Closed-loop approaches for innovative neuroprostheses

The goal of this thesis is to study new ways to interact with the nervous system in case of damage or pathology. In particular, I focused my effort towards the development of innovative, closed-loop stimulation protocols in various scenarios: in vitro, ex vivo, in vivo

Biohybrids for Neural Tracts Regeneration

[ES] Las lesiones del sistema nervioso que implican la interrupción de haces axonales son devastadoras para el individuo. La regeneración autónoma de los tractos axonales dañados o degenerados es poco frecuente, ya que intervienen una gran cantidad de factores que limitan esta recuperación. Hoy en día, la medicina convencional no cuenta con tratamientos efectivos y exitosos para estas lesiones, y el tratamiento de los síntomas suele ser la mejor solución. Para revertirlo y lograr la reconexión funcional de las neuronas, la ingeniería de tejidos actualmente opta por el uso de soportes tridimensionales biocompatibles, células y moléculas bioactivas. Específicamente, una de las estrategias propuestas han sido los conductos nerviosos guiados, no solo para lesiones de nervios periféricos sino también para tractos del sistema nervioso central. En esta Tesis Doctoral, se propone la combinación de un conducto tubular hueco de ácido hialurónico (HA) relleno con fibras de ácido poli-L-lactida (PLA) en su lumen, y con células de Schwann (SC) pre-cultivadas como células de soporte de la extension axonal para superar los obstáculos que limitan la regeneración de axones in vivo. Se ha demostrado que el conducto de HA y las fibras de PLA mantienen la proliferación de las SC, las cuales forman una estructura cilíndica denominada 'vaina de SC' en la pared interna del lumen del conducto y a su vez crecen de forma direccional en las fibras de PLA. El conjunto unidireccional paralelo formado por las fibras PLA y las SC recapitula las características direccionales de los tractos axonales en el sistema nervioso. Al sembrar un explante de ganglio de la raíz dorsal (DRG) en uno de los extremos del conducto, se ha conseguido el crecimiento de los axones del DRG y se ha estudiado las características de las SC, los axones crecidos y su asociación, comprobando que el biohíbrido es capaz de soportar el crecimiento axonal. Además, se propone un concepto multimodular para superar las limitaciones típicas de la regeneración axonal a larga distancia, con la combinación de haces de fibras de PLA en el lumen de varios conductos o módulos de HA individuales más cortos que se posicionan uno detrás del otro, diseñando conductos nerviosos guiados con la longitud deseada, junto con SC pre-cultivadas. El conducto multimodular demostró ser eficaz para promover el crecimiento dirigido de axones. Además, se ha desarrollado un constructo compuesto por la estructura formada por las fibras de PLA y las SC, denominado 'cordón neural', tras eliminar el conducto de HA, lo que abre la puerta a la generación de una estructura neural in vitro para su trasplante.[CA] Les lesions de el sistema nerviós que impliquen la interrupció de feixos axonals són devastadores per a l'individu. La regeneració autònoma dels tractes axonals danyats o degenerats és poc freqüent, ja que intervenen una gran quantitat de factors que limiten aquesta recuperació. Avui dia, la medicina convencional no compta amb tractaments efectius i reeixits per aquestes lesions, i el tractament dels símptomes sol ser la millor solució. Per revertir i aconseguir la reconnexió funcional de les neurones, l'enginyeria de teixits actualment opta per l'ús de suports tridimensionals biocompatibles, cèl·lules i molècules bioactives. Específicament, una de les estratègies proposades han estat els conductes nerviosos guiats, no només per lesions de nervis perifèrics sinó també per tractes de sistema nerviós central. En aquesta tesi doctoral, es proposa la combinació d'un conducte tubular buit d'àcid hialurònic (HA) farcit amb fibres d'àcid poli-L-lactida (PLA) en el seu lumen, i amb cèl·lules de Schwann (SC) pre-cultivades com a cèl·lules de suport de l'extension axonal per superar els obstacles que limiten la regeneració d'axons in vivo. S'ha demostrat que el conducte d'HA i les fibres de PLA mantenen