Dissertations / Theses on the topic 'Neurodegenerative'

Cada tres segons alguna persona al món desenvolupa demència. Les malalties neurodegeneratives, amb la malaltia d’Alzheimer (MA), la malaltia de Parkinson i la demència frontotemporal com les formes més predominants, són un grup de desordres que afecten a milions de persones arreu del món. La neuropatologia de mostres post-mortem humanes encara és necessària per esclarir els mecanismes subjacents alterats en les malalties neurodegeneratives. En aquesta tesis hem combinat immunoassaigs clàssics, tècniques de microscopia òptica i eines computacionals automatitzades per investigar la neuroinflamació i l’acumulació anormal de proteïnes en les malalties neurodegeneratives. En particular, hem investigat alteracions dels astròcits i cèl·lules glials en la MA i altres taupaties. Hem aprofitat les tècniques de super-resolució emergents i hem aplicat i optimitzat l’array tomography (AT) combinant-lo amb stimulated emission depletion microscopy (STED) per estudiar les plaques d’amiloide humanes a nivell nanomètric. Finalment, hem aplicat mètodes digitals i l’AT per analitzar quantitativament els canvis cerebrals en un pacient tractat amb un inhibidor de BACE-1. Aquests tres treballs il·lustren com les eines digitals poden integrar-se amb els mètodes neuropatològics clàssics per la investigació de la MA i altres malalties neurodegeneratives.
Cada tres segundos alguien en el mundo desarrolla demencia. Las enfermedades neurodegenerativas, con la enfermedad de Alzheimer (EA), enfermedad de Parkinson y la demencia frontotemporal como las formas más prevalentes, son un grupo de desórdenes que afectan a millones de personas alrededor del mundo. La neuropatología de muestras post-mortem humanas aún es necesaria para esclarecer los mecanismos subyacentes alterados en las enfermedades neurodegenerativas. En esta tesis hemos combinado immunoensayos clásicos, técnicas de microscopía óptica y herramientas computacionales automatizadas para investigar la neuroinflamación y la acumulación anormal de proteínas en las enfermedades neurodegenerativas. En particular, hemos investigado alteraciones de los astrocitos y células gliales en la EA y otras taupatías. Hemos aprovechado las técnicas de super-resolución emergentes y hemos aplicado y optimizado el array tomography (AT) combinándolo con stimulated emission depletion microscopy (STED) para estudiar las placas de amiloide humanas a nivel nanométrico. Finalmente, hemos aplicado métodos digitales y AT para analizar cuantitativamente los cambios cerebrales en un paciente tratado con un inhibidor de BACE-1. Estos tres trabajos ilustran cómo las herramientas digitales pueden integrarse con los métodos neuropatológicos clásicos para la investigación de la EA y otras enfermedades neurodegenerativas.
Every 3 seconds someone in the world develops dementia. Neurodegenerative diseases (NDDs) with Alzheimer's disease (AD), Parkinson's disease (PD) and frontotemporal dementia (FTD) as the most prevalent forms are a group of disorders affecting millions of people worldwide. Neuropathology of post-mortem human samples is still needed to elucidate the underlying altered mechanisms of NDDs. In this thesis we combined classical immunoassays, light microscopy techniques and automated computational tools in order to investigate neuroinflammation and abnormal protein accumulation in NDDs. In particular, we investigated astrocytic and glial abnormalities in AD and other tauopathies. We took advantage of emerging super-resolution techniques and applied and optimized array tomography (AT) combined with stimulated emission depletion microscopy (STED) to study human amyloid plaques at a nanoscale level. Finally, we applied digital methods and AT to quantitative analyse the brain changes in a patient treated with a BACE-1 inhibitor. These three works illustrate how digital tools can be integrated with classical neuropathological methods in the investigation of AD and other NDDs.
Universitat Autònoma de Barcelona. Programa de Doctorat en Neurociències

El diagnòstic de la malaltia de Parkinson (MP) i de la major part de trastorns del moviment es basa fonamentalment en criteris clínics. No obstant això, existeix un important solapament fenotípic i anatomopatològic que pot dificultar en gran manera el diagnòstic diferencial, especialment en fases inicials de la malaltia. La ultrasonografía transcranial ha sorgit com una tècnica no invasiva, de ràpida execució i baix cost econòmic, de gran utilitat com a eina complementària en el diagnòstic diferencial dels trastorns del moviment. La hipòtesi plantejada en aquesta tesi doctoral és que aquesta tècnica pot ser útil en la pràctica clínica diària d’una unitat de trastorns del moviment (UTM) per a l’estudi de la MP, el seu diagnòstic diferencial amb altres quadres que cursin amb tremolor i/o parkinsonisme i l’estudi d’altres trastorns del moviment hipercinètics com la síndrome de Gilles de la Tourette (SGT). Per a demostrar aquesta hipòtesi s’han dut a terme 4 treballs amb diferents grups mostrals procedents d’una consulta d’UTM i controls sans. Es van analitzar variables sonogràfiques com l’àrea de substància nigra (SN) hiperecogènica, tamany del tercer ventricle i de la banya frontal del ventricle lateral, ecogenicitat dels nuclis del rafe i del nucli lenticular. En el primer treball es va estudiar un grup control sa per a establir els punts de tall patològics per a les variables sonogràfiques quantitatives i es va analitzar la utilitat de les troballes sonogràfiques per al diagnòstic diferencial de la MP enfront d’altres patologies que cursen amb tremolor i/o parkinsonisme. En el segon treball es va intentar identificar si alguna variable sonogràfica servia com a marcador d’un fenotip clínic concret en la MP (major severitat, predomini tremòric o rígid-acinètic, presència de símptomes no motors). En el tercer treball es va comprovar la utilitat de la tècnica, comparada amb tècniques de neuroimatge convencional, per a la comprovació postoperatòria precoç dels elèctrodes d’estimulació cerebral profunda (ECP) en el nucli subtalàmic (NST) en la MP. En el quart treball es van estudiar les troballes sonogràfiques en pacients amb SGT. S’ha demostrat que l’estudi sonogràfic de l’àrea de SN hiperecogènica és útil per al diagnòstic diferencial entre MP, tremolor i altres parkinsonismes (atípics i secundaris), l’estudi del tamany ventricular i l’ecogenicitat del lenticular són útils per a identificar parkinsonismes atípics. Una SN hiperecogènica, una hiperecogenicitat lenticular i un major tamany del ventricle lateral correlacionen amb majors puntuacions en l’escala Hoehn i Yahr; aquest últim s’associa també a una major afectació axial i de la marxa. Pacients amb complicacions motores mostren majors àrees de SN, la grandària del tercer ventricle va ser major si hi havia al·lucinacions visuals i el ventricle lateral va ser major en pacients amb hiposmia, el rafe va ser més sovint hipoecogènic en presència de depressió. La sonografía transcranial és útil per a la comprovació postoperatòria precoç dels elèctrodes d’ECP en el NST. En el SGT s’ha detectat un augment significatiu de la hiperecogenicitat lenticular en comparació amb controls sans. Per tant, els resultats d’aquesta recerca donen suport a l’ús de la ultrasonografía transcranial per a l’estudi dels trastorns del moviment en la pràctica clínica habitual d’una UTM. Són necessàries noves línies de recerca destinades a corroborar aquests resultats i ampliar l’estudi a altres trastorns del moviment poc explorats fins ara.
El diagnóstico de la enfermedad de Parkinson (EP) y de la mayor parte de trastornos del movimiento se basa fundamentalmente en criterios clínicos. Sin embargo, existe un importante solapamiento fenotípico y anatomopatológico que puede dificultar en gran medida el diagnóstico diferencial, especialmente en fases iniciales de la enfermedad. La ultrasonografía transcraneal ha surgido como una técnica no invasiva, de rápida ejecución y bajo coste económico, de gran utilidad como herramienta complementaria en el diagnóstico diferencial de los trastornos del movimiento. La hipótesis planteada en esta tesis doctoral es que esta técnica puede ser útil en la práctica clínica diaria de una unidad de trastornos del movimiento (UTM) para el estudio de la EP, su diagnóstico diferencial con otros cuadros que cursen con temblor y/o parkinsonismo y el estudio de otros trastornos del movimiento hipercinéticos como el síndrome de Gilles de la Tourette (SGT). Para demostrar esta hipótesis se han llevado a cabo 4 trabajos con diferentes grupos muestrales procedentes de una consulta de UTM y controles sanos. Se analizaron variables sonográficas como el área de sustancia negra (SN) hiperecogénica, tamaño del tercer ventrículo y del asta frontal del ventrículo lateral, ecogenicidad de los núcleos del rafe y del núcleo lenticular. En el primer trabajo se estudió un grupo control sano para establecer los puntos de corte patológicos para las variables sonográficas cuantitativas y se analizó la utilidad de los hallazgos sonográficos para el diagnóstico diferencial de la EP frente a otras patologías que cursan con temblor y/o parkinsonismo. En el segundo trabajo se intentó identificar si alguna variable sonográfica servía como marcador de un fenotipo clínico concreto en la EP (mayor severidad, predominio tremórico o rígido-acinético, presencia de síntomas no motores). En el tercer trabajo se comprobó la utilidad de la técnica, comparada con técnicas de neuroimagen convencional, para la comprobación postoperatoria precoz de los electrodoimulación cerebral profunda (ECP) en el núcleo subtalámico en la EP. En el cuarto trabajo se estudiaron los hallazgos sonográficos en pacientes con SGT. Se ha demostrado que el estudio sonográfico del área de SN hiperecogénica es útil para el diagnóstico diferencial entre EP, temblor y otros parkinsonismos (atípicos y secundarios), el estudio del tamaño ventricular y la ecogenicidad del lenticular son útiles para identificar parkinsonismos atípicos. Una SN hiperecogénica, una hiperecogenicidad lenticular y un mayor tamaño del ventrículo lateral correlacionan con mayores puntuaciones en la escala Hoehn y Yahr; éste último se asocia también a una mayor afectación axial y de la marcha. Pacientes con complicaciones motoras muestran mayores áreas de SN, el tamaño del tercer ventrículo fue mayor si había alucinaciones visuales y el del ventrículo lateral fue mayor en pacientes con hiposmia, el rafe fue más frecuentemente hipoecogénico en presencia de depresión. La sonografía transcraneal es útil para la comprobación postoperatoria precoz de los electrodos de ECP en el NST. En el SGT se ha detectado un aumento significativo de la hiperecogenicidad lenticular en comparación con controles sanos. Por tanto, los resultados de esta investigación apoyan el uso de la ultrasonografía transcraneal para el estudio de los trastornos del movimiento en la práctica clínica habitual de una unidad de trastornos del movimiento. Son necesarias nuevas líneas de investigación destinadas a corroborar estos resultados y ampliar el estudio a otros trastornos del movimiento poco explorados hasta la fecha.
The diagnosis of Parkinson's disease (PD) and most movement disorders is based mainly on clinical criteria. However, there is a significant phenotypic and anatomopathological overlap that can make differential diagnosis very difficult, especially in the early stages of the disease. Transcranial ultrasonography has emerged as a non-invasive technique, of rapid execution and low economic cost, very useful as a complementary tool in the differential diagnosis of movement disorders. The hypothesis put forward in this doctoral thesis is that this technique may be useful in the daily clinical practice of a movement disorders unit (MDU) for the study of PD, its differential diagnosis with other conditions with tremor and/or parkinsonism and the study of other hyperkinetic movement disorders such as Gilles de la Tourette syndrome (GTS). In order to demonstrate this hypothesis, four studies have been carried out with different sample groups from a UTM outpatient consultation, and healthy controls. Sonographic variables such as the area of hyperechogenic substantia nigra (SN), size of the third ventricle and the frontal shaft of the lateral ventricle, ecogenicity of raphe nuclei and lenticular nuclei were analysed. In the first work a healthy control group was studied to establish the pathological cut-off points for the quantitative sonographic variables and the usefulness of the sonographic findings for the differential diagnosis of PD against other pathologies with tremor and/or parkinsonism was analysed. In the second work, an attempt was made to identify whether any sonographic variable served as a marker for a particular clinical phenotype in PD (greater severity, predominance of tremor or rigid-akinetic, presence of non-motor symptoms). In the third study, the usefulness of the technique, compared to conventional neuroimaging techniques, for early postoperative testing of deep brain stimulation (DBS) electrodes in the subthalamic nucleus (STN) in PD was tested. In the fourth study, sonographic findings were studied in patients with GTS. Sonographic study of the hyperechogenic SN area has been shown to be useful for the differential diagnosis between PD, tremor and other parkinsonisms (atypical and secondary), the study of ventricular size and lenticular ecogenicity are useful to identify atypical parkinsonisms. Hyperechogenic SN, lenticular hyperechogenicity and larger lateral ventricle size correlate with higher scores on the Hoehn and Yahr scale; the latter is also associated with greater axial and gait involvement. Patients with motor complications show greater areas of SN, the size of the third ventricle was larger if there were visual hallucinations and the lateral ventricle was larger in patients with hyposmia, the lenticular was more frequently hyperechogenic in the presence of depression. Transcranial sonography is useful for early postoperative testing of DBS electrodes in the STN. A significant increase in lenticular hyperechogenicity has been detected in the GTS compared to healthy controls. Therefore, the results of this research support the use of transcranial ultrasonography for the study of movement disorders in the routine clinical practice of a movement disorders unit. New lines of research are needed to corroborate these results and extend the study to other movement disorders that have been little explored to date.
Universitat Autònoma de Barcelona. Programa de Doctorat en Medicina

The P2X7 receptor (P2X7R) is a purinergic receptor that plays a central role in the inflammatory response. Activation of the P2X7R releases pro-inflammatory cytokines such as interleukin 1β (IL-1β), which have been shown to underlie the pathogenesis of a number of neurodegenerative disorders. Consequently, the development of a CNS penetrant P2X7R antagonist is considered a promising target for the inhibition of neurodegenerative diseases. A series of P2X7R antagonists were synthesised to investigate which structural features of the hydrophobic moiety dictated binding site selectivity (orthosteric vs allosteric), and potency data are available for derivatives synthesised; assays to assess binding site selectivity have not currently been undertaken. To assist future pharmacological analyses, fluorescent probes based on lead compounds from the aryl cyanoguanidine and adamantyl benzamide P2X7R antagonist series were synthesised, and antagonist potency and binding affinity data for a number of derivatives are reported. Based on the original structure-activity relationship (SAR) study of the adamantyl cyanoguanidine series, a range of heterobicyclic adamantyl cyanoguanidine analogues were synthesised in order to refine the pharmacophore for potent P2X7R antagonism. The adamantyl indazoles 302 and 303 (IC50 = 18.6 ± 0.5 nM and 22.2 ± 6 nM respectively) were equipotent to the lead 19, and SAR data from this series has identified several structural requirements for potent P2X7R antagonism. Attempts to develop radioligands for visualising P2X7R expression in vivo are reported. The trifluorinated adamantyl benzamide [11C]SMW139 was progressed into first-in-human studies as a radiodiagnostic probe for identifying active areas of neuroinflammation in patients with relapsing-remitting multiple sclerosis (RRMS), with data from the small cohort suggesting it did so successfully. Further studies in larger cohorts are currently in progress.

