Macroautophagy (hereafter referred to as autophagy) is a highly conserved cellular process that promotes cytoplasmic homeostasis via lysosomal degradation of proteins and organelles. Dysfunctional autophagy occurs in numerous human pathologies, including neurodegeneration and cancer. Vinexin (encoded by SORBS3) is a physiologically important adaptor protein for two main reasons: 1. SORSB3 mRNA expression increases in normal human brain ageing, 2. SORBS3 is a candidate tumour suppressor in hepatocellular carcinoma (HCC). This dissertation builds on published data from an siRNA screen for autophagy regulations under basal conditions, which indicates vinexin knockdown upregulates autophagy. I replicate this finding in multiple cell lines, before characterising the impact of siSORBS3 treatment on autophagy; autophagosome biogenesis is increased, while flux through the autophagy pathway remains intact. Having excluded several possible mechanisms suggested by the literature, I focus on the transcriptional coactivators YAP and TAZ. The rationale here is: 1. YAP/TAZ activity is implicated in autophagy, 2. YAP/TAZ and vinexin are both linked to HCC. My data show that YAP/TAZ transcriptional activity is upregulated upon vinexin depletion. Moreover, increased autophagy following siSORBS3 treatment requires YAP and TAZ. A key focus of this dissertation is the mechanism by which vinexin knockdown upregulates YAP/TAZ and hence, autophagy. This centres on altered actin cytoskeleton dynamics; an increase in F-actin structures appears to compete with YAP/TAZ for binding to angiomotins, established sequesterers of YAP/TAZ in the cytosol. In this way, siSORBS3 treatment facilitates YAP/TAZ nuclear localisation and consequent transcriptional activity. Angiomotin overexpression therefore ameliorates the increase in autophagy caused by vinexin depletion. Published RNA sequencing data is used to confirm that SORBS3 mRNA expression increases in normal brain ageing, not only in the frontal cortex (as previously published), but also in the hippocampus. This sits alongside decreased expression of core autophagy genes in both tissues. Accordingly, vinexin could contribute to the decline in autophagic potential thought to occur in normal brain ageing. With regards to SORBS3 as a candidate tumour suppressor in HCC, I show that stably re-expressing vinexin in a HCC cell line downregulates YAP/TAZ and hence, autophagy. These cells also show reduced clonogenicity. My data therefore support the hypothesis that SORBS3 is a tumour suppressor in HCC; YAP and TAZ are well-known to increase proliferation and resistance to apoptosis, while autophagy can enable tumour cells to survive stressors such as nutrient starvation. The conclusions of this dissertation are that vinexin depletion upregulates autophagy in a YAP/TAZ-dependent manner and that this has physiologically important implications, especially with regards to HCC.

Human brain structure can be measured across the lifecourse (“in vivo”) with magnetic resonance imaging (MRI). MRI data are often used to create “atlases” and statistical models of brain structure across the lifecourse. These methods may define how brain structure changes through life and support diagnoses of increasingly common, yet still fatal, age-related neurodegenerative diseases. As diseases such as Alzheimer’s (AD) cast an ever growing shadow over our ageing population, it is vitally important to robustly define changes which are normal for age and those which are pathological. This work therefore assessed existing MR brain image data, atlases, and statistical models. These assessments led me to propose novel methods for accurately defining the distributions and boundaries of normal ageing and pathological brain structure. A systematic review found that there were fewer than 100 appropriately tested normal subjects aged ≥60 years openly available worldwide. These subjects did not have the range of MRI sequences required to effectively characterise the features of brain ageing. The majority of brain image atlases identified in this review were found to contain data from few or no subjects aged ≥60 years and were in a limited range of MRI sequences. All of these atlases were created with parametric (mean-based) statistics that require the assumptions of equal variance and Gaussian distributions. When these assumptions are not met, mean-based atlases and models may not well represent the distributions and boundaries of brain structure. I tested these assumptions and found that they were not met in whole brain, subregional, and voxel-based models of ~580 subjects from across the lifecourse (0- 90 years). I then implemented novel whole brain, subregional, and voxel-based statistics, e.g. percentile rank atlases and nonparametric effect size estimates. The equivalent parametric statistics led to errors in classification and inflated effects by up to 45% in normal ageing-AD comparisons. I conclude that more MR brain image data, age appropriate atlases, and nonparametric statistical models are needed to define the true limits of normal brain structure. Accurate definition of these limits will ultimately improve diagnoses, treatment, and outcome of neurodegenerative disease.