la proliferació de les SC, les quals formen una estructura cilíndica anomenada 'beina de SC' a la paret interna de l'lumen de l'conducte i al seu torn creixen de manera direccional en les fibres de PLA. El conjunt unidireccional paral·lel format per les fibres PLA i les SC recapitula les característiques direccionals dels tractes axonals en el sistema nerviós. A l'sembrar un explantament de gangli de l'arrel dorsal (DRG) en un dels extrems de l'conducte, s'ha seguit el creixement dels axons de l'DRG i s'ha estudiat les característiques de les SC, els axons crescuts i la seva associació, comprovant que el biohíbrido és capaç de suportar el creixement axonal. A més, es proposa un concepte multimodular per superar les limitacions típiques de la regeneració axonal a llarga distància, amb la combinació de feixos de fibres de PLA en el lumen de diversos conductes o mòduls de HA individuals més curts que es posicionen un darrere l'l'altre, dissenyant conductes nerviosos guiats amb la longitud desitjada, juntament amb SC pre-cultivades. El conducte multimodular va demostrar ser eficaç per promoure el creixement dirigit d'axons. A més, s'ha desenvolupat un constructe format per l'estructura formada per les fibres de PLA i les SC, denominat 'cordó neural', després d'eliminar el conducte d'HA, el que obre la porta a la generació d'una estructura neural in vitro per al seu trasplantament.[EN] Injuries to the nervous system that involve the disruption of axonal bundles are devastating to the individual. Autonomous regeneration of damaged or degenerated axonal tracts is infrequent since a large number of factors are involved limiting this recovery. Nowadays, conventional medicine does not have effective and successful treatments for these injuries, and the treatment of symptoms is often the best solution. In order to reverse it and achieve the functional reconnection of neurons, tissue engineering currently opts for the use of biocompatible three-dimensional supports, cells, and bioactive molecules. Specifically, one of the proposed strategies has been nerve guidance conduits, not only for peripheral nerve injuries but also for tracts of the central nervous system. In this Doctoral Thesis, we propose the combination of hyaluronic acid (HA) single-channel tubular conduit filled with poly-L-lactide acid (PLA) fibres in its lumen, with pre-cultured Schwann cells (SC) as cells supportive of axon extension to overcome the obstacles limiting axon regeneration in vivo. We have proved that HA conduit and PLA fibres sustain the proliferation of SC, which form a cylindrical structure named 'SC sheath' on the inner wall of the lumen of the conduit and in turn grow directionally in the PLA fibres. The parallel unidirectional ensemble formed by PLA fibres and SC recapitulates the directional features of axonal pathways in the nervous system. Planting a dorsal root ganglion (DRG) explant on one of the conduit's ends, we have followed axon outgrowth from the DRG and studied the features of SC, the grown axons and their association, checking that the biohybrid is capable of supporting axonal growth. Furthermore, we propose a multimodular concept to overcome the typical limitations of long-distance axonal regeneration, with the combination of PLA fibres bundle in the lumen of several shorter individual HA conduits or modules which positioned themselves one behind the other, designing nerve guided conduits with the desired length, together with pre-cultured SC. The multimodular conduit proved effective in promoting directed axon growth. Moreover, we developed a construct consisting of the structure formed by the PLA fibres and the SC, named 'neural cord', after eliminating the HA conduit, that opens the door to the generation of a neural structure in vitro for transplantation.La presente tesis doctoral se ha realizado con la financiación del Ministerio de Economía y Competitividad a través de los proyectos MAT2015-66666-C3-1-R, DPI2015-72863-EXP, y AEI RTI2018-095872-B-C21-C22/ERDF. Agradezco también la beca FPU15/04975 al Ministerio de Educación Cultura y Deportes.Rodríguez Doblado, L. (2021). Biohybrids for Neural Tracts Regeneration [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/16519