L'AMPc té un paper clau com a missatger intracel.lular regulant la transmissió dels senyals extracel•lulars en diferents teixits i controlant múltiples processos cel lulars. Els nivells intracel.lulars d'AMPc es controlen mitjançant la seva síntesi, catalitzada per l'enzim adenilat ciclasa, i mitjançant la seva degradació a través de l'acció de les fosfodiesterases (PDE) de nucleòtids cíclics. Hi ha 11 famílies de PDEs. La PDE4 representa a una família de fosfodiesterases específiques d'AMPc formada per quatre gens paràlegs (PDE4-D), cadascun dels quals és capaç de generar múltiples variants d’splicing. La PDE4A, la PDE4B i la PDE4D es troben expressades en diferents tipus de cèl lules inflamatòries on tenen un important paper com reguladores dels processos inflamatoris. La inhibició selectiva, tant in vitro com in vivo, de PDEs ha demostrat tenir diferents efectes antiinflamatoris. En aquest treball es mostra la implicació de la isoforma PDE4B, i en concret la seva variant de splicing PDE4B2, durant el procés neuroinflamatori del model animal d’Esclerosis Múltiple, l’Encefalomielitis Autoimmune Experimental (d’EAE). Els resultats mostren un augment de l’expressió de l’ARNm de PDE4B2 a la medul•la espinal de ratolins EAE que correlaciona amb l’expressió d’alguns marcadors inflamatoris de forma dependent a la simptomatologia clínica dels animals. També s’observa que l’enzim PDE4B es trova localitzat principalment en cèl•lules presentadores d’antigen (APCs) com les cèl•lules dendrítques i els macròfags/micròglia. A més, els ratolins PDE4B-/- mostren una aparició temprana dels símptomes clínics en comparació amb els ratolins wildtype, amb alteracions en l’expressió de l’ARNm d’algunes citocines. L’alteració selectiva de la PDE4B2 en el model d’EAE en ratolí i la seva participació en el desenvolupament de la malaltia com s’ha observat en els animals PDE4B-/- presenta noves possibilitats sobre l’ús d’inhibidors selectius per les diverses isoformes (i variants d’splicing) tant per aplicacions terapèutiques com per investigar mecanismes d’inflamació en malalties neurodegeneratives.
Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis that courses with neuroinflammation, axonal damage and demyelination. The model is characterized by T- and B-cell responses to myelin oligodendrocyte glycoprotein which produce a wide range of pro- and anti-inflammatory cytokines. The modulation of cAMP levels through pharmacological manipulation of phosphodiesterases (PDEs) provokes profound anti-inflammatory responses. In the EAE model, amelioration of the clinical signs and delayed onset is observed after PDE4 inhibition and the PDE4B gene has been related to the inflammatory immune response in mice. Here we analyzed post-immunization changes in the expression of mRNA coding for the PDE4B2 splice variant by semiquantitative real-time PCR and in situ hybridization. The results showed an upregulation of PDE4B2 mRNA in the spinal cord of EAE mice which correlates with FoxP3 and TGF-β mRNAs expression in a score-dependent manner. We also found that PDE4B enzyme is mainly localized in antigen-presenting cells (APCs) such as dendritic cells and microglia/macrophages. PDE4B-/- mice show an earlier onset of the disease compared to wildtype mice, with alterations in some cytokine mRNA expression. The results point to a protective role of the PDE4B enzyme and PDE4B2 splice variant in particular, during EAE pathogenesis by modulating cAMP levels in APCs and controlling the cytokine environment for T-cell differentiation.

El gen MC1R (codi OMIM: #155555) codifica pel receptor de la melanocortina 1 (MC1R) que s’expressa majoritàriament a la membrana dels melanòcits i està implicat en la regulació de la síntesi de melanina. El gen MC1R és clau en la regulació del color de la pell i el cabell i és el responsable de controlar la quantitat relativa de síntesi d’eumelanina i feomelanina. Aquest gen és altament polimòrfic, s’hi han identificat més de 100 variants no sinònimes, però l’alteració funcional que provoquen en la proteïna la majoria d’elles és desconeguda. No obstant, hi ha 9 d’aquestes variants: p.V60L, p.D84E, p.V92M, p.R142H, p.R151C, p.I155T, p.R160W, p.R163Q i p.D294H que presenten una freqüència al·lèlica (MAF) > 0,01 en diferents poblacions caucàsiques d’ètnia blanca i que han estat àmpliament estudiades tant a nivell epidemiològic com a nivell funcional. Entre aquestes, hi ha 6 variants al·lèliques que s’han definit com a variants de pèrdua de funció (LOF) (p.D84E, p.R142H, p.R151C, p.I155T, p.R160W i p.D294H), ja que disminueixen notablement la capacitat de senyalització del MC1R a través de la via de l’AMPc i impedeixen la síntesi d’eumelanina. Els individus portadors d’aquestes variants LOF presenten una forta associació amb el fenotip “color de cabell vermell” (cabell pèl-roig, un elevat nombre de pigues, una baixa capacitat per a bronzejar-se i una elevada sensibilitat a la RUV) i un augment del risc a desenvolupar càncer cutani. L’anàlisi del transcriptoma de les cèl·lules cutànies portadores de variants LOF en MC1R va mostrar una desregulació constitutiva de gens implicats en les vies de senyalització de malalties neurodegeneratives i un augment de l’estrès oxidatiu. Donat que l’expressió del MC1R s’ha identificat també en les cèl·lules del sistema nerviós central i que un elevat nivell de dany oxidatiu s’ha observat en les àrees del cervell on hi ha neurodegeneració, varem hipotetitzar que el gen MC1R podria estar implicat en la patogènesi de les malalties neurodegeneratives. Es van dissenyar dos estudis cas-control que incloïen 870 pacients de Parkinson, 525 pacients amb Alzheimer i 736 controls d’origen espanyol. A més, es va avaluar el gen MC1R com a factor modificador de l’edat d’aparició de la malaltia de Huntington, seqüenciant el gen en 600 pacients. Tant els anàlisis d’associació genotípica cas –control com els estudis de regressió múltiple es van dur a terme amb totes aquelles variants genètiques no sinònimes que presentaven una freqüència al·lèlica > 0,01 en els casos avaluats. Complementàriament, amb l’objectiu de posar en context biològic la signatura gènica associada a la pèrdua de funció del MC1R, es va realitzar un anàlisi de xarxes d’interacció proteïna-proteïna (PPI). Els individus portadors de la variant LOF p.R160W presenten un augment del risc a desenvolupar Parkinson (OR=2,10; 95% IC: 1,18-3,73; p-valor ajustat = 0,009) i l’al·lel p.V92M de MC1R s’associa amb un augment del risc a desenvolupar la malaltia d’Alzheimer, especialment en aquells individus on el risc no pot atribuir-se a l’al·lel 4 de l’APOE. A més, el gen MC1R s’ha identificat com un factor modificador de l’edat d’inici de la malaltia de Huntington, ja que els casos portadors de l’al·lel p.R151C presenten una disminució de 5,13 anys respecte a l’edat mitjana d’aparició del Huntington. Concretament, el percentatge de variació en l’edat de debut atribuït a l’al·lel p.R151C és del 1,42%. Els resultats de la present tesi indiquen que el gen MC1R està implicat en la patogènesi de les malalties neurodegeneratives, i que la pèrdua de funció del MC1R pot contribuir a la patogènesi d’aquestes a través d’un augment del dany oxidatiu, una disminució de la resposta antiinflamatòria i una desregulació de l’autofàgia.
The melanocortin 1 receptor (MC1R) gene is a key regulator of skin and hair colour. Certain MC1R polymorphisms, which cause a loss of protein function, have been associated with red hair color (RHC) phenotype (red hair, fair skin, high UV sensitivity and low tanning capacity) and a higher risk of developing skin cancer. Although the MC1R is mainly expressed in melanocytes, it is also detected in several types of brain cells. We have identified that skin cells harbouring loss-of-function MC1R polymorphisms show a deregulation of genes involved in pathways related to neurodegenerative diseases, such as Parkinson’s disease (PD), Alzheimer’s disease (AD) and Huntington’s disease (HD). It has also been shown that skin cells with loss-of-function MC1R polymorphisms show a significantly greater oxidative damage, which is a physiological condition found in areas with neuronal degeneration. Based on these evidences, we hypothesize that MC1R gene could be involved in neurodegenerative diseases pathogenesis. We performed two case-control studies to elucidate the role of MC1R in PD and AD. We sequenced the MC1R gene in 870 PD patients, 525 AD patients and 736 controls from Spain. We assessed the role of MC1R as a modifier factor of age of onset (AOO) in HD, sequencing MC1R in 600 HD patients. We analyzed all non-synonymous MC1R variants with a minor allele frequency of at least 0.01. Additionally, we analyzed the gene signature identified in cells harbouring loss-of-function MC1R polymorphisms by protein-protein interaction network analysis (PPI) to identify biological processes related with loss of MC1R function. We found that the allele p.R160W of MC1R increases risk of PD (OR=2.10, 95% CI: 1.18-3.73, adjusted P = 0.009) and that the p.V92M MC1R allele is associated with AD (OR=1.99, 95% CI: 1.08-3.64, adjusted P = 0 026). Furthermore, the p.R151C MC1R allele modifies AOO in HD (Bonferroni-corrected P = 0.032), its effect explains 1.42% of the variance in AOO that cannot be accounted for by the expanded HD allele. Our results suggest that loss of MC1R function may be involved in neurodegenerative diseases etiology through an increased of oxidative damage, autophagy deregulation and reduced anti-inflammatory response.

La eritropoyetina (EPO) es una glucoproteína inicialmente identificada como una hormona sintetizada y secretada por el riñón que regula la eritropoyesis. Muchos estudios experimentales muestran que la EPO tiene una acción neuroprotectora en el cerebro. En trastornos neurológicos agudos y crónicos, particularmente en apoplejía, lesión cerebral traumática, enfermedad de Alzheimer, enfermedad de Parkinson, esclerosis lateral amiotrófica y en accidente cerebro vascular, tiene efectos neuroprotectores. El objetivo de este estudio, utilizando un modelo in vitro, fue determinar cómo NeuroEPO, una variante de EPO con un bajo contenido de ácido siálico, protege a las neuronas de la acción excitotóxica del glutamato. Se utilizó cultivos neuronales primarios que se obtuvieron de la corteza cerebral de embriones de rata Wistar después de 17 días de gestación. La excitotoxicidad se indujo después de nueve días de cultivo in vitro mediante tratamiento con un medio que contenía glutamato 100 μM durante 15 minutos, para luego ser tratado con 100 ng / ml de NeuroEPO. Después de 24 h, el tratamiento mostró una disminución significativa (p <0,01) en la mortalidad, en comparación con las células tratadas con glutamato solo. El tratamiento NeuroEPO disminuyó la mortalidad y las células tendieron a reproducir las características morfológicas del control. Las imágenes obtenidas mediante microscopía de contraste de fases muestran que las neuronas tratadas con glutamato muestran una contracción corporal, pérdida de dendritas que no hacen contacto con las células vecinas y que NeuroEPO es capaz de preservar las características morfológicas del control. El estrés oxidativo inducido por el glutamato se reduce después del tratamiento con NeuroEPO. Las pruebas por inmunocitoquímica muestran que el cultivo es esencialmente puro en las neuronas, que el glutamato causa mortalidad celular, y que esto se evita parcialmente cuando en el medio de cultivo se añade NeuroEPO. Se analizó también la activación de las vías apoptóticas intrínsecas. La disminución en la relación Bcl-2 / Bax, fue determinante en la liberación de citocromo c desde la mitocondria hacia el citosol y en la expresión y la actividad de caspasa-3 observada en las células tratadas solo con glutamato. Estas alteraciones fueron evitadas en las células bajo tratamiento con NeuroEPO posterior al glutamato. Estos resultados permiten aseverar que NeuroEPO ejerce su función neuroprotectora actuando directamente sobre las neuronas una vez que se ha inducido la excitotoxicidad del glutamato, puede atenuar sustancialmente los cambios morfológicos causados por la excitotoxicidad inducida por glutamato en las neuronas, reduciendo el efecto neurotóxico que éste ejerce sobre la viabilidad neuronal, protege a las neuronas del estrés oxidativo y de la apoptosis inducidos por el glutamato mediante el manteniendo el equilibrio entre proteínas anti-apoptóticas (Bcl-2) y pro-apoptóticas (Bax), evitando la liberación al citosol del citocromo c y reduciendo la actividad de la caspasa-3. Los bajos niveles de ácido siálico de la NeuroEPO comparados con la rhEPO al parecer no afectan a la actividad neuroprotectora de la NeuroEPO.
Erythropoietin (EPO) is a glycoprotein initially identified as a hormone synthesized and secreted by the kidney that regulates erythropoiesis. Many experimental studies show that EPO has a neuroprotective action in the brain. In acute and chronic neurological disorders, particularly in stroke, traumatic brain injury, Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis, it has neuroprotective effects. The objective of this study, using an in vitro model, was to determine how NeuroEPO, a variant of EPO with a low content of sialic acid, protects neurons from the toxic action of glutamate. Primary neuronal cultures that were obtained from the cerebral cortex of Wistar rat embryos after 17 days of gestation were used. The excitotoxicity was induced after nine days of in vitro culture by treatment with a medium containing 100 μM glutamate for 15 minutes. After 24 h, treatment with 100 ng of NeuroEPO / ml showed a significant decrease (p <0.01) in mortality, compared to cells treated with glutamate alone. The NeuroEPO treatment decreased mortality and tended to reproduce the morphological characteristics of the control. The oxidative stress induced by glutamate is reduced after treatment with NeuroEPO. The images obtained by phase contrast microscopy show that neurons treated with glutamate show a body contraction, loss of dendrites that do not make contact with neighboring cells, and NeuroEPO is capable of preserving the morphological characteristics of the control. Immunocytochemistry tests show that the culture is essentially pure in neurons, that glutamate causes cell mortality, and that this is partially avoided when NeuroEPO is added to the culture medium. The activation of the intrinsic apoptotic pathways was also analyzed. The decrease in the Bcl-2 / Bax ratio was decisive in the release of cytochrome from the mitochondria to the cytosol and in the expression and activity of caspase-3 observed in the cells treated with glutamate. These alterations were avoided in the cells under treatment with NeuroEPO. These results confirm that NeuroEPO has a protective effect against the neuronal damage induced by excitotoxicity, improving the antioxidant activity in the neuron and protecting it from oxidative stress; In addition, they show that NeuroEPO protects cortical neurons from glutamate-induced apoptosis by up-regulating Bcl-2 and inhibits the activation of caspase-3 induced by excitotoxicity due to glutamate.

