minha tese, analizei a função de BDNF e do seu receptor TrkB na neurogénese adulta da SVZ. Descobri que a proteína BDNF está presente na SVZ e que TrkB é o receptor de neurotrofinas mais abundante nessa zona do cérebro, mas apenas foi detectada a forma truncada deste receptor (TrkB-TR). TrkB-TR é expresso em células ependimais e astrócitos da SVZ, mas não em neuroblastos. Embora mutantes TrkB exibam proliferação e sobrevivência reduzida na SVZ e tenham menos neurónios novos no OB, células da SVZ knockout para TrkB (TrkB-KO) transplantadas para a SVZ de um cérebro normal (wild type, WT) são capazes de produzir neuroblastos que migram para o OB. Com a excepção de células periglomerulares dopaminérgicas, interneurónios do OB derivados de transplantes de TrkB-KO e WT exibiram propriedades moleculares, morfológicas e de sobrevivência semelhantes. Para estudar o efeito de BDNF na neurogénese da SVZ, infundi esta neurotrofina no ventrículo lateral de ratazanas e ratos adultos. Após 14 dias de infusão, registei uma redução de proliferação na SVZ de ambas as espécies. No entanto, a produção de neurónios foi afectada diferencialmente: infusões de BDNF reduziram o número de novos neurónios no OB de ratazanas mas não tiveram efeito no de ratos. Interessantemente, ratazanas e ratos também diferem na expressão do receptor de neurotrofinas p75. Em ratazanas, detectei p75 em muitas células tipo C (progenitores intermediários da SVZ) e em alguns neuroblastos, enquanto que muito poucas células p75+ foram detectadas na SVZ de ratos. Este é o primeiro estudo a encontrar tais efeitos, uma vez que outros laboratórios relataram um aumento de neurogénese após tratamento com BDNF. Globalmente, os meus resultados indicam que TrkB não é essencial para a produção e diferenciação da maioria dos interneurónios derivados da SVZ e não confirmam a actual visão de que administração de BDNF à SVZ aumenta a neurogénese adulta. Pelo contrário, é possível que TrkB-TR em células ependimais e astrócitos da SVZ sirva para formar uma barreira, protegendo progenitores e neuroblastos da SVZ dos efeitos de uma eventual activação de p75 por BDNF. O meu trabalho deverá promover novos estudos sobre a regulação de neurogénese adulta antes que utilizações terapêuticas sejam possíveis. Para além dos estudos acima descritos, desenvolvi também técnicas para o estudo de expressão genética da SVZ ao nível unicelular baseadas em transcripção reversa e reacção em cadeia de polimerase (RT-PCR). Relato nesta tese resultados preliminares encorajadores, demonstrando que é possível detectar expressão de múltiplos genes em uma única célula da SVZ. No futuro, este método poderá ser usado para caracterizar a heterogeneidade molecular dentro de cada tipo celular da SVZ (astrócitos tipo B, células tipo C e neuroblastos tipo A). Este tipo de análise deverá revelar mais subclasses dentro das células B, C e A, e ajudar a caracterizar as diferenças entre astrócitos neurogénicos e não-neurogénicos bem como entre células de zonas diferentes da SVZ.Neurogenesis continues throughout adulthood in the vertebrate brain. New neurons originate from endogenous neural stem cells which could have important applications for brain repair. In rodents, the adult subventricular zone (SVZ) generates thousands of neuroblasts daily, which migrate to the olfactory bulb (OB) and differentiate into interneurons. Recent findings indicate that the neurotrophin Brain-Derived Neurotrophic Factor (BDNF) can enhance adult SVZ neurogenesis, but the mechanism by which it acts is unknown. In my thesis, I analyzed the role of BDNF and its receptor TrkB in adult SVZ neurogenesis. I found that BDNF protein is present and that TrkB is the most prominent neurotrophin receptor in the mouse SVZ, though only the truncated, kinase-negative isoform (TrkB-TR) was detected. TrkB-TR is expressed in SVZ astrocytes and ependymal cells, but not in neuroblasts. Though TrkB mutants have reduced SVZ proliferation and survival and fewer new OB neurons, grafting SVZ cells from TrkB knockout mice (TrkB-KO) into the SVZ of wild-type mice (WT) showed that neuroblasts are generated and migrate to the OB in the absence of TrkB. With the exception of dopaminergic periglomerular cells, OB interneurons derived from TrkB-KO and WT grafts displayed similar survival, molecular and morphological properties. To study the effect of BDNF on SVZ neurogenesis, I infused this neurotrophin into the lateral ventricle of adult rats and mice. After 14 days of infusion, SVZ proliferation was reduced in both species. However, the neuronal output of the SVZ was differentially affected: BDNF infusions decreased the number of new neurons in the rat OB but had no effect in the mouse. Interestingly, rats and mice also differ in their expression of the neurotrophin receptor, p75. In the rat, I detected p75 receptor in many putative type C cells (SVZ intermediate progenitors) and in some neuroblasts, whereas mice had very few p75+ cells. This is the first study to report such effects, as other laboratories had reported that BDNF increased SVZ neurogenesis. Overall, my results indicate that TrkB is not essential for the production and maturation of most SVZ interneurons and do not support the current view that delivering BDNF to the SVZ can enhance adult neurogenesis. Rather, TrkB-TR in ependyma and glial tubes may form a protective barrier, shielding SVZ progenitors and neuroblasts from an activation of p75 by BDNF. My work should promote new studies on how neurotrophins affect adult neurogenesis and the survival of new neurons before therapeutic applications can be designed. In addition to the research described above, I developed techniques based on reverse transcription and polymerase chain reaction (RT-PCR) to study gene expression in single SVZ cells. I report on encouraging preliminary results, showing that it is possible to detect multiple gene products from a single cell. In the future, this method can be applied to characterize molecular heterogeneity within each cell type of the SVZ (type B astrocytes, type C intermediate progenitors and type A neuroblasts). This type of analysis will likely reveal more subclasses within type B, C and A cells, and will help to characterize differences between neurogenic and non-neurogenic astrocytes and between cells of different regions of the SVZ

Contrasting effects of overexpressing the neurotrophin receptors TrkA and TrkB during development

Neurotrophins and their cognate receptors are part of an important signalling system in the vertebrate nervous system. This includes the control of cell survival during the development of the peripheral nervous system. Whilst the neurotrophins and their respective tyrosine kinase receptors are closely related in structure, unexpected differences have begun to appear with regard to their function. In particular, the tropomyosin receptor kinase A (TrkA) has been reported to cause the death of neurons in the absence of its neurotrophin ligand, nerve growth factor (NGF). By contrast, there have been no indications as of yet that the expression of the closely related brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4) receptor TrkB induces the death of neurons during development. A better understanding of the function of these receptors has important implications as unlike TrkA in the peripheral nervous system, TrkB is widely expressed in the central nervous system where BDNF does not seem to play a significant role as a survival factor. To further explore the role of these receptors, novel in vitro and in vivo models were generated that allowed the conditional overexpression of TrkA and TrkB. It was found that the ubiquitous overexpression of TrkA from the earliest stages of mouse gestation led to a perinatal death phenotype, whilst mice overexpressing TrkB were viable and fertile. Detailed histological examination indicated that the overexpression of TrkA led to the loss of neurons known to depend on NGF for their survival during development. Indeed TrkA-overexpressing mice phenocopy mutants lacking both Ngf alleles. By contrast, the postnatal survival of TrkB-overexpressing mice was unimpaired, despite a loss of cranial sensory neurons approaching what has been reported for mice lacking the genes encoding BDNF and NT4. Potential explanations for these surprising observations are discussed