Different Functions of Recombinantly Expressed Domains of Tenascin-C in Glial Scar Formation

Extracellular matrix glycoprotein tenascin-C (TnC) is highly expressed in vertebrates during embryonic development and thereafter transiently in tissue niches undergoing extensive remodeling during regeneration after injury. TnC's different functions can be attributed to its multimodular structure represented by distinct domains and alternatively spliced isoforms. Upon central nervous system injury, TnC is upregulated and secreted into the extracellular matrix mainly by astrocytes. The goal of the present study was to elucidate the role of different TnC domains in events that take place after spinal cord injury (SCI). Astrocyte cultures prepared from TnC-deficient (TnC-/-) and wild-type (TnC+/+) mice were scratched and treated with different recombinantly generated TnC fragments. Gap closure, cell proliferation and expression of GFAP and cytokines were determined in these cultures. Gap closure in vitro was found to be delayed by TnC fragments, an effect mainly mediated by decreasing proliferation of astrocytes. The most potent effects were observed with fragments FnD, FnA and their combination. TnC-/- astrocyte cultures exhibited higher GFAP protein and mRNA expression levels, regardless of the type of fragment used for treatment. Application of TnC fragments induced also pro-inflammatory cytokine production by astrocytes in vitro. In vivo, however, the addition of FnD or Fn(D+A) led to a difference between the two genotypes, with higher levels of GFAP expression in TnC+/+ mice. FnD treatment of injured TnC-/- mice increased the density of activated microglia/macrophages in the injury region, while overall cell proliferation in the injury site was not affected. We suggest that altogether these results may explain how the reaction of astrocytes is delayed while their localization is restricted to the border of the injury site to allow microglia/macrophages to form a lesion core during the first stages of glial scar formation, as mediated by TnC and, in particular, the alternatively spliced FnD domain

Strategies for Guidance and Electrical and Biological Stimulation in a Neural Regeneration Device

