Impact of Brain Endothelial Senescence on Neurogenesis: Application of Novel Strategies for Age-Related Senescence Detection

Abstract

In vitro methods 2.1. Cellular models of endothelial senescence 2.2. Primary culture of BECs from SAMP8 and SAMR1 mouse brains 2.3. EC senescence detection and characterization 2.4. New fluorogenic probes for SA-β-Gal detection 2.5. Establishment, propagation, and proliferative analysis of neurosphere cultures 2.6. Evaluation of the paracrine effects of EC senescence in NSC cultures 2.7. Adeno-associated vector (AAV2-BR1-p16-GFP) construction for BEC senescence induction Ex vivo methods Histochemical X-gal staining of SEZ whole-mount preparations Analysis of brain endothelial senescence by flow cytometry Measurement of the senescent cell burden in different organs FACS strategy for the analysis of the SEZ neurogenic lineage In vivo methods Longitudinal characterisation of ageingthrough external assessment 4.2. Systemic β-Gal activity assessment with the renally clearable Cy7Gal probe 4.3. Senolytic treatments 4.4. Immunohistochemical analysis of adult neurogenesis in the SEZ and the SGZ 4.5. Analysis of AAV2-BR1-p16-GFP transduction in BECs and its effects on neurogenesis 4.6. Fluorescent tomato lectins for imaging of blood vessels in SEZ whole-mount preparations 5. Statistical analysis and data representation Results 1. Development of novel strategies for longitudinally monitoring ageing-associated senescence in vivo 1.1. Longitudinal characterisation of ageing progression 1.2. In vitro and ex vivo validation of novel molecular probes for senescent cell detection 1.3. Non-invasive monitoring of age-related senescent cell burden in vivo 1.4. Longitudinal measurement of senescence and behaviour associated with accelerated ageing and its reversal by senolytics 1.5. Longitudinal measurement of senescence and behaviour associated with natural ageing and its reversal by senolytics Characterisation of ageing-associated brain endothelial senescence and its impact on adult neurogenesis Paracrine effects of senescent ECs on NSC behaviour Correlative analysis of brain endothelial senescence and neurogenesis during ageing 2.3. Correlative analysis of brain endothelial senescence and neurogenesis after senolytic intervention 2.4. A novel genetic approach to induce senescence in BECs and its impact on neurogenesis DiscussionConclusions Summary Resumen Resum Bibliography Annex I. Antibodies Annex II. Culture media and buffers Annex III. Genetic map of BR1 capsid Annex IV. Submitted publication Material and methods Graphical method 1. BECs primary culture procedure Graphical method 2. New fluorogenic probes for SA-β-Gal detection Graphical method 3. Primary culture, subculture and proliferative analysis of NSCs in vitro Graphical method 4. Isolation protocol of the soluble fraction secreted by ECs Graphical method 5. Evaluation of in vitro viral vector transduction in ECs Graphical method 6. Procedure for phenotyping senescent BECs Graphical method 7. FACS gating strategy for BECs identification Graphical method 8. Analysis of the SEZ neurogenic lineage by flow cytometry (Belenguer et al., 2021) Graphical method 9. Cy7Gal, a re...