El síndrome de Aicardi-Goutières (AGS) es una interferonopatía de tipo I que afecta mayoritariamente al cerebro y a la piel. Las personas afectadas tienen un nivel elevado de IFN-α en el líquido cefalorraquídeo y en el suero, tal y como sucede en una infección vírica congénita, pese a no detectar virus en ninguno de los casos estudiados. Aún no se conocen los detonantes de la aparición de la enfermedad, aunque todo apunta a que podría deberse a un factor exógeno y/o al propio background genético del individuo. En los últimos años se han identificado mutaciones en distintos genes que causan el AGS: TREX1, RNASA H2 (Subunidades A, B y C), SAMHD1, ADAR e IFIH1. Éstas suelen provocar la pérdida de la función de las proteínas, las cuales tienen en común su participación en la señalización y en el procesamiento de los ácidos nucleicos. En nuestro grupo se ha demostrado que el déficit funcional de proteínas que intervienen en la reparación del ADN, como Nbs1 y Trex1, alteran las funciones de los macrófagos, y consecuentemente los procesos donde éstos intervienen. Estas células son capaces de modular la respuesta inflamatoria gracias a sus actividades pro y antiinflamatorias. Por ello, la actividad de los macrófagos debe estar finamente regulada ya que el exceso en la respuesta pro- o antinflamatoria provoca daño en los tejidos y la aparición de enfermedades crónicas. Los macrófagos tienen un papel clave en el mantenimiento de la homeostasis, por lo que las alteraciones producidas en estas células podrían ser la base de la autoinmunidad en el AGS. En esta tesis se muestra el estudio de Samhd1 y del complejo RNasa H2 en los macrófagos y en las enfermedades autoinmunes. Estas proteínas participan en el metabolismo de los ácidos nucleicos y están relacionadas con el AGS. En mamíferos Samhd1 es la fosfohidrolasa encargada de regular negativamente la concentración de dNTPs intracelulares, mientras que el complejo RNasa H2 es la mayor fuente de actividad ribonucleasa H. De hecho, sus actividades son esenciales para el mantenimiento de la integridad genómica. En esta tesis se mostró que Samhd1 y el complejo RNasa H2 forman parte de los mecanismos de reparación del ADN que aseguran la supervivencia de los macrófagos durante la inflamación. Se determinó que los estímulos proinflamatorios inducían la expresión de Samhd1 en los macrófagos mientras que la expresión del complejo RNasa H2 era constitutiva. En ratones deficientes en Samhd1 y en el complejo RNasa H2 se observó un aumento en la expresión de genes estimulados por interferón (ISGs) en el corazón y en el cerebro, tal y como sucede en pacientes con AGS. Además, también se vio incrementada la expresión de estos genes en los macrófagos activados con estímulos proinflamatorios. Por otro lado, no pudimos detectar ningún efecto de las proteínas Samhd1 y RNasa H2 en la diferenciación, proliferación y en la activación de estas células. Sin embargo, cuando fueron activados de forma proinflamatoria, los macrófagos deficientes en estas proteínas presentaron más daño en el ADN respecto a los controles. Además, dicho daño fue causado por las grandes cantidades de especies reactivas de oxígeno (ROS) producidas por los propios macrófagos. Por otro lado, la falta de Samhd1 aumentó la severidad de la respuesta autoinmune la inducción del modelo in vivo de encefalomielitis autoinmune experimental (EAE). En conjunto estas observaciones sugieren que tanto Samhd1 como el complejo RNasa H2 forman parte de los mecanismos de protección de los macrófagos, que previenen el daño en el ADN causado por las grandes cantidades de ROS que estas células producen durante la respuesta inflamatoria.
Aicardi-Goutières Syndrome (AGS) is a type I interferonopathy which affects brain and heart. Patients with AGS show high levels of IFN-α in cerebrospinal fluid and serum, which mimic a congenital virus infection. The trigger of this disease still is unknown but environmental factors and the genetic background could be potentially candidates. Currently, seven mutations have been described in patients with AGS: TREX1, RNASA H2 (Subunit A, B and C), SAMHD1, ADAR and IFIH1. Generally it causes the loss of function of the proteins, which are implicated in the signaling and metabolism of nucleic acids. Macrophages are an essential component of both innate and adaptive immunity. During immune response these cells play a dual role. Firstly macrophages produce pro-inflammatory mediators, including cytokines and reactive oxygen species (ROS), and engulf by phagocytosis pathogens and cell components. Secondly, when this damaging response is unnecessary macrophages acquire anti-inflammatory functions in order to induce tissue repair and the resolution of inflammation. The tightly regulation of pro- and anti-inflammatory activities in macrophages is essential to prevent tissue damage and chronic diseases. In our group we had demonstrated that proteins with DNA repair activities, as Nbs1 and Trex1, were required for macrophage functional activity. Macrophages are essential to maintain homeostasis and its alterations could be the origin of autoimmunity in AGS. In this thesis we showed that Samhd1 and RNase H2 protect macrophages during inflammation against DNA damage, caused by reactive oxygen species that they produce. Also we demonstrated that Samhd1 restrained the autoimmunity response in mice. These results reveal that Samhd1 and RNase h2 confer protection against DNA damage during inflammation in macrophages.

Somatic mutations are those that arise after the zygote is formed and are therefore inherited by a fraction of the cells of an individual. Their relevance in certain skin diseases has been known for almost half a decade and cancer, the most common disease caused by somatic mutations, has been extensively studied. Yet, their prevalence in healthy individuals as well as their putative role in other human disorders such as neurodegenerative diseases are still unanswered questions. Furthermore, accurate detection of somatic variants from bulk sequencing data still poses a technical challenge. This work focuses on detecting and circumventing the biases that hinder their identification. Using this knowledge, we identified somatic point mutations in the exomes of five different tissues from sporadic Parkinson disease patients. We also assessed the detection of somatic copy number variants from array CGH data using two tissues from Alzheimer disease patients. Finally, we participated in the identification of somatic variants in an extensive genomic dataset from a neurotypical individual.
Las mutaciones somáticas son aquellas que surgen tras la formación del cigoto y son, por tanto, heredadas por una fracción de las células de un individuo. Su importancia en algunas enfermedades cutáneas se conoce desde hace casi medio siglo. El cáncer, la enfermedad más común causada por mutaciones somáticas, se ha estudiado extensamente. Sin embargo, su prevalencia en individuos sanos, así como su potencial relevancia en otras afecciones humanas, como las enfermedades neurodegenerativas, son cuestiones todavía por resolver. Asimismo, detectar variantes somáticas con precisión en datos de secuenciación de muestras homogeneizadas sigue siendo complejo técnicamente. Este trabajo se centra en la detección y resolución de los sesgos que dificultan su identificación. Aplicando este conocimiento, identificamos mutaciones somáticas de una sola base en datos de secuenciación del exoma de cinco tejidos diferentes de pacientes de la enfermedad de Parkinson. También evaluamos la detección de variantes de número de copia somáticas en datos de array CGH de dos tejidos de pacientes de Alzheimer. Finalmente, participamos en la identificación de variantes somáticas en un amplio conjunto de datos genómicos de un individuo neurotípico.

EL MK-801 (dizocilpino), además de su conocido efecto como antagonista NMDA, es capaz de potenciar el efecto contráctil de la noradrenalina (NA), tanto exógena como neurogénica, en la porción epididimal del conducto deferente de rata. Se descarta la participación de receptores NMDA en este efecto, así como un favorecimiento de la liberación de NA, y todos los resultados apuntan a que se debe a una inhibición de la recaptación del neurotransmisor. Además del MK-801, otros ligandos PCP y Sigma ensayados potenciaron las contracciones por NA exógena, con un orden: fenciclidina (PCP) mayor que tenociclidina (TCP) mayor que MK-801 mayor que (+)-3-PPP mayor que dextrorfano (DX) mucho mayor que dextrometorfano (DM) y, en lo que respecta a estimulación eléctrica lo hicieron: (+)-3-PPP mayor que PCP mayor que MK-801 mayor que DM mayor que TCP mucho mayor que DX. No se descarta la participación de receptores Sigma en este ultimo efecto. Se confirma, asimismo, la interacción de todos los compuestos ensayados con el transportador de NA en el conducto deferente de rata, ya que inhibieron con CI50 de orden micromolar la union de un marcador especifico como la (3H)nixoxetina, cuyo perfil de unión a este tejido fue caracterizado previamente (KD=1.6 Microm., BMAX=1625 FMoles/mg). Todos ellos también inhibieron, CON CI50 Microm, la captación de (3H)NA en secciones de este órgano. Esta interacción, en el caso del MK-801, se traduce en un ligero incremento de la presión arterial en rata anestesiada con pentobarbital y en una potenciación del efecto presor de la NA como efecto periférico.

The primary tauopathies are a group of neurodegenerative diseases affecting movement and cognition. In this thesis I study Progressive Supranuclear Palsy (PSP) and the Corticobasal Syndrome (CBS), two parkinsonian disorders associated with accumulation of hyperphos- phorylated and abnormally folded tau protein. I contrast these two disorders with Parkinson’s disease (PD), which is associated with the accumulation of alpha-synuclein but has a genetic association with the MAPT gene encoding tau. Understanding the tauopathies to develop effective treatments will require a better grasp of the relationships between clinical syndromes and cognitive measures and how the anatomical and neurochemical networks that underlie clinical features might be altered by disease. I investigate simple clinical biomarkers, showing that a two-minute test of verbal fluency is a potential diagnostic biomarker to distinguish between PD and PSP and that the ACE-R and its subscores could play a role in monitoring cognition over time in PD, PSP and CBS. I assess the implementation of network analysis in Functional Mag- netic Resonance Imaging (fMRI) data, introduce Maybrain software for graphical network analysis and visualisation. I go on to show an overlap between graph theory network measures and I identify three main factors underlying graph network measures of: efficiency and distance, hub characteristics, network community measures. I apply these measures in PD, PSP and the CBS. All three diseases caused a loss of functional connectivity in com- parison to the control group that was concentrated in more highly connected brain regions and in longer distance connections. In ad- dition, widely localised cognitive function of verbal fluency co-varied with the connection strength in highly connected regions across PD, PSP and CBS. To take this further, I investigated specific functional covariance networks. All three disease groups showed reduced connectivity between the basal ganglia network and other networks, and between the anterior salience network and other networks. Localised areas of increased co- variance suggest a breakdown of network boundaries which correlated with motor severity in PSP and CBS, and duration of disease in CBS. I explore the link between gene expression of the tau gene MAPT and its effects on functional connectivity showing that the expression of MAPT correlated with connection strength in highly connected hub regions that were more susceptible to a loss of connection strength in PD and PSP. I conclude by discussing how tau protein aggregates and soluble tau oligomers may explain the changes in functional brain networks. The primary tauopathies are a group of neurodegenerative diseases affecting movement and cognition. In this thesis I study Progressive Supranuclear Palsy (PSP) and the Corticobasal Syndrome (CBS), two parkinsonian disorders associated with accumulation of hyperphos- phorylated and abnormally folded tau protein. I contrast these two disorders with Parkinson’s disease (PD), which is associated with the accumulation of alpha-synuclein but has a genetic association with the MAPT gene encoding tau. Understanding the tauopathies to develop effective treatments will require a better grasp of the relationships between clinical syndromes and cognitive measures and how the anatomical and neurochemical networks that underlie clinical features might be altered by disease. I investigate simple clinical biomarkers, showing that a two-minute test of verbal fluency is a potential diagnostic biomarker to distinguish between PD and PSP and that the ACE-R and its subscores could play a role in monitoring cognition over time in PD, PSP and CBS. I assess the implementation of network analysis in Functional Mag- netic Resonance Imaging (fMRI) data, introduce Maybrain software for graphical network analysis and visualisation. I go on to show an overlap between graph theory network measures and I identify three main factors underlying graph network measures of: efficiency and distance, hub characteristics, network community measures. I apply these measures in PD, PSP and the CBS. All three diseases caused a loss of functional connectivity in com- parison to the control group that was concentrated in more highly connected brain regions and in longer distance connections. In ad- dition, widely localised cognitive function of verbal fluency co-varied with the connection strength in highly connected regions across PD, PSP and CBS. To take this further, I investigated specific functional covariance networks. All three disease groups showed reduced connectivity between the basal ganglia network and other networks, and between the anterior salience network and other networks. Localised areas of increased co- variance suggest a breakdown of network boundaries which correlated with motor severity in PSP and CBS, and duration of disease in CBS. I explore the link between gene expression of the tau gene MAPT and its effects on functional connectivity showing that the expression of MAPT correlated with connection strength in highly connected hub regions that were more susceptible to a loss of connection strength in PD and PSP. I conclude by discussing how tau protein aggregates and soluble tau oligomers may explain the changes in functional brain networks.