Tesis por compendio[ES] Actualmente las lesiones del sistema nervioso periférico que conllevan una pérdida de continuidad de los haces axonales suelen implicar secuelas de tipo permanente. Es cierto que en el sistema nervioso periférico existe una cierta regeneración natural de los tractos axonales dañados, pero solamente cuando el espacio entre ambos extremos de la lesión es pequeño, como máximo de 5 mm. Si el espacio es mayor que esta distancia la regeneración no sucede de forma natural y se crea un neuroma traumático. Por tanto, estas lesiones largas requieren de una intervención quirúrgica para puentear la lesión, normalmente con un nervio autógrafo del propio paciente o un nervio alógrafo de un cadáver. No obstante, su uso presenta diversos inconvenientes, como la morbilidad del sitio donante donde puede ocurrir un neuroma, la necesidad de realizar una segunda cirugía, la diferencia de tamaño entre nervio receptor y donante o la necesidad de inmunosupresión en el caso de los nervios alógrafos. Por ello, la ingeniería de tejidos trabaja en el desarrollo de los conductos de guiado nervioso que incorporan estrategias para guiar topográficamente la regeneración, así como células y moléculas bioactivas. La presente tesis doctoral presenta un nuevo conducto de guiado nervioso con una aproximación multimodular para su aplicación en la regeneración de lesiones nerviosas largas (a partir de 15 mm) que hace uso de conductos tubulares huecos modulares de ácido hialurónico (HA) que contienen en su interior una estructura tubular de microfibras de ácido poliláctico (PLA). La estructura fibrilar aporta un guiado topográfico necesario para guiar el crecimiento axonal durante la regeneración a la vez que mantiene unidos los diferentes módulos de HA. Por su parte, los conductos de HA son un hidrogel que evita adherencias con el tejido circundante. A su vez, proporcionan un soporte sobre el que pueden crecer células presembradas. En concreto se ha optado por presembrar células de Schwann, las cuales son unas células gliales de soporte críticas para la regeneración del sistema nervioso periférico. Se ha observado que dichas células son capaces recubrir por completo las paredes internas de los conductos de HA formando una estructura tipo vaina, así como de recubrir las microfibras de PLA creciendo en dirección longitudinal. Los experimentos in vivo en modelo de nervio ciático de conejo han mostrado que la aproximación multimodular mejora significativamente la regeneración nerviosa gracias a proporcionar una mejor neovascularización. A su vez, gracias a las células de Schwann presembradas se ha logrado una mejora adicional de la regeneración nerviosa gracias a su efecto favorecedor del crecimiento axonal. Además, se han estudiado diferentes mejoras aplicables al conducto de guiado nervioso con el objetivo de mejorar los resultados obtenidos in vivo. Gracias a la incorporación de fibroína de seda a los conductos de HA se ha logrado mejorar sus propiedades mecánicas y biológicas. Asimismo, también se ha desarrollado un sustrato electroconductor de microfibras de PLA recubiertas con el polímero electroconductor Polipirrol gracias al cual se ha observado in vitro que es capaz de mejorar el crecimiento axonal al aplicar una estimulación eléctrica. Además, mediante un sistema de modificación génica de las células de Schwann por electrotransfección se ha logrado aumentar su secreción del factor neurotrófico derivado del cerebro (BDNF), gracias a lo cual se ha observado que se incrementa la velocidad de crecimiento axonal in vitro.[CA] Actualment les lesions del sistema nerviós perifèric que comporten una pèrdua de continuïtat dels feixos axonals solen implicar seqüeles de tipus permanent. És cert que al sistema nerviós perifèric hi ha una certa regeneració natural dels tractes axonals danyats, però només quan l'espai entre ambdós extrems de la lesió és petit, com a màxim de 5 mm. Si l'espai és més gran que aquesta distància la regeneració no succeeix de manera natural i es crea un neuroma traumàtic. Per tant, aquestes lesions llargues requereixen una intervenció quirúrgica per pontejar la lesió, normalment amb un nervi autògraf del pacient o un nervi al·lògraf d'un cadàver. No obstant això, el seu ús presenta diversos inconvenients, com la morbilitat del lloc donant on pot ocórrer un neuroma, la necessitat de fer una segona cirurgia, la diferència de mida entre nervi receptor i donant o la necessitat d'immunosupressió en el cas dels nervis al·lògrafs . Per això, l'enginyeria de teixits treballa en el desenvolupament dels conductes de guiatge nerviós que incorporen estratègies per guiar topogràficament la regeneració, així com cèl·lules i molècules bioactives. Aquesta tesi doctoral presenta un nou conducte de guiatge nerviós amb una aproximació multimodular per a la seva aplicació en la regeneració de lesions nervioses llargues (a partir de 15 mm) que fa ús de conductes tubulars buits modulars d'àcid hialurònic (HA) que contenen al seu interior una estructura tubular de microfibres d'àcid polilàctic (PLA). L'estructura fibril·lar aporta un guiatge topogràfic necessari per guiar el creixement axonal durant la regeneració alhora que manté units els diferents mòduls d'HA. Per part seva, els conductes d'HA són un hidrogel que evita adherències amb el teixit circumdant. Alhora, proporcionen un suport sobre el qual poden créixer cèl·lules presembrades. En concret s'ha optat per