The Whitehall II (Stress and Health) study is a prospective study based on originally 10,308 British civil servants. Since 1985, rich socio-demographic, health and life-style measures have been acquired every 2-5 years. Eight hundred participants were randomly selected from the remaining 6,308 participants of Phase 11 for magnetic resonance imaging (MRI) and further examination in Oxford (2012-14). There is evidence of changes in brain anatomy and mental health following traumatic events, such as combat stress or sexual abuse, in patients with posttraumatic stress disorder, mood or personality disorders, but little is known about the association between everyday stress, brain structure and function, and mental health in the general population. The secondary stress markers, Allostatic Load (AL) index, Framingham Stroke Risk score (FSRS) and Metabolic Syndrome (MetS) were selected as potential predictors of brain changes at follow-up. This thesis focuses on measures of structural grey and white matter (GM, WM) integrity, collected with the Siemens 3T Verio scanner in the first 563 participants of the Oxford study. As hypothesised, stress markers measured as early as 20 years prior to the scan, predicted lower GM and WM integrity in older age. Unique linear relationships remained even after controlling for socio-demographic confounders between each stress marker and GM density but not with measures of WM integrity. This suggests that some, if not most variance is shared between the stress markers and the usual correlates of general health, such as age and employment class. The three markers did not have equal power to predict brain measures: AL added more unique predictive variance to GM density than MetS and FSRS. On the other hand, FSRS was the more powerful predictor of poor WM integrity compared with AL and MetS, and after removing confounding variable effects. This thesis thus provides some empirical support for the concept of allostatic load, linking 'everyday' stress and features of the ageing human brain.

Lipids are important for structural and physiological functions of neuronal cell membranes. They exhibit a range of biological effects many are bioactive lipid mediators derived from polyunsaturated fatty acids such as sphingolipids, fatty acid ethanolamides (FA-EA) and endocannabinoids (EC). These lipid mediators and oxysterols elicit potent bioactive functions in many physiological and pathological processes of the brain and neuronal tissues. They have been investigated for biomarker discovery of ageing, neuroinflammation and neurodegenerative disorders. The n-3 fatty acids EPA and DPA are thought to exhibit a range of neuroprotective effects many of which are mediated through production of such lipid mediators. The aims of this study were to evaluate the effects of n-3 EPA and n-3 DPA supplementation on RBC membranes and in this way assess dietary compliance and to investigate brain sphingomyelin species of adult and aged rats supplemented with n-3 EPA and n-3 DPA to evaluate the effects and benefits on age-related changes in the brain. Furthermore, to study the effects of lithium on the brain FA-EAs and ECs to further understand the neuroprotective effects of lithium neuroprotective action on neuroinflammation as induced by LPS. Finally to examine if circulating oxysterols are linked to the prevalence of ALS and whether RBC fatty acids are markers of this action in relation to age and disease stages. These analytes were extracted from tissue samples and analysed with GC, LC/ESI-MS/MS and GC-MS. It was found that aged rats exhibited a significant increase in brain AA and decrease in Σn-3 and Σn-6 PUFAs when compared to adult animals. The observed increase of brain AA was reversed following n-3 EPA and n-3 DPA supplementation. Sphingomyelin was significantly increased when aged animals were supplemented with n-3 DPA. LPS treatment following lithium supplementation increased LA-EA and ALA-EA, while it decreased DHA-EA. Both oxysterols 24-OH and 27-OH increased in ALS patients and SOD1-mice. Eicosadienoic acid was different in ASL-patients compared to aged SOD1-mice. These studies demonstrated that dietary intake of n-3 EPA and n-3DPA significantly altered RBC fatty acids and sphingolipids in rat brain. They suggest that n-3 DPA can be a potential storage form for EPA, as shown by retro-conversion of n-3 DPA into EPA in erythrocyte membranes, ensuring supply of n-3 EPA. Also, n-3 EPA and n-3 DPA supplementation can contribute to an increase in brain sphingomyelin species with implications for age effects and regulation of brain development. Effects of lithium highlight novel anti-neuroinflammatory treatment pathways. Both 24-hydroxycholesterol and eicosadienoic acid may be used as biomarkers in ALS thereby possibly helping to manage the progressive stages of disease.