There is growing recognition that accurate assessment of brain function includes activity at multiple temporal and spatial scales. In this thesis, we explored ways to combine clinically-relevant imaging information derived from subjects with neurodegenerative disease. In the first work, we investigated a two-step framework to determine both joint and unique biomarkers from structural and functional MRI in 18 healthy control (HC) and 12 Parkinson’s disease (PD) subjects. Three matrices (structural, functional, and structural/functional interactions) were derived from a subset of features in both modalities that were likely candidates for discrimination between PD and HC subjects. Finally, Least Absolute Shrinkage and Selection Operator (LASSO) regression was performed to determine if subjects’ clinical characteristics such as gender, smoking history, smell performance, Hoehn and Yahr Scale (H&Y Stage), and Unified Parkinson’s Disease Rating Scale (UPDRS) values, could be accurately predicted based on the imaging features. The results revealed that complementary biomarkers were most informative in predicting clinical scores in both groups. In the second work, for analyzing imaging data from subjects with Multiple Sclerosis (MS), we employed a joint Multimodal Statistical Analysis Framework, a data fusion approach that used Latent Variables (LV). We studied fusion of information from seven different imaging modalities: Myelin Water Imaging (MWI), Diffusion Tensor Imaging (DTI), resting state functional MRI (rsfMRI), cortical thickness of the right and left hemisphere, MS lesion load, and normalized brain volume from 47 subjects with MS. Decomposed common and unique information in each modality were acquired and their relationships with disease duration (DD), the Expanded Disability Status Scale (EDSS), and age, were analyzed through LASSO regression. We noted that common components of the seven modalities were the most accurate in predicting clinical indices. Results further revealed the regional importance of each modality by indicating a unique pattern of degeneration in MS and an asymmetry between the cortical thickness components in the two hemispheres. Our results demonstrate the power of utilizing multimodal imaging biomarkers in neurodegenerative diseases. Since structural imaging data is acquired along with functional data, we propose that fusion of information from both types of data should become part of routine analysis.
Applied Science, Faculty of
Graduate

This thesis is broken into four chapters. The first two chapters summarize two separate lines of investigation into the role of a putative neurotoxin in the pathogenesis of Huntington's Disease (HD). The third chapter outlines an investigation of the putative role of beta-N-methylamino-L-alanine (BMAA) in the pathogenesis of amyotrophic lateral sclerosis (ALS), while the final chapter details a post-mortem investigation of the contents of biogenic amines and amino acids in the brain of a man who died of a familial form of parkinsonism. Chapter I is a description of a chromatographic technique developed to isolate quinolinic acid (QA), an endogenous compound implicated in the pathogenesis of HD, from deproteinized human sera. A cation exchange column was used to selectively isolate QA, which was eluted with 10 mM HCl. The eluted fractions were analyzed by UV spectrometry to isolate and quantify QA. Once the fractions corresponding the elution of authentic QA were isolated, concentrated and the excess HCl removed, the fractions were added to growing fetal rat striatal explant cultures as an assay of neurotoxicity. Since HD involves the selective degeneration of GABAergic neurons in the striatum, the activity of glutamic acid decarboxylase, the final enzyme in the synthesis of GABA, was used to determine the viability of the cultures. Unfortunately, the method was confounded by the contamination of all effluents by compounds originating from the cation exchange resin, which were discovered to be neurotoxic to the striatal cultures, and as a result the investigation had to be abandoned. Chapter II describes an investigation designed to further characterize the nature of neurotoxicity observed in the sera obtained from patients with HD (Perry et al. 1987). Compounds with the capacity to selectively stimulate neurons at the N-methyl-D-aspartate (NMDA) receptor have been implicated in a variety of neurodegenerative disorders, including HD. Selective antagonists at the NMDA receptor have been shown to protect neurons from the degenerative effects of such "excitotoxins". The investigation described used MK-801, a potent noncompetitive NMDA antagonist, in an attempt to protect fetal rat striatal cultures from the neurodegenerative effects of the sera obtained from HD patients. The results obtained were equivocal. No evidence was obtained to support a role of the NMDA receptor in the mediation of the neurotoxicity, and in addition the neurodegenerative effects of HD sera were not reproduced in the present investigation. A variety of possible explanations for the apparent discrepancy are suggested. Chapter III describes an experiment intended to produce an animal model of ALS based on the observations by Spencer et al. 1987 that chronic oral administration of BMAA in monkeys produced the histological and behavioural characteristics of this disease. In the present investigation synthetic D,L-BMAA was given by gavage to mice over an eleven week period. Since BMAA is known to act at the NMDA receptor, a subset of the mice were also given MK-801 in an effort to protect them from any deleterious effects based on the action of BMAA at this receptor. The animals were sacrificed at the end of the experiment, and biochemical analyses were performed on the striata and cortices of the animals. In addition, neuropathological studies were performed on the spinal cords, basal ganglia and related structures. The results indicated no biochemical or neuropathological abnormality as a result of BMAA administration. Chapter IV describes a post-mortem investigation of a man who was a member of a well described pedigree which carries an autosomal dominant form of parkinsonism. The object of the investigation was to determine post-mortem levels of dopamine, noradrenaline, serotonin and their metabolites, in addition to amino acids in various regions of brain. Although conflicting evidence was obtained during life, neuropathological findings and the present neurochemical analyses confirm the degeneration of the nigrostriatal dopaminergic tract, characteristic of parkinsonism, in this man.
Medicine, Faculty of
Anesthesiology, Pharmacology and Therapeutics, Department of
Graduate

Our global population is ageing and an ever increasing number of elderly are affected with neurodegenerative diseases, including the subjects of the studies in this work, Alzheimer's disease (AD), Parkinson's disease (PD), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). On strong evidence that several genes may influence the development of sporadic neurodegenerative diseases, the genetic association approach was used in the work of this thesis to identify the multiple variants of small effect that may modulate susceptibility to common, complex neurodegenerative diseases. It has been shown that the common genetic variation of one of these susceptibility genes, MAPT, that of the microtubule associated protein, tau, is an important genetic risk factor for neurodegenerative diseases. There are two major MAPT haplotypes at 17q21.31 designated as H1 and H2. In order to dissect the relationship between MAPT variants and the pathogenesis of neurodegenerative diseases, the architecture and distribution the major haplotypes of MAPT have been assessed. The distribution of H2 haplotype is almost exclusively in the Caucasian population, with other populations having H2 allele frequencies of essentially zero. A series of association studies of common variation of MAPT in PSP, CBD, AD and PD in different populations were performed in this work with the hypothesis that common molecular pathways are involved in these disorders. Multiple common variants of the H1 haplotypes were identified and one common haplotype, H1c, showed preferential association with PSP and AD. A whole-genome association study of PD was also undertaken in this study in order to detect if common genetic variability exerts a large effect in risk for disease in idiopathic PD. Twenty six candidate loci have been found in this whole-genome association study and they provide the basis for our investigation of disease causing genetic variants in idiopathic PD.

Mitochondrial diseases affect the core energy generating pathways and can result in significant cognitive impairment and neurodegeneration. The mechanisms underlying the neurological and pathological consequences of mitochondrial disease are not understood and current treatment of mitochondrial diseases is limited in scope and efficacy. pyruvate dehydrogenase (PDH) and Complex I are key mitochondrial enzymes pivotal for cellular energy metabolism, and mutations in genes coding for PDH and Complex I proteins are common causes of mitochondrial disease. This thesis describes the use oftwo techniques, gene targeting, and RNA interference, to generate in vitro models of Complex I and PDH deficiency in pluripotent cell lines. Gene targeting of the ndufal gene was unsuccessful, however, a number of cell lines were isolated with significant reductions in PDH activity mediated by RNAi targeted against the Pdhal transcript. These cells lines were neurodifferentiated in vitro and used to characterise the developmental and degenerative consequences of PDH deficiency on neural cultures. The cultures were found to successfully differentiate into neural cells, but were developmentally abnormal with defective neuronal migration and neurite extension, and neuritic varicosities, an indication of neuritic degeneration in many neurological disease models. These features were activity-dependent with the most severe phenotype found in the cell line with the least residual PDH activity. These cultures were used to explore the nature of energy metabolism in PDH deficient neurons, and therapeutic strategies were successfully tested. This research has successfully established a reproducible and practical model to assess neurodegeneration in PDH deficiency, to test t reatments and to model theories of disease mechanisms. As such it provides promise for the improvement of the understanding of and treatment of disorders of brain energy metabolism in the future.

The 14-3-3 family of proteins are important signalling proteins involved in a number of cellular processes. These include cell cycle regulation, apoptosis, signal transduction and cell signalling. There is also considerable evidence in the literature that 14-3-3 proteins play a vital role in the pathology of neurodegenerative diseases, including Alzheimer’s, Parkinson’s, Huntington’s and Prion disease. The neurodegenerative disease of focus in this research is Spinocerebellar Ataxia Type 1 (SCA1). SCA1 is a polyglutamine-repeat disease and the interaction of the disease protein ataxin-1 with 14-3-3 proteins leads to the toxic accumulation and subsequent protein aggregation which is characteristic of this disease. This study focused on attempting to elucidate the structure of various domains of the disease protein and also in identifying potential inhibitors of this deleterious interaction. Unfortunately, structural studies were not successful due to a number of caveats encountered in the expression and purification of the ataxin-1 protein domains. By utilising computational methods and small molecule inhibitors, a number of potential lead compounds which possess the ability to at least partly disrupt the interaction of 14- 3-3ζ have been identified. As 14-3-3 proteins play roles in other neurodegenerative diseases, successful identification of potential drug lead treatments can have far reaching benefits in a number of neurodegenerative diseases including SCA1. Lipid rafts are also involved in neurodegenerative disease pathology. Lipid rafts are cholesterol and sphingolipid rich domains which organise the plasma membrane into discrete microdomains and act as signalling platforms and processing centres which attach specific proteins and lipids. A number of disease proteins are processed at these membrane regions, including those involved in Alzheimer’s, Parkinson’s and Prion disease. This processing is a step which is critical in the pathology of disease and abnormal processing leads to the formation of toxic protein aggregates. Previous research in the lab identified the association of low levels of the five main brain isoforms of 14-3-3 proteins with rafts. This study expanded on this to positively identify the presence of the two phospho-forms of 14-3-3, α and δ. The mechanism by which 14-3-3 proteins associate with rafts was also investigated, indicating that 14-3-3 associates with rafts via an unidentified raftbound protein(s). In addition, the phosphorylation status and quaternary structure of 14-3-3 in the presence of sphingolipids has been explored.

Apathy is characterised by a lack of motivation towards goal directed behaviour and is a symptom of various neurodegenerative diseases. There are various tools that can be used to assess apathy but a caveat of these is that they usually assess it as a unidimensional concept. Apathy has been recognised to have a multidimensional substructure. The Dimensional Apathy Scale is the only comprehensive measure designed to quantify neurobiologically-based subtypes, called Executive, Emotional and Initiation apathy. The first aim of this study was to explore multidimensional apathy, and its associations with demographic variables, in healthy, community dwelling adults. Secondly, multidimensional apathy was explored in neurodegenerative diseases, specifically Amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD) and Alzheimer’s disease (AD). For each disease group, the validity and reliability of both the self rated and carer rated DAS were also determined. Finally, the association between specific apathy subtype impairments and executive dysfunction was explored in ALS patients. Four hundred healthy community dwelling adults, eighty-three ALS patients (seventy-five carers), thirty-four PD patients (thirty carers) and forty-nine AD patients (eighty-nine carers) were recruited for the questionnaire study. In the healthy community dwelling adults, Executive apathy decreased with age, whereas Emotional increased with age. Gender differences were also shown with higher apathy in males on Emotional apathy. There were also employment differences, in that Executive apathy was higher in unemployed individuals compared to those who were employed. Emotional apathy showed difference in type of employment, where full time employed individuals were significantly more apathetic than those employed part time. These findings were taken into account in selecting the appropriate control samples to match our patient groups. In the patient groups, ALS patients were found to be significantly more impaired on the Initiation subscale when compared to controls. Furthermore, Initiation apathy was found to be the most frequent impairment above abnormality cut-off on the carer rated DAS. PD patients were significantly more impaired on Executive and Initiation apathy when compared to controls. These two subscales were most frequently above abnormality cut-off in the carer rated DAS. Finally, AD patients were significantly more impaired on all subscales when compared to controls and, on the carer rated DAS, global impairment over all subscales was most often reported as above abnormality cut-off. Additionally in AD, there was a significant disparity between carer and patient ratings on Executive and Initiation apathy, indicating patients’ impaired awareness. When comparing patient groups, there was a significant difference between carer rated apathy subtype impairments for each patient group. Validity and reliability of the DAS was found to be robust when compared to standard measures of apathy and depression. In the experimental study, a sample of ALS patients (and their carers) and healthy controls (and their informants) were recruited to complete a battery of neuropsychological tests, the DAS, other apathy and depression measures. ALS patients were impaired on tasks of executive functioning when compared to controls. Furthermore, apathy subtype deficits were associated with executive dysfunction in ALS. In conclusion, apathy is a multidimensional concept that manifests in different subtype profiles dependent on neurodegenerative disease. This has further implications for understanding and assessment of cognitive dysfunction and neuropsychiatric symptoms, such as apathy, in ALS and other neurodegenerative disease patient groups.