Structure and function of GDNF receptor alpha splice variants

The growth factors of the glial cell line-derived neurotrophic factor (GDNF) family consisting of GDNF, neurturin (NRTN), artemin (ARTN) and persephin (PSPN), are involved in the development, differentiation and maintenance of many types of neurons. They also have important functions outside the nervous system in the development of kidney, testis and thyroid gland. Each of these GFLs preferentially binds to one of the glycosylphosphatidylinositol (GPI)-anchored GDNF family receptors α (GFRα). GDNF binds to GFRα1, NRTN to GFRα2, ARTN to GFRα3 and PSPN to GFRα4. The GFLs in the complex with their cognate GFRα receptors all bind to and signal through the receptor tyrosine kinase RET. Alternative splicing of the mouse GFRα4 gene yields three splice isoforms. These had been described as putative GPI-anchored, transmembrane and soluble forms. My goal was to characterise the function of the different forms of mouse GFRα4. I firstly found that the putative GPI-anchored GFRα4 (GFRα4-GPI) is glycosylated, membrane-bound, GPI-anchored and interacts with PSPN and RET. We also showed that mouse GFRα4-GPI mediates PSPN-induced phosphorylation of RET, promotes PSPN-dependent neuronal differentiation of the rat pheochromocytoma cell line PC6-3 and PSPN-dependent survival of cerebellar granule neurons (CGN). However, although this receptor can mediate PSPN-signalling and activate RET, GFRα4-GPI does not recruit RET into lipid rafts. The recruitment of RET into lipid rafts has previously been thought to be a crucial event for GDNF- and GFL-mediated signalling via RET. I secondly demonstrated that the putative transmembrane GFRα4 (GFRα4-TM) is indeed a real transmembrane GFRα4 protein. Although it has a weak binding capacity for PSPN, it can not mediate PSPN-dependent phosphorylation of RET, neuronal differentiation or survival. These data show that GFRα4-TM is inactive as a receptor for PSPN. Surprisingly, GFRα4-TM can negatively regulate PSPN-mediated signalling via GFRα4-GPI. GFRα4-TM interacts with GFRα4-GPI and blocks PSPN-induced phosphorylation of RET, neuronal differentiation as well as survival. Taken together, our data show that GFRα4-TM may act as a dominant negative inhibitor of PSPN-mediated signaling. The most exciting part of my work was the finding that the putative soluble GFRα4 (GFRα4-sol) can form homodimers and function as an agonist of the RET receptor. In the absence of PSPN, GFRα4-sol can promote the phosphorylation of RET, trigger the activation of the PI-3K/AKT pathway, induce neuronal differentiation and support the survival of CGN. Our findings are in line with a recent publication showing the GFRα4-sol might contribute to the inherited cancer syndrome multiple endocrine neoplasia type 2. Our data provide an explanation to how GFRα4-sol may cause or modify the disease. Mammalian GFRα4 receptors all lack the first Cys-rich domain which is present in other GFRα receptors. In the final part of my work I have studied the function of this particular domain. I created a truncated GFRα1 construct lacking the first Cys-rich domain. Using binding assays in both cellular and cell-free systems, phosphorylation assays with RET, as well as neurite outgrowth assays, we found that the first Cys-rich domain contributes to an optimal function of GFRα1, by stabilizing the interaction between GDNF and GFRα1.GDNF (glial cell line-derived neurotrophic factor) -perheen hermokasvutekijät GDNF, neurturiini (NRTN), artemiini (ARTN) ja persefiini (PSPN) osallistuvat useiden hermosolutyyppien kehittymiseen, erilaistumiseen ja ylläpitoon. Niillä on myös tärkeitä toimintoja hermoston ulkopuolella munuaisen, testiksen sekä kilpirauhasen kehityksessä. Kukin näistä GFL:istä (GDNF family ligand) sitoutuu ensisijaisesti yhteen glykosyylifosfatidyyli-inositoli (GPI) -ankkuroituun GFRα (GDNF family receptor α) -reseptoriin: GDNF sitoutuu GFRα1:een, NRTN GFRα2:een, ARTN GFRα3:een ja PSPN GFRα4:een. Jokainen GFL sitoutuu yhdessä siihen kuuluvan GFRα-reseptorin kanssa reseptorityrosiinikinaasi RET:iin ja signaloi sen kautta. Hiiren GFRα4-geenin vaihtoehtoisen silmikoinnin tuloksena on oletettu syntyvän kolmenlaisia GFRα4-reseptoreja - GPI-ankkuroituja, solukalvon lävistäviä sekä liukoisia. Havaitsin ensin, että mahdollinen GPI-ankkuroitu GFRα4 (GFRα4-GPI) on glykosyloitu sekä todella sitoutunut solukalvoon GPI-ankkurilla ja interaktoi PSPN:n sekä RET:n kanssa. Osoitimme myös, että hiiren GFRα4-GPI välittää PSPN:n indusoimaa RET:n fosforylaatiota ja edistää PSPN:stä riippuvaa rotan feokromosytoomaperäisen PC6-3-solulinjan solujen erilaistumista neuronaalisiksi sekä CGN (cerebellar granule neuron) -solujen PSPN:stä riippuvaa eloonjäämistä. Vaikka tämä reseptori pystyy välittämään PSPN:n signalointia ja aktivoimaan RET:n, GFRα4-GPI ei kuitenkaan vedä RET:ä mukanaan solukalvon lipidilautoille. Tämän tapahtuman on aikaisemmin oletettu olevan ratkaisevassa asemassa GDNF- ja GFL-välitteisessä RET-signaloinnissa. Seuraavaksi osoitin, että mahdollinen transmembraani-GFRα4 (GFRα4-TM) on todella solukalvon läpäisevä GFRα4-proteiini. Vaikka se sitoo heikosti PSPN:ä, se ei kuitenkaan pysty välittämään PSPN:stä riippuvaa RET:n fosforylaatiota, neuronaalista erilaistumista tai eloonjäämistä. Tämä aineisto osoittaa, että GFRα4-TM on PSPN-reseptorina inaktiivinen. Yllättäen GFRα4-TM pystyy kuitenkin säätelemään negatiivisesti PSPN:stä riippuvaa GFRα4-GPI:n kautta tapahtuvaa signalointia. GFRα4-TM interaktoi GFRα4-GPI:n kanssa ja estää PSPN:stä riippuvan RET-fosforylaation, neuronaalisen erilaistumisen sekä eloonjäämisen. Yhdessä tuloksemme osoittavat, että GFRα4-TM saattaa toimia dominant negative -tyyppisenä inhibiittorina PSPN-välitteiselle signaloinnille. Mielenkiintoisin osa työstäni oli havainto, että mahdollinen liukoinen GFRα4 (GFRα4-sol) voi muodostaa homodimeerejä ja toimia RET-reseptorin agonistina. Ilman PSPN:ä GFRα4-sol pystyy edistämään RET:n fosforylaatiota, laukaisemaan PI-3K/AKT-reitin aktivaation, indusoimaan neuronaalista erilaistumista sekä tukemaan CGN-solujen eloonjäämistä. Löydöksemme sopivat hyvin yhteen vastikään julkaistujen tulosten kanssa, jotka osoittavat, että GFRα4-sol saattaa edesauttaa perinnöllisen syöpäsyndrooman, multippelin endokriinisen neoplasia tyyppi 2:n, puhkeamista. Tuloksemme antavat selityksen sille, miten GFRα4-sol voi aiheuttaa tai muunnella tätä sairautta. Kaikkien nisäkkäiden GFRα4-reseptoreista puuttuu ensimmäinen Cys-pitoinen domeeni, joka on muissa GFRα-reseptoreissa. Työni viimeisessä osassa tutkin tämän domeenin toimintaa. Tein GFRα1-konstruktin, jota oli lyhennetty niin, että siitä puuttui ensimmäinen Cys-pitoinen domeeni. Käyttäen sitomiskokeita sekä solupohjaisissa että soluttomissa systeemeissä, RET:n fosforylaatiokokeita, sekä neuriittien uloskasvukokeita havaitsimme, että tämä ensimmäinen Cys-pitoinen domeeni edesauttaa GFRα1-reseptorin optimaalista toimintaa stabiloimalla GDNF:n ja GFRα1:n välistä interaktiota

Characterisation of MYB and TRKB as candidate targets in CYLD cutaneous syndrome

PhD ThesisCYLD cutaneous syndrome is a rare, autosomal dominant inherited disease in which an individual is predisposed to developing multiple cutaneous tumours; namely cylindromas, spiradenomas and trichoepitheliomas, due to germline mutations in the tumour suppressor gene CYLD. However at present there are no curative treatments other than repetitive surgery to control tumour burden. Therefore, an overarching theme of this thesis was to identify potential therapeutic targets that could possibly translate into treatment strategies in the clinic. The first strategic target, c-MYB, was explored, as overexpression of c-MYB was previously shown in sporadic cylindromas due to the presence of the fusion protein MYB-NFIB. It was demonstrated that inherited cylindromas did not express the MYBNFIB fusion protein. However, using immunohistochemistry c-MYB was