Lipids are important for structural and physiological functions of neuronal cell membranes. They exhibit a range of biological effects many are bioactive lipid mediators derived from polyunsaturated fatty acids such as sphingolipids, fatty acid ethanolamides (FA-EA) and endocannabinoids (EC). These lipid mediators and oxysterols elicit potent bioactive functions in many physiological and pathological processes of the brain and neuronal tissues. They have been investigated for biomarker discovery of ageing, neuroinflammation and neurodegenerative disorders. The n-3 fatty acids EPA and DPA are thought to exhibit a range of neuroprotective effects many of which are mediated through production of such lipid mediators. The aims of this study were to evaluate the effects of n-3 EPA and n-3 DPA supplementation on RBC membranes and in this way assess dietary compliance and to investigate brain sphingomyelin species of adult and aged rats supplemented with n-3 EPA and n-3 DPA to evaluate the effects and benefits on age-related changes in the brain. Furthermore, to study the effects of lithium on the brain FA-EAs and ECs to further understand the neuroprotective effects of lithium neuroprotective action on neuroinflammation as induced by LPS. Finally to examine if circulating oxysterols are linked to the prevalence of ALS and whether RBC fatty acids are markers of this action in relation to age and disease stages. These analytes were extracted from tissue samples and analysed with GC, LC/ESI-MS/MS and GC-MS. It was found that aged rats exhibited a significant increase in brain AA and decrease in ¿n-3 and ¿n-6 PUFAs when compared to adult animals. The observed increase of brain AA was reversed following n-3 EPA and n-3 DPA supplementation. Sphingomyelin was significantly increased when aged animals were supplemented with n-3 DPA. LPS treatment following lithium supplementation increased LA-EA and ALA-EA, while it decreased DHA-EA. Both oxysterols 24-OH and 27-OH increased in ALS patients and SOD1-mice. Eicosadienoic acid was different in ASL-patients compared to aged SOD1-mice. These studies demonstrated that dietary intake of n-3 EPA and n-3DPA significantly altered RBC fatty acids and sphingolipids in rat brain. They suggest that n-3 DPA can be a potential storage form for EPA, as shown by retro-conversion of n-3 DPA into EPA in erythrocyte membranes, ensuring supply of n-3 EPA. Also, n-3 EPA and n-3 DPA supplementation can contribute to an increase in brain sphingomyelin species with implications for age effects and regulation of brain development. Effects of lithium highlight novel anti-neuroinflammatory treatment pathways. Both 24-hydroxycholesterol and eicosadienoic acid may be used as biomarkers in ALS thereby possibly helping to manage the progressive stages of disease. / Libyan Government

[ES] El envejecimiento se concibe como un proceso progresivo de deterioro funcional que se produce a lo largo de la vida. En general, el avance de la edad va acompañado del deterioro de múltiples tejidos, la alteración de la homeostasis y el aumento de la fragilidad. La edad se considera el principal factor de riesgo de un gran número de enfermedades con elevadas tasas de mortalidad, como las patologías cardiovasculares, el cáncer, la fibrosis pulmonar, la esteatosis hepática y los trastornos neurodegenerativos, entre otros. A medida que aumenta la población de edad avanzada, estas patologías son cada vez más frecuentes en nuestra sociedad y constituyen actualmente una de las principales preocupaciones sanitarias en atención primaria. Por consiguiente, comprender los mecanismos que causan el envejecimiento y el consiguiente deterioro de la salud es un reto importante para la comunidad científica, respaldado por un significativo interés socioeconómico. Alcanzar este objetivo permitiría realizar intervenciones específicas contra las enfermedades relacionadas con la edad, promover un envejecimiento saludable y prolongar la vida humana. En este contexto, la senescencia celular se ha convertido en un importante objetivo de investigación, ya que este destino celular se considera un factor que contribuye al deterioro funcional y estructural de los tejidos con el aumento de la edad, dando lugar a enfermedades crónicas. Los trastornos