Analysis of Reelin function in the molecular mechanisms underlying Alzheimer’s disease

Abstract

Alzheimer’s disease (AD) is the most common cause of dementia in the elderly, with more than 25 million people affected worldwide. First described in 1907 by the German physician Alois Alzheimer, AD is a neurodegenerative disease characterized by a dramatic progressive loss of synapses and neuronal populations, initially affecting medial temporal lobe structures and finally resulting in diffuse cortical atrophy. The earliest clinical symptoms are episodic memory loss, especially in remembering new items (anterograde amnesia). As the disease progresses, amnesia occurs in conjunction with major executive dysfunctions, such as impairment of language (aphasia), object use (apraxia), recognition of faces or objects (agnosia), abstract reasoning, step-by-step planning, and decision making. This gradual erosion of cognition slowly increases in severity until the symptoms eventually become incapacitating, and at the histological level it is reflected by the progressive spread of specific pathological lesions in a non-random manner across various brain regions. At the histopathological level AD shows two main hallmarks: amyloid plaques and neurofibrillary tangles, occurring in a context of vascular damage, inflammation, oxidative stress, synaptic loss and neurodegeneration. Reelin is an extracellular matrix protein crucial for neural development. In addition, recent studies have shown that in the adult brain Reelin controls distinct plasticity events, including synaptic plasticity and adult neurogenesis. The Reelin signalling cascade has been previously related to Alzheimer Disease (AD) pathogenesis. However, the exact role that Reelin itself plays on the disease is not fully understood. The main aim of this thesis is to investigate the involvement of Reelin in AD aetiology, addressing the question of whether Reelin could act as a protective factor, with eventual therapeutic implications, or rather if it might favour the onset of the pathology. To this end established an strategy to purifiy Reelin protein and to study in vitro its involvement in the process of amyloid fibrils formation. By this approach we found in vitro that Reelin delays amyloid- (Aβ42) fibril formation until it is recruited into amyloid fibrils. Further, Reelin protects against both Aβ42-oligomer induced neuronal death and synaptic loss. We also generated AD mouse models overexpressing Reelin, to address in vivo the impact of Reelin overexpression on the main hallmarks of the disease, such as amyloid fibril formation and phosphorylation of Tau. In detail, we crossed a model of conditional Reelin overexpression (TgRln) from our lab (Pujadas et al., 2010) with two models of AD pathology: J20 and Tet/GSK-3β mice. J20 mice constitutionally express a mutated form of human APP, bearing both the Swedish and the Indiana mutation (hAPP we/Ind) (Mucke et al., 2000), thus being a good model to analyse Aβ pathology. The generated J20 mice overexpressing Reelin are referred to as TgRln/J20. Tet/GSK-3β is a conditional transgenic model of overexpression of GSK-3β, the main kinase for Tau protein, thus being a good model of Taupathology. In TgRln/J20 we showed that Reelin transgene expression delays amyloid plaque formation in vivo. The generated Tet/GSK-3β mice overexpressing Reelin are referred to as TgRln/GSK-3β. Moreover, we show that overexpression of Reelin in J20 AD mice rescues the dendritic spine loss documented for this mouse strain with consequent rescue of the cognitive deficits associated to this AD model, such as non-spatial recognition tasks (assessed by Novel Object Recognition test). Additionally, both aged TgRln and TgRln/J20 mice behaved better than aged wild-type mice, indicating that Reelin protects also from the impairments due to physiological aging. In TgRln/GSK-3β mice we show that Reelin decreases Tau phosphorylation and reduces cognitive deficits in a non-spatial recognition task (Novel Object Recognition). All together these data indicate that by delaying Aβ plaque formation, protecting against neuronal death and synaptic loss, reducing Tau phosphorylation and preventing AD cognitive deficits the Reelin pathway should be considered a therapeutic strategy for the treatment and amelioration of AD pathogenesis and cognitive deficits in normal aging.

La Reelina es una proteína de la matriz extracelular crucial para el desarrollo neural. Además, estudios recientes han demostrado que en el cerebro adulto Reelina controla eventos de plasticidad sináptica y neurogénesis. La cascada de señalización de Reelina ha sido previamente relacionada con la enfermedad de Alzheimer (EA). Sin embargo, aún no se conoce exactamente el papel que Reelina juega en la enfermedad. La EA es la forma más común de demencia en personas mayores y se caracteriza por una dramática pérdida progresiva de sinapsis y poblaciones neuronales. A nivel histopatológico se han descrito dos características principales: los ovillos neurofibrilares (paquetes intracelulares de la proteína Tau hiperfosforilada ) y las placas amiloides (depósitos extracelulares de péptido Aβ). El péptido Aβ se genera por el corte proteolítico de la proteína transmembrana APP (Amyloid precursor protein) por la acción secuencial de las proteasas de membrana β- y γ-secretasa. El Aβ monomérico comienza a agregarse en especies oligoméricas solubles, capaces de provocar disfunción sináptica en la EA. El proceso de agregación termina con la formación de fibrillas amiloides, los constituyentes principales de las placas. En esta tesis se demuestra que Reelina retrasa in vitro la formación de fibrillas amiloides, hasta que queda secuestrada en las mismas, perdiendo su actividad biológica. Además, Reelina protege contra la muerte neuronal y el daño sináptico inducidos por oligómeros de Aβ. También se generan modelos murinos de EA sobreexpresantes de Reelina. En uno de estos modelos (TgRln/J20) se demuestra que la expresión transgénica de Reelina disminuye in vivo la deposición de placas amiloides, previene la pérdida de espinas dendríticas y rescata el deterioro cognitivo asociado a este modelo de EA. En otro modelo (TgRln/GSK-3β) la sobreexpresión de Reelina reduce la fosforilación de Tau. El conjuntos de datos presentados en esta tesis indica que, al disminuir la formación de placas amiloides, al proteger contra la muerte neuronal y la pérdida sináptica, al reducir la fosforilación de Tau y al prevenir el deterioro cognitivo de la EA, la vía de Reelina debe ser considerada una estrategia terapéutica para el tratamiento y la mejora de la patogénesis de la EA.