A Role for Tctp in Neural Circuitry Formation

Tese de doutoramento em Biociências, ramo de especialização em Biologia Celular e Molecular, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de CoimbraWith 86 billion neurons in the human brain, the ordered array of neuronal pathways is an exceptionally complex web of accurately connected axonal and dendritic processes, ultimately allowing us to perceive the world and respond consciously to it. Precise wiring of the nervous system is therefore the cornerstone of its intricate functions. Neuronal connectivity begins to take shape during embryonic development, when post-migratory newborn neurons send out a single threadlike axon that extends in a highly directed manner to the vicinity of its appropriate target region. Axon pathfinding relies on molecular ‘guideposts’ presented in the embryonic landscape and integrated by the growth cone, an amoeboid, sensory structure at the tip of developing axons first identified by the legendary Ramón y Cajal. Studies in the past three decades have revealed that a certain degree of functional autonomy is endowed upon this cellular outpost, perhaps best exemplified by the demonstration that axons separated from their cell bodies can still navigate correctly in vivo. It is now evident that this flexibility partly arises from the local regulation of the axonal proteome in response to extracellular cues. The local translation of new proteins elicited by these factors allows for rapid alterations in cytoskeleton dynamics, guidance receptor expression, substrate adhesion, as well as promoting axonal and mitochondrial maintenance. The recent appreciation of the complexity of the axonal transcriptome, with thousands of mRNAs identified, clearly illustrates the functional significance of this homeostatic mechanism. Here, I pioneered the study of translationally controlled tumor protein (Tctp) in the context of neural connectivity using the Xenopus laevis (African clawed frog) retinotectal projection as an in vivo model system. Tctp is an evolutionary conserved pro-survival protein implicated in cell growth and particularly well-studied in cancer pathogenesis, where its expression is often found upregulated and correlates with indicative markers of aggressive disease. Significantly, across diverse neuronal populations, including retinal ganglion cells, the tpt1 transcript, which encodes for Tctp, is ranked among the most enriched in the axonal compartment, suggestive of an unexplored relevant role in neurobiology. My most significant original contribution to knowledge is the identification of Tctp as a cell-autonomous checkpoint for axon development through its support of mitochondrial homeostasis. Specifically, Tctp deficiency during embryogenesis results in shorter retinotectal projections that fail to reach their target at the appropriate developmental window. Tctp-depleted axons exhibit mitochondrial dysfunction, decreased mitochondrial density and defects in mitochondrial dynamics, but Tctp knockdown embryos have intact mitochondrial biogenesis and mass, arguing for a phenotype with pre- dominantly axonal repercussions. Furthermore, I document that axonal Tctp interacts with Bcl-2 pro-survival oncoproteins myeloid cell leukemia 1 (Mcl1) and Bcl-2-like protein 1 (Bcl-XL), and that Caspase-3 activation and increased P53 levels are found in growth cones depleted of Tctp. Overall, the data I have collected over the course of my research indicate that Tctp regulates axon development by impacting on the homeostatic mechanisms of the neuron. My findings thus suggest a novel and fundamental role for Tctp in vertebrate neural circuitry formation.Com os seus cerca de 86 mil milhões de neurónios [1], o cérebro humano é constituído por uma rede intrincada de processos axonais e dendríticos complexamente interligados, e que, como um todo coerente, nos permite reconhecer sinais com origem no meio externo e responder de forma consciente sobre este. A formação de ligações precisas entre as diversas áreas do sistema nervoso assume, portanto, um papel fundamental no garante das suas funções. Numa primeira fase, os programas de conectividade neuronal desenvolvem-se no decurso da embriogénese. Em concreto, após a formação e subsequente migração do neurónio, este estende um axónio que se expande de modo estereotipado até à vizinhança do seu alvo pós-sináptico. Os mecanismos de navegação axonal dependem de uma sequência de marcos moleculares distribuídos pelo sistema nervoso embrionário, os quais são integrados ao nível do cone de crescimento, uma estrutura ameboide com capacidades sensoriais e motoras, existente no extremo distal do axónio. Ao longo das últimas três décadas, um vasto leque de estudos foi permitindo perceber que o cone de crescimento é dotado de um certo grau de autonomia funcional [2], facto claramente ilustrado pela capacidade inalterada revelada por axónios embrionários seccionados – isto é, separados dos seus corpos celulares – para se orientarem corretamente in vivo [3]. Sabe-se hoje que esta flexibilidade de atuação advém, em parte, da regulação local do proteoma axonal promovida pelos sinais moleculares presentes no meio extracelular embrionário. A tradução local de novas proteínas despoletada por estes factores permite, por exemplo, alterações rápidas ao nível do citoesqueleto [4-6] e da expressão de receptores na membrana celular do cone de crescimento [7], assim como potenciar mecanismos de manutenção axonal e mitocondrial [8-10]. É, no entanto, o carácter transversal do transcriptoma axonal – o número de espécies de ARNm localizados no compartimento axonal situa-se na ordem dos milhares [11-15] – que porventura nos dá verdadeiramente conta da importância funcional deste processo celular. Nos estudos laboratoriais conducentes a esta tese, foi estudada a participação da Tctp (do acrónimo inglês, translationally controlled tumor protein) nos processos de conectividade neuronal, usando como modelo a projeção retinotectal da rã-de-unhas -africana (Xenopus laevis). A Tctp é uma proteína conservada filogeneticamente [16], relevante ao nível de processos de sobrevivência [17] e de crescimento celular [18, 19], e bem caracterizada em particular no âmbito da oncogénese [20]. A motivação inicial para estudar a Tctp neste contexto surgiu da identificação do ARNm que codifica a Tctp entre os mais abundantemente expressos no compartimento axonal de diversas populações neuronais [11-14], incluindo em células ganglionares da retina, indicando que esta proteína detém um papel local relevante, mas por explorar, no campo da neurobiologia. A minha contribuição original mais significativa para o conhecimento prende-se com a identificação do envolvimento da Tctp na regulação do desenvolvimento axonal através da sua função de suporte à homeostasia mitocondrial. Especificamente, a depleção da Tctp durante a embriogénese resulta em projeções retinotectais que não granjeiam alcançar a zona-alvo no mesencéfalo aquando do estádio de desenvolvimento normal. Os axónios deficitários em Tctp apresentam uma disfunção e menor densidade mitocondrial, bem como dinâmicas de transporte mitocondrial alteradas; contudo, tais manifestações não se traduzem em decréscimos globais da biogénese ou da massa destes organelos, pelo que se infere um fenótipo com repercussões predominantemente axonais. Documento ainda a interação intra-axonal da Tctp com duas oncoproteínas anti-apoptóticas da família Bcl-2 – a Mcl1 (do acrónimo inglês, myeloid cell leukemia 1) e a Bcl-XL (Bcl-2-like protein 1) – e aumentos nos níveis de expressão da P53 e da forma ativada da Caspase-3 no cone de crescimento de axónios desprovidos da Tctp. Estes resultados indicam que a Tctp regula o desenvolvimento axonal pela sua ação nos mecanismos de homeostasia celular. O meu estudo sugere, portanto, que à Tctp cabe uma função de fundamental relevância nos mecanismos de formação dos circuitos neuronais em vertebrados.FCT- SFRH/BD/33891/200