shown to be overexpressed in inherited tumours and that gene silencing of MYB caused a reduction in cell viability. A novel finding, transcriptomic and protein analysis also revealed that keratinocytes expressed the alternate MYB 9B isoform. A second strategic target, Trk, was explored using RNA-seq of CYLD defective tumours. Exploration of Trk signalling in these tumour cells found that the truncated TrkB.T1 isoform of the TrkB receptor is overexpressed in the tumours, alongside the receptors cognate ligand BDNF. Overexpression of TrkB.T1 in the HaCaT, keratinocyte cell line, indicated that TrkB.T1 increased cell survival. BDNF stimulation of TrkB.T1 overexpressing cells caused Stat3 phosphorylation, and in primary cylindroma tumour cells BDNF stimulation increased the nuclear localisation of Stat3. Finally, a three-dimensional spheroid cell culture method was established and characterised using immunofluorescence, as 3D in vitro models better reflect the in vivo environment. The cultures were found to have an expression pattern similar to their in vivo counterparts and were shown to be sensitive to inhibitory Stat3 targeting, supporting the translational relevance of this work in the hope of bringing targeted therapies for CYLD cutaneous syndrome to the clinic

Cellular and Molecular Characterisation of Familial Haemophagocytic Lymphohistiocytosis Type 1

Haemophagocytic lymphohistiocytosis (HLH) is a severe hyperinflammatory condition in which absent or markedly reduced T cell and natural killer (NK) cell cytotoxicity results in uncontrolled proliferation of T cells, activation of macrophages, hypercytokinaemia, pancytopaenia, and hepatosplenomegaly. Familial HLH (FHL), an autosomal recessive disorder affecting young infants and children, is fatal unless treated with chemotherapy and allogeneic haemopoietic stem cell transplantation. Mutations in the gene encoding perforin, a lytic protein involved in cell cytotoxicity, account for 30% of cases of FHL (FHL II). Mutations in genes encoding hMunc 13-4, syntaxin 11, and syntaxin binding protein 2, all essential for perforin release and T/NK cell cytotoxicity, have also been identified in FHL (FHL III IV and V respectively). A previous report form our group showed that in the UK, in consanguineous families of Pakistani descent, FHL (designated FHL I) maps to 9q21.3-22 in an area spanning 5Mb and containing 14 genes. The aim of my project was to identify the gene responsible for FHL I and to study the function of the corresponding protein. Based on the premise that similar to other types of FHL, the protein product of FHL I candidate gene would function in the perforin-dependent cytotoxic pathway, 4 out of the 14 genes emerged as strong functional candidates: KIF27, RASEF, UBQLN1 and FRMD3. My mutation screening strategy aimed at amplifying and sequencing all exons and intronexon boundaries of the candidate genes, followed by screening the remaining genes of the FHL I locus. The material used was either genomic DNA extracted from 4 parental and 1 patient B cell line, or cDNA from the latter. In 2 newly identified patients, genomic DNA from granulocytes was used. PCR amplification of genomic DNA and direct sequencing or cloning and sequencing of individual clones or in some cases direct sequencing of the full length cDNA from the patient B cell line did not reveal any mutations in any of the known genes in the FHL I locus. For KIF27, mutations were excluded in gene regulatory areas by amplification and direct sequencing of the whole promoter region, up to 1kb upstream of the transcription start site. KIF27 protein expression was also studied by immunoblotting and immunostaining followed by confocal microscopy but no abnormality was identified at protein level. Promoter area mutation was also excluded in UBQLN1, the other strong candidate. The gene responsible for FHL I remains unidentified. Further refinement of the locus with high-density SNP arrays in newly diagnosed patients demonstrating linkage to the FHL I locus might help identify the gene responsible for FHL I