presembrar cèl·lules de Schwann, les quals són unes cèl·lules glials de suport crítiques per a la regeneració del sistema nerviós perifèric. S'ha observat que aquestes cèl·lules són capaces de recobrir completament les parets internes dels conductes d'HA formant una estructura tipus beina, així com de recobrir les microfibres de PLA creixent en direcció longitudinal. Els experiments in vivo en model de nervi ciàtic de conill han mostrat que l'aproximació multimodular millora significativament la regeneració nerviosa gràcies a proporcionar una millor neovascularització. Alhora, gràcies a les cèl·lules de Schwann presembrades s'ha aconseguit una millora addicional de la regeneració nerviosa gràcies al seu efecte afavoridor del creixement axonal. A més, s'han estudiat diferents millores aplicables al conducte de guiatge nerviós per tal de millorar els resultats obtinguts in vivo. Gràcies a la incorporació de fibroïna de seda als conductes d'HA s'ha aconseguit millorar les seues propietats mecàniques i biològiques. També s'ha desenvolupat un substrat electroconductor de microfibres de PLA recobertes amb el polímer electroconductor Polipirrol gràcies al qual s'ha observat in vitro que és capaç de millorar el creixement axonal quan s'aplica una estimulació elèctrica. A més, mitjançant un sistema de modificació gènica de les cèl·lules de Schwann per electrotransfecció s'ha aconseguit augmentar la secreció del factor neurotròfic derivat del cervell (BDNF), gràcies a la qual cosa s'ha observat que s'incrementa la velocitat de creixement axonal in vitro.[EN] Currently, lesions of the peripheral nervous system that lead to a loss of continuity of the axonal bundles usually involve permanent sequelae. It is true that in the peripheral nervous system there is some natural regeneration of damaged axonal tracts, but only when the space between the two ends of the lesion is small, at most 5 mm. If the gap is greater than this distance, regeneration does not occur naturally, and a traumatic neuroma is created. Therefore, these long injuries require surgical intervention to bridge the injury, usually with an autograph nerve from the patient or an allograph nerve from a cadaver. However, its use has various drawbacks, such as the morbidity of the donor site where a neuroma can occur, the need to perform a second surgery, the difference in size between the recipient and donor nerves, or the need for immunosuppression in the case of allograft nerves. For this reason, tissue engineering works on the development of nerve guidance conduits that incorporate strategies to topographically guide the regeneration, as well as cells and bioactive molecules. This doctoral thesis presents a new nerve guidance conduit with a multimodular approach for its application in the regeneration of long nerve lesions (from 15 mm) that makes use of modular hollow tubular conduits of hyaluronic acid (HA) that contain in their inside a tubular structure of microfibers of polylactic acid (PLA). The fibrillar structure provides the necessary topographic guidance to guide axonal growth during regeneration while keeping the different HA modules together. For their part, the HA conduits are a hydrogel that prevents adhesions with the surrounding tissue. In turn, they provide a support on which preseeded cells can grow. Specifically, it has been decided to pre-seed Schwann cells, which are glial support cells that are critical for the regeneration of the peripheral nervous system. It has been observed that these cells are capable of completely covering the inner walls of the HA conduits, forming a sheath-like structure, as well as covering the PLA microfibers by growing in a longitudinal direction. In vivo experiments in a rabbit sciatic nerve model have shown that the multimodular approach significantly improves nerve regeneration by providing better neovascularization. In turn, thanks to the pre-seeded Schwann cells, an additional improvement in nerve regeneration has been achieved thanks to its promoting effect on axonal growth. In addition, different improvements applicable to the nerve guidance conduit have been studied with the aim of improving the results obtained in vivo. Thanks to the incorporation of silk fibroin into HA conduits, their mechanical and biological properties have been improved. Likewise, an electroconductive substrate of PLA microfibers coated with the electroconductive polymer Polypyrrole has also been developed, thanks to which it has been observed in vitro that it is capable of improving axonal growth by applying electrical stimulation. In addition, by means of a gene modification system of Schwann cells by electrotransfection, it has been possible to increase their secretion of brain-derived neurotrophic factor (BDNF), thanks to which it has been observed that the speed of axonal growth is increased in vitro.Agradezco la ayuda de los diferentes proyectos del Ministerio de Economía y Competitividad del Gobierno de España que han hecho posible la financiación de esta tesis doctoral: MAT2015-66666-C3-1-R, DPI2015-72863-EXP, AEI RTI2018-095872-B-C21-C22/ERDF y FPU16/01833 del Ministerio de Universidades del Gobierno de España, sin la cual no hubiera podido realizar esta tesis doctoral.Gisbert Roca, F. (2022). Strategies for Guidance and Electrical and Biological Stimulation in a Neural Regeneration Device [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/189937Compendi