In this thesis, the haemodynamic, functional and structural changes in Alzheimer's Disease, Huntington's Disease and Multiple Sclerosis are assessed at 7T. Across all chapters, there is a focus on the use of Arterial Spin Labelling (ASL) to provide haemodynamic measures of perfusion (or cerebral blood flow) and transit time (TT) to provide a useful marker of disease. Arterial Spin Labelling (ASL) has the advantage that it is a non-invasive method to measure perfusion using magnetic resonance imaging (MRI). Clinically, perfusion is assessed using contrast-enhanced techniques which requires the intravenous administration of an exogenous gadolinium-based contrast agent, such as Prohance-TM and Gadovist-TM. Contrast-enhanced techniques typically provide higher SNR than ASL methods, however the non-invasive nature of ASL makes it a safe method suited for repeated measures in any subjects, including those with poor renal clearance. Additionally, gadolinium contrast agents have been shown to accumulate in neuronal tissue, and until the clinical significance of this is determined, contrast-enhanced scans should be performed with caution. In Chapter 5, arterial spin labelling is used to assess cerebral perfusion in a patient group with Alzheimer's Disease (AD) and compared with an age-matched healthy control group (HC). Functional MRI (fMRI) is used to assess functional connectivity within the default mode network (DMN) and measures compared between the AD and HC group. In addition, high resolution structural data is acquired to assess the effects of atrophy in AD. Results demonstrate a significant decrease in grey matter perfusion and a significant increase in grey matter transit time in the AD group compared the HC group. A trend showing a decrease in functional connectivity in the DMN was found in the AD group as compared to the HC group. As expected, significant grey matter loss and cortical thinning were observed in the AD group compared to the HC group. Secondly, haemodynamic and vascular changes in a Huntington's Disease (HD) patient group are assessed and compared with healthy age matched controls (HC). Phase contrast angiography is used to assess vessel density and vessel radius distributions between the two groups. Structural data was also acquired to assess grey matter volume and cortical thickness differences between the two groups. A significant reduction in perfusion was found in grey matter, putamen and the caudate in the HD group compared to the HC group. The ASL transit time was found to be significantly increased in the caudate and putamen in the HD group compared to the HC group. Phase contrast angiography data showed an increase in the frequency of smaller vessels (0.15-0.35mm) in the HD group compared to the HC group, whereas larger vessels appeared more frequently in the HC group. A significant reduction in grey matter volume was also observed in the HD group compared to the HC group, which manifested as thinning of the cortical ribbon. In the final study of this thesis, high spatial resolution arterial spin labelling is used to assess perfusion inside cortical lesions and compare with perfusion in surrounding normal appearing grey matter in a Multiple Sclerosis (MS) patient group. Grey matter perfusion as a function of distance from the cortical lesions was also assessed. It was found that cortical lesions have reduced perfusion compared to surrounding normal appearing grey matter. Perfusion increased and stabilised immediately outside of the cortical lesion itself, suggesting that the perfusion deficit observed is highly spatially localised.

Neurodegeneration is the progressive loss of the structure or the function of the neurons. Indeed, the dopaminergic function in Parkinson’s disease (PD) is strongly damaged. Currently, L-DOPA is the most effective treatment for PD. However, the wearing off and on-off phenomena represent a limit of this therapy. Moreover, events like a stroke can cause severe impairments and neural death. In this context, the sirtuin family has shown to have a significant influence on the extent of neuronal death. In this work, firstly, we highlighted the ability of sulforaphane, an isothiocyanate, in counteracting and preventing the neurotoxicity induced by L-DOPA. This effect is mediated by the NRF2 pathway through the increase of cellular antioxidant defenses. Secondly, in C. elegans, we found that knock-out of mitochondrial sirtuin sir-2.3, homologous to mammalian SIRT4, is protective in both chemical ischemia and hyperactive channel induced necrosis. Furthermore, the block of glycolysis enhances the protective effect of sir-2.3 knock-out both in vivo and in culture, suggesting that this occurs independently of the insulin/IGF pathway. Analysis of ROS in sir-2.3 knock-out reveals that ROS become elevated in this mutant under dietary deprivation (DD) and early on in dietary deprived animals under ischemic conditions, suggesting that mitohormetic elevation of ROS is operative in this mutant. In conclusion, this work suggests novel pathways that can be targeted for the design of therapies aimed at protecting neurons from age linked damages.

A worldwide demographic shift is currently occurring, with rapidly increasing numbers of elderly individuals. Since the incidence of neurodegenerative disease generally increases with age, this entails an increase in dementia prevalence. There are several strong incentives for establishing robust and widely available imaging methods for the early diagnosis of these diseases. Atrophy patterns are evident only late in the disease process, and the distinction from healthy ageing can often be elusive. For early diagnosis, physiologic parameters such as perfusion or metabolism must be assessed. The available modalities all have restricted clinical usefulness. The main aim of this thesis was to advance the clinical usefulness of perfusion and metabolism imaging in patients with neurodegenerative dementia, with a focus on visual assessment. A cohort of patients with neurodegenerative dementia was included, along with an age-matched control group. All subjects underwent MRI, including a pseudocontinuous ASL sequence and FDG-PET. In papers II and III, a subgroup containing both patients and controls underwent a second FDG-PET with reduced dose. In paper IV, the material was combined with a similar cohort from Amsterdam. Paper I showed that spatial smoothing increased the correlation between visually assessed perfusion and metabolism levels as displayed with FDG-PET. However, the distinction between patients and healthy controls was less satisfactory due to false positives. Paper II showed that differences in regional standard uptake value ratios between normal- and low-dose FDG-PET were small and without clinically significant bias. Paper III showed that the diagnostic performance of Z-score maps showing regions of significant deficits in metabolism was highly similar in normal- and low-dose FDG-PET images.  Paper IV showed that ASL perfusion-based Z-score maps can be used for diagnostic purposes with high specificity, but inferior sensitivity, compared to FDG-PET. In conclusion, the included studies address aspects of the visual assessment of perfusion and metabolism neuroimaging, with a focus on clinical usefulness in diagnosing neurodegenerative dementia.

The incidence of neurodegenerative diseases, such as Alzheimer's disease, increases every year due to population aging. Therefore, the development of new therapeutic strategies, such as biodegradable nanostructured systems like poly-lactic-co-glycolic acid functionalized with polyethylene glycol (PLGA-PEG) is extremely necessary in order to provide greater therapeutic adherence as well as to reduce the adverse effects of the drugs. Glaucoma is currently considered a neurodegenerative disease since there is a direct connection between the brain and the last ocular structures sharing many characteristic features with Alzheimer's disease. The only drug approved for moderate to severe phases of Alzheimer's is Memantine Hydrochloride (MEM). Clinical studies with Ibuprofen have shown that the levels of markers associated with the disease decrease with this drug. Therefore, the main objective of this thesis is the development and characterization of PLGA-PEG nanostructured systems encapsulating MEM drugs and Dexibuprofen (DXI, active enantiomer of ibuprofen) in order to increase the passage through the blood-brain barrier (BBB) and hematoretinal barrier (BHR). The developed nanostructured systems were optimized in order to obtain homogeneous nanoparticles with an average size below 200 nm with a high encapsulation efficiency (80-99%). These systems were characterized by spectroscopic methods (X-ray diffraction or infrared spectroscopy) and thermal methods (differential scanning calorimetry), confirming the correct incorporation of the drug into the particles without the formation of new covalent bonds. The in vitro release profile of these systems showed a sustained release, more prolonged than the free drug with two phases (an initial rapid phase due to the drug attached to the surface followed by a slower release of the encapsulated drug). The corneal and scleral permeation of the systems showed a tropism of the nanoparticles towards the corneal tissues. In vitro studies confirmed that the nanoparticles developed were neither cytotoxic in brain nor retinal cells and that they are able cross trough the BBB. Although free MEM did affect cell viability by acting the nanoparticles as protection. The systems developed were tested in vivo for Alzheimer's disease, glaucoma and ocular inflammation. The experiments regarding Alzheimer's disease, were carried out using a double transgenic mouse model and by administering the nanoparticles orally. It was shown that both MEM and DXI nanoparticles decreased the level of β-amyloid plaques and the associated inflammatory brain process. The results of MEM nanoparticles assessed for glaucoma disease in rats using the ocular hypertension model by administering two drops per day of these systems demonstrated that, despite not decreasing intraocular tension, the systems prevent the death of the retinal ganglion cells. In addition, ocular administration of DXI nanoparticles demonstrated to be more effective in preventing ocular inflammation than the free drug, for these reason it would be indicated in the prophylaxis of inflammation secondary to ocular surgery.
La incidencia de enfermedades neurodegenerativas, como la enfermedad de Alzheimer, aumenta cada año debido al envejecimiento de la población. Por ello es necesaria la búsqueda de nuevas estrategias terapéuticas, como los sistemas nanoestructurados de ácido poli-láctico-co-glicólico funcionalizados con polietilenglicol (PLGA-PEG), para proporcionar mayor adherencia terapéutica así como reducir los efectos adversos de los fármacos. El glaucoma se considera actualmente una enfermedad neurodegenerativa ya que existe una conexión directa entre en cerebro y las últimas estructuras oculares compartiendo muchos rasgos característicos con la enfermedad de Alzheimer. El único fármaco aprobado para las fases moderadas-severas de Alzheimer es el hidrocloruro de Memantina (MEM). Estudios clínicos desarrollados con Ibuprofeno demuestran que este disminuye los niveles de marcadores asociados a la enfermedad. Por lo tanto, el principal objetivo de esta tesis es el desarrollo y caracterización de sistemas nanoestructurados de PLGA-PEG encapsulando los fármacos MEM y Dexibuprofeno (DXI, enantiómero activo del ibuprofeno) con la finalidad de incrementar el paso a través de la barrera hematoencefálica (BHE) y barrera hematoretiniana (BHR). Los sistemas nanoestructurados desarrollados fueron optimizados con el objetivo de obtener nanopartículas homogéneas de tamaño inferior a 200 nm con una elevada eficiencia de encapsulación (80-99 %). Éstos fueron caracterizados mediante métodos espectroscópicos (difracción de rayos X o espectroscopia de infrarrojo) y métodos térmicos (calorimetría diferencial de barrido), confirmando la correcta incorporación del fármaco en las partículas sin la formación de nuevos enlaces covalentes. El perfil de liberación in vitro de estos sistemas presentó una liberación prolongada, más sostenida que el fármaco libre sujeta a dos fases (una fase inicial de rápida debido al fármaco en superficie seguida de una liberación más lenta del fármaco encapsulado). La permeación corneal y escleral de los sistemas mostró un tropismo de las nanopartículas desarrolladas por los tejidos corneales. Los estudios in vitro confirmaron que las nanopartículas desarrolladas no eran citotóxicas ni en células cerebrales ni retinales y que son capaces de atravesar la BHE. Por el contrario, la MEM libre disminuyó la viabilidad celular de manera significativa, demostrando que la matriz polimérica actuaba como protección. Los sistemas desarrollados fueron ensayados in vivo para la enfermedad de Alzheimer, glaucoma e inflamación ocular. Los estudios para la enfermedad de Alzheimer se llevaron a cabo usando un modelo de ratón doble transgénico y administrando las nanopartículas por vía oral y se observó que tanto las nanopartículas de MEM como las de DXI disminuían el nivel de placas de β-amiloide y la inflamación cerebral asociada. Los resultados de las nanopartículas de MEM ensayadas para la enfermedad de glaucoma en ratas usando el modelo de hipertensión ocular administrando dos gotas al día de estos sistemas, pusieron de relieve que, pese a no disminuir la tensión intraocular, si impedían la muerte de las células ganglionares de la retina. Además, la administración ocular de los sistemas de DXI demostró ser más efectiva para prevenir la inflamación ocular que el fármaco libre, por lo que estaría indicada en la profilaxis de la inflamación secundaria a cirugía ocular.

Aberrant protein phosphorylation is a hallmark of neurodegenerative diseases, such as Alzheimer's disease (AD) and tauopathies. Pathological phosphorylation is commonly observed as intracellular tau containing deposits correlating with neuronal death. Tau is a cytoskeletal protein, key to the stabilisation of the microtubules (MT), important ,for structural integrity and protein trafficking. Tau's ability to promote MTs is regulated by protein phosphorylation by kinases/phosphatases. In spontaneous AD, the activity/expression of kinases are enhanced w$ilst phosphatases are decreased. Alterations in phosphorylation have significant regulatory effects on neuronal signalling which may lead to deficits in neuronal signalling prior to neuronal death. Using cultured rat hippocampal neurons and pharmacological inhibitors of serine/threonine (Okadaic acid and Cantharidin) and tyrosine (Sodium orthovanadate) phosphatases, the consequences of increased phosphorylation for neuronal signalling were investigated. Using Fura-2 Ca2+ imaging, serine/threonine phosphatase inhibition was determined to exert bidirectional modulation on neuronal excitability. Low level short duration inhibition enhanced, whilst str9nger or more prolonged inhibition suppressed Ca2 + responses towards stimulation. Suppression of excitability was coincident with' a reduction in receptor expression and an increase in cytoskeletal phosphorylation. The data mimic changes in signalling utilising alternative means of modelling AD, suggesting that common ' phosphorylation cascades may be active within these models and in the disease process. Acute inhibition of tyrosine phosphatases evoked a prolonged Ca2 + response dependent on the activation of trans-plasma membrane Ca2+ channels. Prolonged inhibition of tyrosine phosphatases enhanced neuronal excitability. A novel sponge toxin polymeric 1,3-alkylpyridinium salts was employed as an agent for inducing tau over-expression in cultured neurons and further Ca2 + imaging studies conducted. Modulation on excitability was observed to be dependent on the level oftau expression. Overall, this thesis furthers the understanding of phospho-regulation of neuronal signalling ~oth physiologically, and pathologically and provides preliminary data for novel roles of tau in neuronal signalling regulation.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) characterised pathologically by inflammation, demyelination and variable degrees of axonal loss and gliosis. Remyelination, axonal and synaptic plasticity have been identified as mechanisms underlying functional recovery. MS typically follows a chronic course, in the sense that new episodes or steady progression are the rule. Currently radiological surrogates of inflammation, demyelination and axonal loss are available, but these correlate only modestly with the development of cumulative disability. In this thesis, potential brain specific protein (BSP) markers for these pathological processes are identified and compared to existing magnetic resonance (MR) markers. OBJECTIVES: Levels of the nervous system proteins S100B (astrocytic activation), glial fibrillary acidic protein (GFAP astrogliosis), neurofilaments (axonal marker) and ferritin (microglial activation) and other inflammatory markers (i.e. anti-myelin antibodies) were measured. These values were correlated with both histopathological measurements in post-mortem specimens and MR imaging measures in patients with MS. Histology and immunochemistry of tissue sections characterised normal and pathological CNS and localized the extent of demyelinating plaques.