neurodegenerativos son en su mayoría de etiología desconocida, y en la actualidad sólo se dispone de tratamientos paliativos. La neurodegeneración puede deberse a la disminución de la neurogénesis que se produce con el envejecimiento. A este respecto, la vasculatura cerebral representa un componente esencial de los nichos neurogénicos, donde residen las células con capacidad para generar nuevas neuronas. Los microvasos cerebrales contribuyen sustancialmente a preservar la homeostasis y el potencial neurogénico de estas regiones, pero también experimentan cambios estructurales y funcionales con la edad. Sin embargo, aún no está claro si estas alteraciones están relacionadas con la senescencia celular. De ahí que el propósito de esta tesis sea profundizar en los entresijos de la senescencia relacionada con la edad y su impacto en el proceso de envejecimiento cerebral. En este sentido, hemos desarrollado una sonda fluorogénica para la detección in vivo de la actividad ß-Gal, un marcador ampliamente utilizado de senescencia celular. Hemos estimado la carga de células senescentes de forma no invasiva en estudios longitudinales utilizando esta sonda durante el envejecimiento natural y acelerado o durante tratamientos senolíticos. Estos resultados nos han permitido establecer una correlación real entre envejecimiento y senescencia. Por otro lado, hemos centrado nuestros esfuerzos en crear estrategias para detectar la senescencia en las células endoteliales cerebrales y estudiar sus efectos sobre la neurogénesis durante el envejecimiento. Los resultados mostraron que las células endoteliales senescentes a nivel cerebral tienen un impacto perjudicial sobre la neurogénesis y las células madre neurales, lo que sitúa a estas células como diana para futuros estudios sobre el envejecimiento cerebral. / [CA] L'envelliment es concep com un procés progressiu de deterioració funcional que es produeix al llarg de la vida. En general, l'avanç de l'edat va acompanyat de la deterioració de múltiples teixits, l'alteració de l'homeòstasi i l'augment de la fragilitat. L'edat es considera el principal factor de risc d'un gran nombre de malalties amb elevades taxes de mortalitat, com les patologies cardiovasculars, el càncer, la fibrosi pulmonar, la esteatosis hepàtica i els trastorns neurodegeneratius, entre altres. A mesura que augmenta la població d'edat avançada, aquestes patologies són cada vegada més freqüents en la nostra societat i constitueixen actualment les principals preocupacions sanitàries en atenció primària. Per consegüent, comprendre els mecanismes que causen l'envelliment i la consegüent deterioració de la salut és un repte important per a la comunitat científica, recolzat per un significatiu interés socioeconòmic. Aconseguir aquest objectiu permetria realitzar intervencions específiques contra les malalties relacionades amb l'edat, promoure un envelliment saludable i prolongar la vida humana. En aquest context, la senescència cel·lular s'ha convertit en un important objectiu d'investigació, ja que aquest destí cel·lular es considera un factor que contribueix a la deterioració funcional i estructural dels teixits amb l'augment de l'edat, donant lloc a malalties cròniques. Els trastorns neurodegeneratius són en la seua majoria d'etiologia desconeguda, i en l'actualitat només es disposa de tractaments pal·liatius. La neurodegeneración pot deure's a la disminució de la neurogènesi que es produeix amb l'envelliment. Referent a això, la vasculatura cerebral representa un component essencial dels nínxols neurogènics, on resideixen les cèl·lules amb capacitat per a generar noves neurones. Els microvasos cerebrals contribueixen substancialment a preservar l'homeòstasi i el potencial neurogènic d'aquestes regions, però també experimenten canvis estructurals i funcionals amb l'edat. No obstant això, encara no és clar si aquestes alteracions estan relacionades amb la senescència cel·lular. D'aquí ve que el propòsit d'aquesta tesi siga aprofundir en els secrets de la senescència relacionada amb l'edat i el seu impacte en el procés d'envelliment cerebral. En aquest sentit, hem desenvolupat una sonda fluorogénica per a la detecció in vivo de l'activitat ß-Gal, un marcador àmpliament utilitzat de senescència cel·lular. Hem estimat la càrrega de cèl·lules