Substrate-bound and soluble domains of tenascin-C regulate differentiation, proliferation and migration of neural stem and progenitor cells

IntroductionThe lack of regenerative capacity of the central nervous system is one of the major challenges nowadays. The knowledge of guidance cues that trigger differentiation, proliferation, and migration of neural stem and progenitor cells is one key element in regenerative medicine. The extracellular matrix protein tenascin-C (Tnc) is a promising candidate to regulate cell fate due to its expression in the developing central nervous system and in the adult neural stem cell niches. Of special interest are the alternatively spliced fibronectin type III (FnIII) domains of Tnc whose combinatorial diversity could theoretically generate up to 64 isoforms in the mouse. A total of 27 isoforms have already been discovered in the developing brain, among others the domain combinations A1D, CD, and A124BCD.MethodsIn the present study, these domains as well as the combination of the constitutively expressed FnIII domains 7 and 8 (78) were expressed in Chinese hamster ovary cells as pseudo-antibodies fused to the Fc-fragment of a human immunoglobulin G antibody. The fusion proteins were presented to primary mouse neural stem/progenitor cells (NSPCs) grown as neurospheres, either as coated culture substrates or as soluble additives in vitro. The influence of the domains on the differentiation, proliferation and migration of NSPCs was analyzed.ResultsWe observed that the domain combination A124BCD promoted the differentiation of neurons and oligodendrocytes, whereas the domain A1D supported astrocyte differentiation. The constitutively expressed domain 78 had a proliferation and migration stimulating impact. Moreover, most effects were seen only in one of the presentation modes but not in both, suggesting different effects of the Tnc domains in two- and three-dimensional cultures.DiscussionThis knowledge about the different effect of the Tnc domains might be used to create artificial three-dimensional environments for cell transplantation. Hydrogels spiked with Tnc-domains might represent a promising tool in regenerative medicine

Clinical Management and Evolving Novel Therapeutic Strategies for Patients with Brain Tumors

A dramatic increase in knowledge regarding the molecular biology of brain tumors has been established over the past few years, and this has lead to the development of novel therapeutic strategies for these patients. In this book a review of the options available for the clinical management of patients with these tumors are outlined. In addition advances in radiology both for pre-operative diagnostic purposes along with surgical planning are described. Furthermore a review of newer developments in chemotherapy along with the evolving field of photodynamic therapy both for intra-operative management and subsequent therapy is provided. A discussion of certain surgical management issues along with tumor induced epilepsy is included. Finally a discussion of the management of certain unique problems including brain metastases, brainstem glioma, central nervous system lymphoma along with issues involving patients with a brain tumor and pregnancy is provided