Neurodegenerative diseases are fatal disorders in which disease pathogenesis results in the progressive degeneration of the central and/or the peripheral nervous systems. These diseases currently affect -2% of the population but are expected to increase in prevalence as average life expectancy increases. The majority of these diseases have a complex genetic basis. The work presented in this thesis aimed to investigate the genetic basis of two neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and the human prion diseases kuru and sporadic Creutzfeldt-Jakob disease (sCJD), using novel complex genetic approaches. ALS is a fatal neurodegenerative disease in which motor neurons are seen to degenerate. It is a complex disease with 10% of individuals having a family history and the remaining 90% of non-familial cases having some genetic component. The gene DYNC1H1 is involved in retrograde axonal transport and is a good candidate for ALS. In this thesis the genetic architecture of DYNC1H1 was elucidated and a mutation screen of exons 8, 13 and 14 was undertaken in familial forms of ALS and other motor neuron diseases. No mutations were found. A linkage disequilibrium (LD) based association study was conducted using two tagging single nucleotide polymorphisms (tSNPs) which were identified as sufficient to represent genetic variation across DYNC1HI. These tSNPs were tested for an association with sporadic ALS (SALS) in 261 cases and 225 matched controls but no association was identified. Kuru is a devastating epidemic prion disease which affected a highly geographically restricted area of the Papua New Guinea highlands, predominantly affected adult women and children. Its incidence has steadily declined since the cessation of its route of transmission, endocannibalism, in the late 1950's. Kuru imposed strong balancing selection on codon 129 of the prion gene (PRNP). Analysis of kuru-exposed and unexposed populations showed significant deviations from Hardy-Weinberg equilibrium (HWE) consistent with the known protective effect of codon 129 heterozygosity. Signatures of selection were investigated in the surviving populations, such as deviations from HWE and an increasing cline in codon 129 valine allele frequency, which covaried with disease exposure. A novel PRNP G127V polymorphism was detected which, while common in the area of highest kuru incidence, was absent from kuru patients and unexposed population groups. Genealogical analysis revealed that the heterozygous PRNP G127V genotype confers strong prion disease resistance, which has been selected by the kuru epidemic. Finally, PRNP copy number was investigated as a possible genetic mechanism for susceptibility to kuru and sCJD. No conclusive copy number changes were identified.

Inherited metabolic diseases (IMD’s) are a large class of heterogeneous genetic disorders caused by dysfunction within a single pathway of intermediary metabolism. In many of these diseases, the dysfunction of metabolic enzymes leads to the accumulation of toxic metabolites which disrupts the normal development of the central nervous system. With the advent of treatments that positively influence neuropsychological outcomes, there is a need for sensitive and objective neuropsychological measures that allow patients to be systematically tracked in order to understand the efficacy of existing treatments. In this thesis, a neuropsychological test battery consisting of attention, language and oculomotor measures was developed to accurately describe individual and developmental differences between IMD patients and healthy developing controls. The functioning of five diseases was examined: Morquio syndrome (\(N\) = 12), Hurler syndrome (\(N\) = 3), Maroteux-Lamy syndrome (\(N\) = 2), Tyrosinemia type I (\(N\) = 13) and Tyrosinemia type III (\(N\) = 5). Findings indicated that disease effects were not homogeneous across tasks, and that performance on the same tasks was not uniform across diseases. The obtained data offers a promising basis for understanding how biological factors influence the severity and timecourse of developmental effects in future research.

Inflammation is thought to be a pathological feature that drives the neuronal degeneration in Alzheimer (AD) and Parkinson's diseases (PD). This is normally associated with classically activated microglia, which release pro-inflammatory cytokines. Recently, the heterogeneous nature of microglial activation states has been acknowledged and the focus has been shifting to the alternative activation state, an immunosuppressive state that promotes tissue maintenance and repair. This project examined microglia phenotypes in AD and PD, focusing on alternative activation and phagocytosis. Traumatic brain injury (TBI) cases were also studied to provide a comparison between an acute inflammatory reaction and chronic inflammation observed in AD and PD. An initial test using a battery of antibodies against microglia e.g. MHCII, Iba1, CD68, MRC1 was used to identify interesting targets for characterization in AD and PD. CD163 and CD14, both of which are thought to be exclusive to perivascular macrophages, were detected in parenchymal microglia in AD and PD, with a much more florid reaction observed in AD. Many of these microglia were associated with extracellular pathology i.e. Abeta plaques and extracellular Lewy Bodies. Experimental evidence from AD, PD and TBI cases suggest that these microglia were of local and systemic origin. A number of clinicopathological correlations were found in PD cases. Upregulation of CD14+ microglia in the substantia nigra was significantly correlated with absence of gait and balance problems as an onset symptom. Upregulation of CD163+ microglia in the cingulate cortex, entorhinal cortex, and locus coeruleus was significantly correlated with a lack of anxiety. In TBI cases, positive association was found between the upregulation of CD163+ microglia and survival time. My study demonstrates differential microglial activation in AD and PD, emphasizes the heterogeneity of microglia phenotype, and provides an insight into the influence of systemic inflammation on microglial activation.

Neurodegenerative disease is an umbrella term for pathologies that primarily damage neurons. As their incidence increases with age it is becoming of a greater concern for the west, due to its aging population. Due to their chronic nature and the difficulty to create reliable and reproducible animal models of these diseases their pathophysiologies are still poorly understood. For all these reasons, a mathematical modelling approach is suggested. The methodology of the work here consisted of identifying the state of the art models that describe the healthy behaviour of cells (e.g. metabolism and ionic regulation) and adapting them for pathological environments. With these models hypotheses provided by clinicians and pathologists were tested. The work focuses on developing models of mechanisms common to neurodegenerative diseases, which include: glutamate excitotoxicity, aquaporin water kinetics, inflammatory complement lysis and acute inflammation. Glutamate excitotoxicity was modelled by creating a compartmental model of glutamate exchange between neurons and astrocytes. This model was the first model of glutamate kinetics validated in an ischaemic stroke context. The aquaporin water kinetics and complement lysis models were developed in the context of the autoimmune disease Neuromyelitis Optica. Through this project a hypothesised trigger for the pathology was confirmed. Additionally, the first model of astrocytic cytotoxic oedema due to complement lysis was developed. Finally, a preventative drug for complement lysis was simulated. Acute inflammation was explored in the context of understanding the potential of chemerin as a pro-resolving cytokine. To that effect, a model of acute inflammation was developed where pro-resolving mechanisms were included. This model was the first to attempt model the effects of an intervention in inflammation. The results indicated that there is a maximum inhibitory effect of chemerin on inflammation. Additionally, two preventive avenues for chronic inflammation were found. With this work, the first attempts of capturing relevant mechanisms of neurodegenerative diseases were presented. These models can now be further developed and adapted to other pathological environments.

Neurodegeneration is a chronic, progressive and debilitating condition that affects majority of the World's elderly population who are at greater risk. Numerous scientific studies suggest that there could be a common underlying molecular mechanism that promotes the degeneration and the subsequent neuronal loss, however so far the progress in this direction is rather limited. Abnormal protein misfoldings, as well as protein plaque formations in the brain, are some of the hallmark characteristic features of neurodegenerative disorders (NDDs). Genetic and environmental factors, oxidative stress, excessive reactive oxygen species formation, mitochondrial dysfunction, energy depletion and autophagy disruption etc. are some of the widely suspected mechanisms that manifest the cognitive, motor and emotional symptoms of these NDDs. Motivated by some molecular traits found in common in several NDDs, network-based systems biology tools and techniques were used in this study to identify critical molecular players and underlying biological processes that are common for Parkinson's, Alzheimer's and Huntington's disease. Utilizing multiple microarray gene expression datasets, several biomolecular networks such as direct interaction, shortest path, and microRNA regulatory networks were constructed and analyzed for each of the disease conditions. The network-based analysis revealed 26 genes of potential interest in Parkinson's, 16 in Alzheimer's and 30 in Huntington's disease. Many new microRNA-target regulatory interactions were identified. For each disorder, several routes for possible disease initiation and protection scenarios were uncovered. A unified neurodegeneration mechanism network was constructed by utilizing the significantly differentially expressed genes found in common in Parkinson's, Alzheimer's and Huntington's microarray datasets. In this integrated network many key molecular partakers and several biological processes that were significantly affected in all three NDDs were uncovered. The integrated network also revealed complex dual-level interactions that occur between disease contributing and protecting entities. Possibilities of microRNA-target interactions were explored and many such pairs of potential interest in NDDs were suggested. Investigating the integrated network mechanism, we have identified several routes for disease initiating, as well as alleviating ones that could be utilized in common for Parkinson's, Alzheimer's and Huntington's disease. Finding such crucial and universal molecular players in addition to maintaining a delicate balance between neurodegeneration promoters and protectors is vital for restoring the homeostasis in the three NDDs.

The Ataxia telangiectasia (A-T) syndrome is a multisystem disorder exhibiting a complex array of clinical features for which there is no effective therapy. The earliest and most debilitating aspect of this disease is progressive ataxia resulting from neurodegeneration of the cerebellum. Several lines of evidence suggest a potential function for ATM in the maintenance of cellular homeostasis, and disruptions in functional ATM appear to result in alterations cholesterol homeostasis. This association may provide some insight into the neurodegenerative aspect of this disease. Cholesterol homeostasis is particularly significant in the central nervous system (CNS) with several neurodegenerative disorders involving disturbances in cholesterol homeostasis. The goal of this thesis was to attempt to establish a potential relationship between ATM and cholesterol homeostasis. As well it was hoped the establishment of a mechanism for such a relationship may help contribute to the establishment of therapies for this debilitating aspect of A-T...
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Science, Environment, Engineering and Technology
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Orientador: Satoshi Kitamura
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: A Doença de Parkinson (DP) é uma das doenças neurodegenerativas mundialmente mais prevalentes. Dentre as doenças do neurônio motor, a Esclerose Lateral Amiotrófica (ELA) é a mais frequente. A qualidade de vida e o prolongamento da expectativa de vida dos sujeitos com doenças neurodegenerativas, como ELA e DP, são foco da intervenção fonoaudiológica, visto que uma das maiores causas de morte são as pneumonias aspirativas. Esta pesquisa teve por objetivo analisar e descrever aspectos relacionados à disfagia e à sua progressão em sujeitos diagnosticados com ELA e DP. Ao todo, participaram 49 sujeitos com ELA e 24 sujeitos com DP. Todos foram avaliados e acompanhados no ambulatório de Otorrinolaringologia/Disfagia do Hospital de Clínicas da Universidade Estadual de Campinas. Foram incluídos no estudo, apenas os sujeitos que estavam em acompanhamento periódico no ambulatório de neurologia do referido hospital e em tratamento medicamentoso. Foram excluídos os sujeitos sem queixa de deglutição ou que apresentassem outras doenças que pudessem causar alteração na deglutição. Todos foram submetidos à entrevista estruturada, videoendoscopia da deglutição, avaliação clínica da deglutição e intervenção fonoaudiológica, além de terem a funcionalidade de ingestão oral classificada pela Functional Oral Intake Scale. Foi realizada uma análise descritiva dos dados com apresentação de frequência das variáveis categóricas e medidas de tendência central e dispersão das variáveis numéricas. Na análise exploratória foram utilizados: Regressão de Cox, teste Exato de Fisher, teste de Kruskal-Wallis, Qui-quadrado, teste de Mann-Whitney e análise de sobrevivência de Kaplan-Meier. As análises foram realizadas por meio do software SPSS versão 13.0 para Windows, tendo sido adotado o nível de significância para os testes estatísticos de 0,05. Na ELA, foi identificado como fator associado à disfagia moderada ou grave, a odinofagia (p=0,01). Foram identificados como fatores que influenciaram na progressão da disfagia na ELA a idade de início da doença (p=0,02) e o início bulbar (p=0,04). A idade de início avançada (p=0,03) e o menor tempo de doença até a primeira avaliação (p=0,004) foram identificados como fatores que levaram à necessidade de indicação de uma via alternativa de alimentação em menor tempo na ELA. Não foram identificados fatores que influenciassem a progressão da disfagia na DP. Observou-se melhora e estabilização da função de deglutição na maioria dos sujeitos com DP estudados. Conclui-se que a idade de início e o início bulbar da ELA são fatores associados à piora rápida da disfagia. Não houve fatores associados à progressão da disfagia na PD e a funcionalidade na deglutição destes pacientes foi caracterizada por melhora e manutenção
Abstract: Parkinson's disease (PD) is one of the most prevalent neurodegenerative diseases worldwide. Among the motor neuron diseases, the Amyotrophic Lateral Sclerosis (ALS) is the most common. The quality of life and longer life expectancy for these individuals with neurodegenerative diseases are the purpose of speech-language therapy, as the leading cause of death are aspirative pneumonias. The objective of this study was to analyze and describe aspects related to dysphagia and its progression in patients diagnosed with ALS and PD. Altogether, 49 patients with ALS and 24 patients with PD participated in the study. All patients were evaluated and followed by at the Otolaryngology/Dysphagia services of the Clinical Hospital of the University of Campinas. The study included only patients who have been regularly monitored at the neurology service and undergoing drug treatment. Patients who had other conditions that could cause changes in swallowing or with no complaints concerning swallowing were excluded. All patients underwent a structured interview, Fiberoptic Endoscopic Evaluation of Swallowing, clinical evaluation of swallowing and swallowing management. Furthermore, they had the swallowing functionality classified by the Functional Oral Intake Scale. We performed a descriptive analysis with presenting the frequency of categorical variables, central measures tendency and dispersion of numerical variables. In exploratory data analysis were applied Cox Regression, Kruskal-Wallis, Chi-square, Mann-Whitney and survival analysis of Kaplan-Meier. The analyses were performed using SPSS version 13.0 for Windows and the significance level for statistical tests was 5%. Odynophagia was identified as an associated factor with moderate or severe dysphagia in ALS. The onset age of ALS (p = 0.02) and the bulbar onset ALS (p = 0.04) were identified as factors that influence the progression of dysphagia in ALS. Advanced onset age (p = 0.03) and shorter disease duration (p = 0.004) were identified as factors that lead to sooner need for non-oral feeding. We did not identify any associated factors with the progression of dysphagia in PD. We noticed an improvement and stabilization of the swallowing function in most patients with PD. We conclude that the onset age and bulbar onset of ALS are factors associated with rapid worsened dysphagia
Doutorado
Epidemiologia
Doutora em Saúde Coletiva