senescentes de forma no invasiva en estudis longitudinals utilitzant aquesta sonda durant l'envelliment natural i accelerat o durant tractaments senolíticos. Aquests resultats ens han permés establir una correlació real entre envelliment i senescència. D'altra banda, hem centrat els nostres esforços a crear estratègies per a detectar la senescència en les cèl·lules endotelials cerebrals i estudiar els seus efectes sobre la neurogènesi durant l'envelliment. Els resultats van mostrar que les cèl·lules endotelials *senescentes a nivell cerebral tenen un impacte perjudicial sobre la neurogènesi i les cèl·lules mare *neurales, la qual cosa situa a aquestes cèl·lules com a diana per a futurs estudis sobre l'envelliment cerebral. / [EN] Ageing is conceived as a progressive process of functional decline that occurs over the course of life. In general, advancing age is accompanied by the deterioration of multiple tissues, altered homeostasis and increased frailty. Age is considered to be the main risk factor for a large number of diseases with high mortality rates, such as cardiovascular pathologies, cancer, pulmonary fibrosis, hepatic steatosis, and neurodegenerative disorders, among others. As the elderly population grows, these pathologies are becoming increasingly prevalent in our society and are now the leading health concerns in primary care. Consequently, comprehending the mechanisms that cause ageing and resulting health decline is a major challenge for the scientific community, backed by significant socio-economic interest. Achieving this goal would allow for targeted interventions against age-related diseases, promote healthy ageing, and extend human lifespan. In this context, cellular senescence has emerged as an important research target, as this cellular fate is considered a contributing factor to the functional and structural deterioration of tissues with increasing age, leading to chronic diseases. Neurodegenerative disorders are mostly of unknown etiology, and only palliative treatments are currently available. Neurodegeneration may be prompted by the decline in neurogenesis that occurs with ageing. In this regard, the brain vasculature represents an essential component of the neurogenic niches, where cells with the capacity to generate new neurons reside. Brain microvessels contribute substantially to preserving the homeostasis and neurogenic potential of these regions, but they also undergo structural and functional changes with age. However, whether these alterations are linked to cellular senescence it is not yet clear. Hence, the purpose of this thesis is to delve deeper into the intricacies of age-related senescence and its impact on the process of brain ageing. In this regard, we have developed a fluorogenic probe for the in vivo detection of ß-Gal activity, a widely used marker of cellular senescence. We have estimated the senescent cell burden non-invasively in longitudinal studies using this probe during natural and accelerated ageing or during senolytic treatments. These results have allowed us to establish a real correlation between ageing and global senescence. On the other hand, we have focused our efforts on creating strategies to detect senescence in brain endothelial cells and to study its effects on neurogenesis during ageing. The results showed that senescent endothelial cells at the brain level have a detrimental impact on neurogenesis and neural stem cells, positioning these cells as a target for future studies on brain ageing. / This PhD thesis has been supported by a pre-doctoral scholarship from the Spanish Ministry of Universities, “Programa de Formación del Profesorado Universitario (FPU)”, and a technical expert contract funded by the European Commission. The research has been funded by the following research projects led by Ramón Mártinez Máñez: - Gobierno de España (RTI2018-100910-B-C41 and PID2021-126304OB-C41). - Generalitat Valenciana (PROMETEO 2018/024 and CIPROM/2021/007). - CIBER-BBN- Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (CB06/01/2012). And by the following research projects led by Isabel Fariñas: - 2014-2018. Estudio de células madre en el ámbito de las investigaciones básicas en terapia celular. Fundación Botín-Banco de Santander. - 2018-2021. Regulación del