Identificação de complexos proteicos na doença de Alzheimer

Doutoramento em Ciências BiomédicasA Doença de Alzheimer (AD) é a maior doença neurodegenerativa a nível mundial, e a principal causa de demência na população idosa. O processamento da proteína precursora de amilóide (APP) pelas β- e g- secretases origina o peptídeo Aβ, que agrega em oligómeros neurotóxicos e em placas senis. Estes são eventos-chave na patogénese da DA que levam à rutura da neurotransmissão sináptica, morte neuronal e inflamação neuronal do hipocampo e córtex cerebral, causando perda de memória disfunção cognitiva geral. Apesar dos grandes avanços no conhecimento do papel do processamento da APP na DA, a sua função fisiológica ainda não foi totalmente elucidada. Os mapas de interações proteína-proteína (PPI) humanos têm desempenhado um papel importante na investigação biomédica, em particular no estudo de vias de sinalização e de doenças humanas. O método dois-híbrido em levedura (YTH) consiste numa plataforma para a produção rápida de redes de PPI em larga-escala. Neste trabalho foram realizados vários rastreios YTH com o objetivo de identificar proteínas específicas de cérebro humano que interagissem com a APP, ou com o seu domínio intracelular (AICD), tanto o tipo selvagem como com os mutantes Y687F, que mimetizam o estado desfosforilado do resíduo Tyr-687. De facto, a endocitose da APP e a produção de Aβ estão dependentes do estado de fosforilação da Tyr-687. Os rastreios YTH permitiram assim obter de redes proteínas que interagem com a APP, utilizando como “isco” a APP, APPY687F e AICDY687F. Os clones positivos foram isolados e identificados através de sequenciação do cDNA. A maior parte dos clones identificados, 118, correspondia a sequências que codificam para proteínas conhecidas, resultando em 31 proteínas distintas. A análise de proteómica funcional das proteínas identificadas neste estudo e em dois projetos anteriores (AICDY687E, que mimetiza a fosforilação, e AICD tipo selvagem), permitiram avaliar a relevância da fosforilação da Tyr-687. Três clones provenientes do rastreio YTH com a APPY687F foram identificados como um novo transcrito da proteína Fe65, resultante de splicing alternativo, a Fe65E3a (GenBank Accession: EF103274), que codifica para a isoforma p60Fe65. A p60Fe65 está enriquecida no cérebro e os seus níveis aumentam durante a diferenciação neuronal de células PC12, evidenciando o potencial papel que poderá desempenhar na patologia da DA. A RanBP9 é uma proteína nuclear e citoplasmática envolvida em diversas vias de sinalização celulares. Neste trabalho caracterizou-se a nova interação entre a RanBP9 e o AICD, que pode ser regulada pela fosforilação da Tyr-687. Adicionalmente, foi identificada uma nova interação entre a RanBP9 e a acetiltransferase de histonas Tip60. Demonstrou-se ainda que a RanBP9 tem um efeito de regulação inibitório na transcrição mediada por AICD, através da interação com a Tip60, afastando o AICD dos locais de transcrição ativos. O estudo do interactoma da APP/AICD, modelado pela fosforilação da Tyr-687, revela que a APP poderá estar envolvida em novas vias celulares, contribuindo não só para o conhecimento do papel fisiológico da APP, como também auxilia a revelar as vias que levam à agregação de Aβ e neurodegeneração. A potencial relevância deste trabalho relaciona-se com a descoberta de algumas interações proteicas/vias de sinalização que podem que podem ser relevantes para o desenvolvimento de novas estratégias terapêuticas na DA.Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder worldwide and the leading cause of dementia in the elderly. Processing of amyloid-β precursor protein (APP) by β- and g-secretases produces Aβ, which aggregates into neurotoxic oligomers and senile plaques. These are key events in the pathogenesis of AD that lead to the disruption of synaptic neurotransmission, neuronal cell death, and inflammation in the hippocampus and cerebral cortex, thus causing memory loss and global cognitive dysfunction. Despite advances in understanding the role of APP processing in AD, the normal physiological function of this protein has proven more difficult to elucidate. Human protein-protein interaction (PPI) maps play an increasingly important role in biomedical research and have been shown to be highly valuable in the study of a variety of human diseases and signaling pathways. The yeast twohybrid (YTH) system provides a platform for the rapid generation of large scale PPI networks. Several YTH screens were performed to identify human brainspecific proteins interacting with APP, or with its intracellular domain (AICD), either the wild-type or the Y687F mutant, which mimics the dephosphorylated residue. In fact, APP endocytosis and Aβ generation are dependent upon Tyr- 687 phosphorylation. A human APP network comprised of the protein interactions was assembled through YTH screening, using as baits APP, APPY687F and AICDY687F. Positive clones were isolated and identified by DNA sequencing and database searching. The majority of these clones, 118, matched to a protein coding sequence, yielding 31 different proteins. Functional proteomics analysis of the proteins identified in this study, and two additional screens from previous projects (phospho-mutant AICDY687E and wild-type AICD), allowed to infer the relevance of Tyr-687 phosphorylation. Three clones from YTH with APPY687F were identified as a new splice variant of the APP binding protein Fe65, Fe65E3a (GenBank Accession EF103274), encoding the p60Fe65 isoform. Fe65E3a is expressed preferentially in the brain and the p60Fe65 protein levels increased during PC12 cell differentiation. This novel Fe65 isoform and the regulation of the splicing events leading to its production, may contribute to elucidating neuronal specific roles of Fe65 and its contribution to AD pathology. RanBP9 is an evolutionarily conserved nucleocytoplasmic protein implicated as a scaffolding protein in several signaling pathways. In this work a novel interaction between RanBP9 and AICD, which can be regulated by Tyr-687 phosphorylation, was characterized. Moreover, a novel interaction between RanBP9 and the histone acetyltransferase Tip60 was identified. RanBP9 was demonstrated to have an inhibitory regulatory effect on AICD-mediated transcription, through physical interaction with Tip60, relocating AICD away from transcription factories. Overall, the APP/AICD interactome shaped by the phosphorylation state of Tyr- 687 provided clues to elucidate APP pathways leading to amyloid deposition and neurodegeneration. As such the work here described brings us nearer to unravelling the physiological functions of APP. This in turn is of potential significant relevance in the pathology of AD, and for the design of effective novel therapeutic strategies