Treballs publicats recentment demostren que el factor de transcripció CCAAT/enhancer binding protein beta (C/ EBPbeta) regula l'activació microglial. Aquests resultats suggereixen que la inhibició de EBPbeta a les cèl.lules de la micròglia podria resultar neuroprotectora en trastorns del SNC on la inflamació crònica resulta ésser patològica. Amb l'objectiu de corroborar aquesta hipòtesi en models animals de neurodegeneració, durant el desenvolupament d'aquesta tesi doctoral s'ha generat la colònia LysMCre-C/ EBPbeta (fl/fl) amb deficiència selectiva de C/EBPbeta a les cèl.lules de la micròglia. Aquests animals són fèrtils, viables i l'estudi histològic no mostra cap alteració als òrgans i teixits, contràriament a allò observat als animals on el factor de transcripció ha estat eliminat a tots els tipus cel.lulars. Cultius primaris de micròglia mostren que la deleció de C/EBPbeta s'ha produït al 100% de les cèl.lules, mentre que l'expressió del factor de transcripció als astròcits no es veu alterada. La deleció de C/EBP8 a les cèl.lules microglials inhibeix la producció de NO alhora que augmenta la capacitat d'aquestes cèl.lules per a fagocitar i eliminar bacteris. A més, la seqüenciació de l'ARN de la micròglia primària mostra una gran afectació del transcriptoma d'aquest tipus cel.lular en resposta als tractaments amb LPS i LPS+IFNy. En aquestes mostres, la deleció de C/EBPbeta afecta l'expressió de 1068 gens involucrats principalment en les respostes inflamatòria i immune, fet que suggereix la implicació del factor de transcripció en aquests processos biològics. Per tal de corroborar aquests resultats en ratolins adults, s'ha aïllat la micròglia dels SNC d'aquests animals. Les dades obtingudes mostren que el 90% de les cèl.lules micròglials no expressen el factor de transcripció, alhora que l'expressió de gens proinflamatoris es veu clarament redu•la. Finalment, l'expressió de C/EBPbeta augmenta marcadament al model animal d'esclerosi múltiple, EAE, per aquesta raó s'ha induït la patologia als animals LysMCre- EBPbeta (fl/fl) ". Aquests ratolins mostren un retard en l'aparició dels primers símptomes, així com una marcada atenuació de la simptomatologia patològica. Així doncs, els resultats obtinguts en aquest treball demostren que C/EBPbeta és un factor clau en la regulació de l'activació microglial i que la seva inhibició resulta ésser una estratègia terapèutica.
Recently published reports demonstrate that the transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) regulates microglia activation. This suggests that microglial C/EBPbeta inhibition could have therapeutical potential in CNS disorders with a pathogenic neuroinflammatory component. To test this hypothesis in animal models of neurodegenerative diseases, we have therefore generated mice with specific microglial C/EBPbeta deficiency, crossing mice with Cre expression under the microglial/macrophage promoter LysM with C/EBPbetaflm mice. Double mutants LysMCre-C/EBPbetaflm were selected. These animals showed normal fertility, survival and histology in contrast to C/EBPbeta deficient mice. In primary microglial cultures, lack of C/EBPbeta was observed in virtually 100% of microglial cells, whereas astrocytes showed normal C/EBPbeta expression. Microglial C/EBPbeta absence resulted in the almost blockade of NO production induced by LPS+IFNy and in an altered bacterial phagocytic function. Furthermore, RNA sequencing of cultured microglia shows dramatic changes in the microglia transcriptome in response to LPS and LPS+IFNy. In these samples C/EBPbeta deletion alters the expression of 1068 mainly involved in immune and inflammatory responses, suggesting and important role of this transcription factor in this biological processes. To corroborate these findings in adult mice, microglia was acutely isolated from the CNS and analyzed. Data revealed a very high (90%) proportion of C/EBPbeta negative microglial cells also in vivo, as well as, a remarkably reduction of proinflammatory genes expression. Finally, because C/EBPbeta was markedly upregulated by experimental autoimmune encephalomyelitis (EAE) in wild-type mice, control and LysMCre-C/EBPbetaflm mice were subjected to EAE. LysMCre-C/EBPbetaflm mice presented a delayed EAE onset and EAE severity was markedly attenuated. Alltogether, these findings support the hypothesis that C/EBPbeta is a key regulator of proinflammatory gene expression in microglial cells and that its inhibition has therapeutical potential.

Protein misfolding diseases represent a large burden to human health for which only symptomatic treatment is generally available. These diseases, such as Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis, and the systemic amyloidoses, are characterized by conversion of globular, nativelyfolded proteins into pathologic β-sheet rich protein aggregates deposited in affected tissues. Understanding the thermodynamic and kinetic details of protein misfolding on a molecular level depends on accurately appraising the free energies of the folded, partially unfolded intermediate, and misfolded protein conformers. There are multiple energetic and entropic contributions to the total free energy, including nonpolar, electrostatic, solvation, and configurational terms. To accurately assess the electrostatic contribution, a method to calculate the spatially-varying dielectric constant in a protein/water system was developed using a generalization of Kirkwood Frohlich theory along with brief all-atom molecular dynamics simulations. This method was combined with previously validated models for nonpolar solvation and configurational entropy in an algorithm to calculate the free energy change on partial unfolding of contiguous protein subsequences. Results were compared with those from a minimal, topologically-based Gō model and direct calculation of free energies by steered all-atom molecular dynamics simulations. This algorithm was applied to understand the early steps in the misfolding mechanism for β₂-microglobulin, prion protein, and superoxide dismutase 1 (SOD1). It was hypothesized that SOD1 misfolding may follow a template-directed mechanism like that discovered previously for prion protein, so misfolding of SOD1 was induced in cell culture by transfection with mutant SOD1 constructs and observed to stably propagate intracellularly and intercellularly much like an infectious prion. A defined minimal assay with recombinant SOD protein demonstrated the sufficiency of mutant SOD1 alone to trigger wtSOD1 misfolding, reminiscent of the “protein-only” hypothesis of prion spread. Finally, protein misfolding as a feature of disease may extend beyond neurodegeneration and amyloid formation to cancer, in which derangement of protein folding quality control may lead to antibodyrecognizable misfolded protein present selectively on cancer cell surfaces. The evidence for this hypothesis and possible therapeutic targets are discussed as a future direction.

The accumulation of misfolded proteins to form inclusion bodies and other protein aggregates outside of cells is a pathological hallmark of neurodegenerative diseases. These aggregating proteins represent potential biomarkers of disease that may facilitate disease diagnosis and the development of therapeutic treatments. Currently neurodegenerative diseases are diagnosed clinically using physical, behavioural and memory based tests along with brain imaging techniques. These methods are associated with variable sensitivity and specificity, and at the point of development of physical symptoms a large amount of neuronal damage has already occurred. Protein biomarkers in easily accessible fluids such as plasma and CSF, can provide additional information to aid in earlier diagnosis, therefore allowing treatments to have a better chance of not only alleviating symptoms but slowing disease progression or perhaps halting disease development.

This thesis aimed to improve the methodology of disease-modification clinical trials in neurodegenerative disorders, with particular reference to Parkinson's disease (PD) and Alzheimer's disease. A systematic review was undertaken to determine what biomarkers for disease progression in PD exist, and whether any have sufficient evidence to be used in clinical trials. Included studies (n=183) were generally of poor quality, being cross-sectional with small numbers of participants, applying excessive inclusion/exclusion criteria, having flawed methodologies and applying simplistic statistical analyses. Insufficient evidence was, therefore, found to recommend the use of any disease progression biomarker in PD clinical trials. A subsequent review in Alzheimer's disease (n=59) demonstrated that these issues were not unique to PD. A 'roadmap' was, therefore, developed to improve future disease progression biomarker studies. The sensitivity to change of a range of PD clinical outcome measures was analysed using data from a follow-up study of an incident cohort of patients with parkinsonism. The MMSE, total UPDRS and PDQ-39 summary index were the most sensitive to change of the continuous outcome measures examined. Amongst binary outcome measures, a new 'dead or dependent' outcome measure was most sensitive to change, and was shown to be a feasible outcome measure for future PD RCTs. Finally, a systematic review was undertaken to examine the validity of differing clinical trial designs used in Alzheimer's disease and PD to demonstrate disease-modification. A variety of design strategies, including wash-in and wash-out analyses, delayed-start designs and long-term follow-up studies, have been used but have methodological limitations. No evidence was found of novel clinical trial designs having been used previously or planned for use in the future. Final recommendations are made that future disease-modification trials should be long-term follow-up studies involving newly diagnosed patients. 'Dead or 'dependent' is highlighted as an efficacious measure to use in such trials.

L'âge, les facteurs de risque vasculaire, l'inactivité physique sont connus comme facteurs de risque des syndromes démentiels.Le sommeil joue un rôle majeur dans la physiologie cérébrale. Le syndrome d'apnée du sommeil (SAS) est une pathologie fréquente chez les sujets âgés, surtout chez ceux souffrant de pathologie démentielle. Les études chez les patients atteints d'hypertension, et/ou de fibrillation atriale montrent une prévalence 2 à 3 fois plus élevée de SAS. Le cerveau et sa vascularisation forment un ensemble avec le coeur et les poumons qui fournissent l'apport en énergie et en oxygène. Une dynamique harmonieuse entre ces trois organes est nécessaire au fonctionnement physiologique du cerveau et un dysfonctionnement de cet ensemble pourrait engendrer une altération cognitive. Ce travail a pour but d'analyser les liens entre l'altération du débit vasculaire de la macro-circulation témoin de l'activité cardiaque, la pulsatilité du liquide céphalospinal reflétant l'hydrodynamique cérébrale, les paramètres respiratoires en lien avec les apnées du sommeil, et le statut cognitif du sujet âgé. En nous basant sur les résultats des bilans clinique, biologique, neuropsychologique, de l'imagerie par résonnance magnétique (morphologie et flux), des holters tensionnel et rythmique dans une population de 95 patients âgés cognitivement altérés, nous avons analysé les relations entre les paramètres concernant le cerveau, le coeur et les poumons. Cette approche révèle une relation entre ces trois systèmes et la cognition. La découverte d'une prévalence de plus de 70% de SAS dans cette population associée à d’autres résultats inattendus devrait faire l'objet de travaux ultérieurs
Age, vascular disorders, and lack of physical activity are known risk factors for dementia syndromes. Sleep has an important role in cerebral physiology. Sleep apnea syndrome (SAS) is common in elderly patients, especially in those with dementia. It was reported that the prevalence of SAS is 2 to 3 times higher in patients with arterial hypertension and/or atrial fibrillation. The brain and its vascular system cannot be considered separately from the heart and the lungs, which provide energy and oxygen supply. Cognitive alterations do not reflect the function of the brain only, and balanced dynamics between all these organs is necessary to maintain neurological functions. Therefore, the aim of this dissertation was to analyze the impact of altered cerebral blood flow in macrocirculation reflecting cardiac activity, pulsatility of the cerebrospinal fluid reflecting cerebral hydrodynamics, and SAS reflecting respiratory function on the cognitive status of elderly patients. Based on a clinical examination, geriatric and neuropsychological assessment, blood tests, structural magnetic resonance imaging, phase-contrast magnetic resonance imaging, 24-hour ambulatory blood pressure measurement, and 24-hour electrocardiogram in the population of 95 elderly patients aged 75 years and older, and suffering from neurodegenerative diseases, we analyzed the correlations between the neurological, cardiac, and respiratory parameters. This approach allowed an identification of associations between the abnormalities in these 3 systems and cognitive function. An unexpected finding, among some other abnormalities, was the prevalence of SAS exceeding 70%, which will be the subject of future research

MicroRNAs (miRNAs) are non-coding small RNAs, which have been found to regulate gene expression at the post-transcriptional and translational levels. A lot of studies demonstrated that miRNAs regulate various cellular processes, including differentiation, development, aging, apoptosis, oncogenesis and metabolism. Moreover, dysregulation of specific miRNAs is associated with a variety of diseases, including neurodegenerative disorders. Identification of differenzial pattern expression of miRNAs could be of value for development of novel biomarkers and discovery of new pharmacological targets for human diseases. The aim of our research was to investigate miRNAs regulation in neurodegenerative diseases. Glaucoma is a progressive optic nerve neuropathy and it is one of the leading cause of blindness in the industrialized countries. Age related macular degeneration (AMD) is the leading cause of blindness among people aged 50 and over. Signs of irreversible neurodegeneration in glaucoma, AMD and Alzheimer s disease (AD), are usually evident at least a decade after onset of disease; thus early diagnosis is an urgent need in order to start effective therapy against neurodegenerative process. Identification of deregulated miRNA and associated pathways common to glaucoma, AMD and AD might help in the challenging search of biomarkers and novel therapeutic strategies. We found, from literature search, 8 deregulated miRNAs in glaucoma, 9 and 23 in AMD and AD, respectively. One miRNA was found to be commonly deregulated in glaucoma and AMD (miR-23a), two miRNA (miR-29a, miR-29b) in glaucoma and AD, and four miRNAs in AMD and AD (miR-9, miR-31, miR-21, miR-34a, miR-146a). Predicted miRNAs common to the three neurodegenerative diseases were 9 (miR-107, miR-137, miR-146a, miR-181c, miR-197, miR-21, miR-22, miR-590, miR-9), which demonstrate to be involved in the regulation of inflammation pathways. Based on prediction of miRNA and associated biochemical pathways, inflammation could represent a therapeutic target common to glaucoma, AMD and AD. Then we evaluated the differential expression profile of miRNAs in a rat model of AMD and in patients with AMD. Analysis of rat retina revealed that miR-27a, miR-146a and miR-155 are up-regulated in comparison to control rats. Seven miRNA (miR-9, miR-23a, miR-27a, miR-34a, miR-126, miR-146a and miR-155) have been found to be dysregulated in serum of AMD patients in comparison to control group. Dysregulated miRNAs, both in the AMD animal model and in AMD patients, can target genes regulating pathways linked to neurodegeneration and inflammation. Our findings support the assessment of specific miRNAs as potential biomarkers and therapeutic targets in retinal neurodegenerative diseases by means of preclinical and clinical studies.