comportamiento de las células madre neurales por el medio sistémico: el nicho extendido. MINECO (SAF2017-86690-R). - 2016-ongoing. CIBER en Enfermedades Neurodegenerativas (CIBERNED). ISCIII (Programa de Investigación Cooperativa, CB06/05/0086). - 2017-2021. Efectos directos y remotos de la respuesta inflamatoria sobre las células madre neurales. Generalitat Valenciana (Proyectos de Excelencia, PROMETEO/2017/030). - 2017-2021. RETIC de terapia celular ISCIII (RD16/0011/0017). - 2021-2024 Regulación molecular de la heterogeneidad celular en los nichos neurogénicos adultos MICINN (PID2020- 117937GB-I00). - 2021-2025. Una aproximación multidisciplinar al estudio de la respuesta al daño genómico en células madre neurales: de levadura a mamíferos y vuelta. Generalitat Valenciana. Programa Prometeo de Proyectos de Excelencia (PROMETEO/2021/028). / Rojas Vázquez, S. (2023). Impact of Brain Endothelial Senescence on Neurogenesis: Application of Novel Strategies for Age-Related Senescence Detection [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/194629

The physical complications of Type 1 diabetes mellitus (T1DM) have been understood as an accelerated ageing process (Morley, 2008). Do people with T1DM also experience accelerated cognitive and brain ageing? Using findings from research of the normal cognitive and brain ageing process and conceptualized in theories of the functional brain changes in cognitive ageing, a combination of cognitive testing and functional magnetic resonance imaging (fMRI) techniques were used to evaluate evidence of accelerated cognitive and brain ageing in middle-aged adults with T1DM. The first part of this thesis comprises a cognitive study of 94 adults (≥ 45 years of age) with long duration (≥ 10 years) of T1DM. Participants completed cognitive assessment and questionnaires on general mood and feelings about living with diabetes. Findings highlighted the importance of microvascular disease (specifically retinopathy) as an independent predictor of cognitive function. The incidence and predictors of mild cognitive impairment (MCI) were then explored. Results indicate a higher percentage of the group met criteria for MCI than expected based on incidence rates in the general population, providing initial evidence of accelerated cognitive ageing. Psychological factors were explored next. The relationship between the measures of well-being, diabetes health, and cognitive function highlighted the need for attention to patient's psychological well-being in diabetes care. Finally, a subgroup of 30 participants between the ages of 45 and 65 who differed on severity of retinopathy were selected to take part in an fMRI study. Blood oxygen level dependent (BOLD) activity was evaluated while participants were engaged in cognitive tasks and during rest. The findings provided evidence that the pattern of BOLD activation and functional connectivity for those with high severity of retinopathy are similar to patterns found in adults over the age of 65. In line with the theories of cognitive ageing, functional brain changes appear to maintain a level of cognitive function. Evidence of accelerated brain ageing in this primarily middle-aged group, emphasizes the importance of treatments and regimens to prevent or minimize microvascular complications.

Durant l'envelliment es dónen canvis estructurals i funcionals al cervell, especialment a l'escorça prefrontal, un dels substractes anatòmics responsables del control atencional. Aquest es va mesurar emprant tècniques neuropsicològioques i neurofuncionals durant l'execució de tasques de canvi amb senyals implícites (tipus WCST). 80 subjectes majors sans es varen dividir segons la seva edat i el seu nivell de control cognitiu. El baix control cognitiu (però no l'edat) s'associà a un augment dels costos residuals de resposta, en paral.lel amb una major amplitud del component P2 davant els senyals. L'edad avançada, en conjunt amb un baix nivell de control s'associà a un increment dels costos locals de resposta durant el canvi de tasca, paral.lelament amb l'augment de les ones lentes durant la fase de senyalització. Mantenir dues tasques en memòria es més difícil per als subectes amb baix control, reflexat per una reducció en l'amplitud de les ones lentes fronto-parietals