Tctp in Neuronal Circuitry Assembly.

Although tctp expression in many areas of the human brain was reported more than 15 years ago, little was known about how it functions in neurons. The early notion that Tctp is primarily expressed in mitotic cells, together with reports suggesting a relative low abundance in the brain, has perhaps potentiated this almost complete disregard for the study of Tctp in the context of neuron biology. However, recent evidence has challenged this view, as a number of independent genome-wide profiling studies identified tctp mRNA among the most enriched in the axonal compartment across diverse neuronal populations, including embryonic retinal ganglion cells. Considering the emerging parallels between axon guidance and cancer cell invasion, the axonal expression of cancer-associated tctp was suggestive of it holding an unexplored role in the wiring of neuronal circuits. Our study revealed that Tctp is necessary for the accurate and timely development of axon projections during the formation of vertebrate retinal circuits via its association with the survival machinery of the axon. Globally, the findings indicate that compromised pro-survival signaling in Tctp-deficient axons results in mitochondrial dysfunction and a subsequent decrease in axonal mitochondrial density. These effects likely translate into a metabolic state inadequate to support the normal guidance and extension processes of a developing axon

Tumor protein Tctp regulates axon development in the embryonic visual system.

The transcript encoding translationally controlled tumor protein (Tctp), a molecule associated with aggressive breast cancers, was identified among the most abundant in genome-wide screens of axons, suggesting that Tctp is important in neurons. Here, we tested the role of Tctp in retinal axon development in Xenopus laevis We report that Tctp deficiency results in stunted and splayed retinotectal projections that fail to innervate the optic tectum at the normal developmental time owing to impaired axon extension. Tctp-deficient axons exhibit defects associated with mitochondrial dysfunction and we show that Tctp interacts in the axonal compartment with myeloid cell leukemia 1 (Mcl1), a pro-survival member of the Bcl2 family. Mcl1 knockdown gives rise to similar axon misprojection phenotypes, and we provide evidence that the anti-apoptotic activity of Tctp is necessary for the normal development of the retinotectal projection. These findings suggest that Tctp supports the development of the retinotectal projection via its regulation of pro-survival signalling and axonal mitochondrial homeostasis, and establish a novel and fundamental role for Tctp in vertebrate neural circuitry assembly.This work was supported by Fundação para a Ciência e Tecnologia (C.G.R.; fellowship SFRH/BD/33891/2009), Sir Edward Youde Memorial Fund, Croucher Foundation, Cambridge Commonwealth–European & International Trust (H.W.), Gates Cambridge Scholarship (J.Q.L.), and a Wellcome Trust Programme Grant (C.E.H.; grant 085314/Z/08/Z).This is the final version of the article. It first appeared from The Company of Biologists via http://dx.doi.org/10.1242/dev.13106

Evaluation of Juvenile Animal Studies for Pediatric CNS-Targeted Compounds: A Regulatory Perspective

Central nervous system (CNS)-targeted products are an important category of pediatric pharmaceuticals. In view of the significant postnatal maturation of the CNS, juvenile animal studies (JAS) are performed to support pediatric development of these new medicines. In this project, the design and results of juvenile toxicity studies from 15 drug compounds for the treatment of neurologic or psychiatric conditions were analyzed. Studies were conducted mostly in rats; sometimes in addition in dogs and monkeys. The study design of the pivotal JAS was variable, even for compounds with a similar therapeutic indication. Age of the juvenile animals was not consistently related to the starting age of the intended patient population. Of 15 compounds analyzed, 6 JAS detected more severe toxicities and 6 JAS evidenced novel CNS effects compared to their adult counterparts. The effects of CNS on acoustic startle and learning and memory were observed at high dosages. Reversibility was tested in most cases and revealed some small effects that were retained or only uncovered after termination of treatment. The interpretation of the relevance of these findings was often hampered by the lack of matching end points in the adult studies or inappropriate study designs. Detailed clinical observation and motor activity measures were the most powerful end points to detect juvenile CNS effects. The need for more detailed behavioral examinations in JAS, for example, on learning and memory, should, therefore, be decided upon on a case-by-case basis, based on specific concerns in order to avoid overloading the studies