O presente relatório pretende descrever as atividades desenvolvidas durante o estágio curricular de seis meses no Hospital Veterinário do Restelo, referindo a casuística acompanhada e incluindo uma monografia sobre a síndrome de disfunção cognitiva canina (DCC). A área com maior representatividade foi a clínica médica, com 75,23% do total de casos acompanhados. A DCC é uma síndrome neurodegenerativa, de etiologia multifatorial, que afeta cães, sobretudo a partir dos 11 anos, estando muitas vezes associada a outras afeções, o que pode dificultar o seu diagnóstico. Os sinais clínicos seguem a sigla DISHA, do inglês disorientation (desorientação), interaction (interação), sleep (sono), house soiling (eliminação errática) e activity (atividade). É necessário excluir outras patologias que possam assumir uma sintomatologia semelhante para se poder estabelecer o diagnóstico de DCC. A terapêutica disponível não reverte os sinais clínicos, pelo que o diagnóstico precoce se torna particularmente importante; Abstract: Small animal clinics The present report aims to describe the activities developed during the six-month period traineeship at Hospital Veterinário de Restelo, referring the accompanied casuistry and including a monography about the canine cognitive dysfunction syndrome (DCC). The most represented field was medical clinic, with 75,23% from the total cases. The DCC is a neurodegenerative syndrome, with multifactorial aetiology, that affects dogs mainly above 11 years old. It is often associated to other affections, which may difficult the diagnosis. The clinical signs follow the acronym DISHA, disorientation, sleep, house soiling and activity. It is necessary to exclude other pathologies that may assume a similar symptomatology to establish the DCC diagnosis. The available therapeutics do not revert the clinical signs, making the early diagnosis particularly important.

Spinal bulbar muscular atrophy (SBMA) is a member of the polyglutamine (polyQ) expansion diseases family; the most famous of which is Huntington disease (HD). SBMA is caused by the expansion of the coding region for the polyQ tract in the exon 1 of androgen receptor (AR), which represents the N-terminal intrinsically disordered transactivation domain (NTD). AR is the nuclear receptor sensible to testosterone and aggregates of this polyQ-expanded protein are observed in the motor neurons of SBMA patients. The aggregation mechanism of polyQ proteins depends both on the length of the tract and on the chemical properties of the regions flanking it, that can increase or decrease the rate of aggregation depending on their secondary structure. In order to study the structure of the polyQ tract in AR and the mechanism by which this protein forms aggregates, we developed recombinant proteins designed over the N-terminal fragment of cleavage of a caspase (Caspase 3) associated to the onset of the toxicity in SBMA. We also developed a set of biophysical tools for rendering these aggregation-prone proteins monomeric and to monitor their evolution from the monomer level to the fibril. These methodologically challenging endeavor allowed us to study the secondary structure of this intrinsically disordered protein as a monomer and then to monitor what regions are important in its oligomerization and aggregation. Bulk biophysical experiments and NMR indicated that the polyQ tract of AR is in α-helical conformation, unlike other polyQ tracts described in literature, and we demonstrated that this conformation is caused by the nucleating effect of an N-terminal flanking sequence of four Leu residues (54LLLL58). We also showed that the helical conformation of this tract prevents the polyQ to acquire the ß-sheet conformation and to progress as a fibril, as a deletion mutant of the 54LLLL58 motif aggregates and forms fibrils faster than the wild type. By measuring the aggregation rates of three different AR recombinant proteins with progressively higher polyQ length (4Q, 25Q and 51Q) emerged that the polyQ is not the only region responsible for oligomerization and we identified by NMR that a second region, N- terminal and far apart from the polyQ is responsible of the early oligomerization. By analysis of the chemical shifts in different NMR experiments we obtained that this region (23FQNLF27) however not entirely helical, is prone to interact and acquire secondary structure. Furthermore, this sequence is known to bind to the ligand binding domain (LBD) of AR in an interaction critical for its dimerization and subsequent translocation into the nucleus, which is called N/C interaction. The crystal structure of this complex shows 23FQNLF27 in α-helical conformation when bound to LBD. We then investigated what amino-acids were important in the interaction stabilizing the intereaction of 23FQNLF27. By mutational analysis and measurements of aggregation rates we demonstrated that the helicity of this region is important for the aggregation and mutations that increase the helicity also an increase the aggregation propensity of the protein. We also identified that the residues responsible for the contact are the Gln in position 2, 28 and 36 which form a ‘spine’ of polar residues in register along the α-helix. This polar side of the helix is not the one in contact with LBD during the N/C interaction and it is possible that the two events occur in parallel. In the complex, we characterized the early oligomerization of AR in the aggregation process associated to SBMA with the perspective to provide valuable information for the development of drugs for this diseases that has currently no treatment.
Les malalties neurodegeneratives són una de les malediccions de la civilització moderna i es troben estretament lligades a l’augment de l’esperança de vida de la població mundial. La majoria d’aquestes malalties estan associades a la deposició de material proteic, altrament conegut com a fibres amiloides, a les neurones i el cervell en general. Les fibres amiloides són conjunts supramoleculars lineals, composats per proteïnes disposades en fulla beta, que mostren una alta rigidesa i estabilitat termodinàmica. Exemples famosos de proteïnes amiloides són la beta amiloide (Aβ), associada a la malaltia d’Alzheimer, i l’α-­‐sinucleïna i la proteïna tau, més estretament lligades a la malaltia de Parkinson. Una altra família de desordres neurodegeneratius associats a la deposició de proteïnes és la de les malalties poliglutamines (poliQ). Aquesta família està formada per nou patologies, entre les que es troben sis atàxies espinocerebrals diferents (de les sigles en anglès, SCA 1, 2, 3, 6, 7, 17), la atròfia dentatorubral-­‐pallidoluysian (de les sigles en anglès, DRPLA) i la atròfia muscular espinal bulbar (de les sigles en anglès, SBMA), històricament la primera en ser descrita. Totes elles són hereditàries, dominants i es manifesten en edat avançada. D’altra banda, totes elles estan associades a l’adquisició de neurotoxicitat degut a l’agregació de la proteïna causant de la malaltia, que s’acumula progressivament a les neurones amb el temps. La mutació responsable de la malaltia és una expansió genètica a la regió polimòrfica de l’ADN que és comuna a totes le proteïnes associades en aquests enfermetats. Aquesta regió polimòrfica és un conjunt de repeticions CAG que codifiquen l’aminoàcid glutamina a nivell d’expressió de proteïna, és per això que es coneix com a tram de poliglutamines. Aquest tram pot tenir diverses longituds, però l’efecte tòxic només té lloc quan es supera un determinat límit d’allargada. Aquest límit fluctua entre 30 i 40 repeticions i varia de malaltia a malaltia, però en tots els casos el número de repeticions influencia la severitat i l’edat en la que s’inicia la malaltia. La raó que explica aquesta inestabilitat genètica resulta de la propensitat de les seqüències d’ADN altament repetitives (com ara els hairpins) que en determina el slippage de la cadena principal durant la replicació de l’ADN. Les expansions més llargues són causades per la reiteració d’aquesta petita mutació i s’ha observat una reducció progressiva de l’estabilitat genètica amb l’increment del número de repeticions, que en última instància determina un avançament temporal i empitjorament dels símptomes. Considerant l’estreta relació entre la presència d’agregats en els teixits dels pacients malalts i l’estadiatge de la malaltia, és fonamental entendre les propietats biofísiques dels trams de poliQ, com aquestes seqüències determinen l’agregació de la proteïna i el tipus d’estructura que presenten els agregats.

El terme cossos de poliglucosà (PGBs) fa referència a agregats complexos formats per polímers de glucosa que arriben a fer diàmetres de desenes de micròmetres. Aquestes estructures s’han observat en el sistema nerviós central, però també en altres òrgans i teixits, com ara al cor, al múscul esquelètic i al fetge. Els PGBs han estat descrits en humans i altres espècies, però sobretot s’han estudiat més àmpliament en els mamífers. Diferents formes de PGBs estan relacionades amb determinades situacions o malalties. Durant el procés d'envelliment, el cervell humà acumula un tipus de PGBs anomenat cossos amilacis o corpora amylacea (CA). Encara que els CA han estat estudiats durant diversos anys, el seu origen i la seva funció encara romanen incerts. Els PGBs també es troben associats a la malaltia de Lafora, un trastorn neurodegeneratiu que es caracteritza per la presència d'estructures de tipus de poliglucosà denominades cossos de Lafora (LBs). D'altra banda, l'envelliment cerebral en ratolins condueix a l’aparició i l’expansió progressiva d’uns PGBs granulars degeneratius anomenats grànuls periodic acid Schiff (PAS). Aquests grànuls, que es troben principalment a l'hipocamp, s'originen en processos astrocítics i solen agrupar-se formant clústers. Cada clúster correspon al conjunt de grànuls d'un astròcit determinat. Recentment, el nostre grup de recerca ha descrit la presència d’uns neo-epítops en aquestes estructures que són reconeguts per IgMs. Aquestes IgMs, que es troben com a contaminants en alguns anticossos comercials, són les responsables de produir falsos marcatges positius d’aquests cossos quan s'utilitzen procediments immunohistoquímics. Aquesta tesi pretén estudiar l'origen, la composició i la funció dels PGBs que apareixen amb l'edat i en condicions neurodegeneratives, com la malaltia d'Alzheimer i la malaltia de Lafora. Els resultats obtinguts mostren que els CA són estructures similars als grànuls PAS de ratolí i que els CA també contenen neo-epítops. En ambdós casos, els neo-epítops poden ser reconeguts per IgMs naturals, fet que suggereix una nova relació entre els PGBs i el sistema immunitari innat. A més a més, els falsos marcatges positius que es produeixen degut a les IgMs naturals que es troben com a contaminants en alguns anticossos comercials aclareixen alguns resultats controvertits en quant a la presència d'alguns components descrits prèviament als CA. També s’ha determinat que els CA acumulen productes no degradables generats durant el procés d'envelliment i augmentats en malalties neurodegeneratives, com per exemple la malaltia d'Alzheimer. Tenint en compte aquests resultats, es planteja la hipòtesi que els CA són uns elements encarregats de l’eliminació de productes perjudicials o residuals a través de l'acció del sistema immunitari innat. D'altra banda, s’ha observat que els ratolins deficients de malina, un model de la malaltia de Lafora, presenten un elevat nombre de dos tipus diferents de PGBs: els grànuls PAS o CA-like, originats en astròcits i que contenen neo-epítops i els neuronal LBs, que són exclusius de la malaltia de Lafora i no contenen neo-epítops. Així doncs, l'absència de malina provoca la formació de PGBs a les neurones, però també augmenta el seu desenvolupament en astròcits. Postulem, en contra el que s’havia descrit prèviament, que els astròcits podrien intervenir en l'etiopatogènesi de la malaltia de Lafora. En global, els resultats obtinguts mostren que, fins ara, els diferents PGBs han estat estudiats amb una perspectiva específica i limitada. Una imatge global o principal és necessària per obtenir el coneixement de la seva importància fisiopatològica.
The term polyglucosan bodies (PGBs) refers to complex aggregates composed of relatively large glucose polymers reaching diameters of tens of micrometres. PGBs have been reported in the central nervous system, but also in other organs and tissues. During the ageing process, human brain accumulates one type of PGBs called corpora amylacea (CA). Although CA have been studied for several years, their origin and function remain unclear. PGBs are also associated with Lafora disease, a neurodegenerative condition that is characterized by the presence of PGBs structures called Lafora bodies (LBs). On the other hand, brain ageing in mice leads to the progressive appearance and expansion of degenerative granular PGBs frequently referred to as Periodic Acid Schiff (PAS) granules. These granules, which are present mainly in the hippocampus, originate in astrocytes processes and tend to appear in clusters. Recently, our research group have reported the presence of neo-epitopes on these structures, responsible of numerous false positive staining on these bodies when immunohistochemical procedures are used. This thesis aimed to study the origin, composition and function of PGBs that appear with age and in neurodegenerative conditions such as Alzheimer’s disease and Lafora disease, focusing on the possible presence of neo-epitopes on these structures. The results obtained in this thesis show, firstly, that CA are structures similar to PAS granules from mice brain and that CA also contain neo-epitopes. In both cases, the neo-epitopes can be recognized by natural IgM antibodies, suggesting a new relation between PGBs and the natural immune system. This fact explains the controversial results previously described about the presence of some components in CA. In this work, some of these components have been discarded. Secondly, malin deficient mice, a mouse model of Lafora disease, present distinct types of PGBs: PAS granules or CA-like granules, originated in astrocytes, and neuronal LBs. This last type of PGBs is specific of this condition and does not contain neo-epitopes. Overall results suggest that CA are PGBs related to the aggregation of non-degradable products produced during ageing process and increased in neurodegenerative conditions, whereas LBs are pathogenic structures responsible of the neurodegeneration observed in Lafora disease.