Periodic Reporting for period 1 - NEUROSTAD (Characterization of the gene and protein networks regulated by Staufen 2 in neurogenesis and their alterations in AD)
Berichtszeitraum: 2021-06-01 bis 2023-05-31
Alzheimer's disease (AD) is a devastating neurodegenerative disorder affecting 60% of the 55 million people with dementia globally. It leads to a gradual loss of cognitive functions, posing a significant global health challenge. One critical aspect of AD is the impact on hippocampal neurogenesis, which contributes to the progressive loss of recent memory.The urgent need to address this global health challenge posed by AD drive us to propose NEUROSTAD project, which explores the intricate world of neurogenesis. Neurogenesis is the process by which new neurons form in the brain, and we aim to decode the role of Staufen2 (STAU2) in this fundamental process, exploring its implications for AD. Deeper knowledge will help unravel the complexities of AD and other neurodegenerative diseases, paving the way for innovative treatment approaches. This increased understanding has the potential to significantly improve the healthcare system, offering enhanced prevention, accurate diagnostics, and more personalized treatment options for AD and other related disorders.
The overall objectives of this Marie Skłodowska Curie Action (MSCA) have been (i) generating and analyzing the transcriptome of a STAU2 Knockout (KO) hiPSC cell line at the single-cell level during various stages of neuronal differentiation; (ii) compare the transcriptome of the STAU2 KO differentiation with the single-cell transcriptomic profiles from AD patients hiPSCs undergoing neuronal differentiation; (iii) unraveling the pivotal role of STAU2 in neurogenesis and (iv) exploring its potential implications in AD. In parallel, the goal of the MSCA Individual Fellowship is the personal growth of the Fellow towards scientific independence and to enrich the European community with excellent researchers to face the forthcoming challenges of the future.
The overall objectives of this Marie Skłodowska Curie Action (MSCA) have been (i) generating and analyzing the transcriptome of a STAU2 Knockout (KO) hiPSC cell line at the single-cell level during various stages of neuronal differentiation; (ii) compare the transcriptome of the STAU2 KO differentiation with the single-cell transcriptomic profiles from AD patients hiPSCs undergoing neuronal differentiation; (iii) unraveling the pivotal role of STAU2 in neurogenesis and (iv) exploring its potential implications in AD. In parallel, the goal of the MSCA Individual Fellowship is the personal growth of the Fellow towards scientific independence and to enrich the European community with excellent researchers to face the forthcoming challenges of the future.
The NEUROSTAD project conducted under this MSCA Research Fellowship, has made significant progress in unravelling the role of Stau2 in neurogenesis and its implications in AD. The project was organized into six work packages (WPs), each contributing to the overall objectives.
WP1 included deleting STAU2 from hiPSC lines and investigating the impact of Stau2 depletion on neuron's development. The genome editing CRISPR/Cas9 system was employed for precise STAU2 knockout, resulting in a stable hiPSC cell line. The differentiation potential of these cells was assessed, revealing accelerated neurogenesis in STAU2 knockout conditions. This work represents a scientific milestone, providing a robust platform for exploring Stau2's role in neurogenesis.
WP2 comprised the single-cell library formation and sequencing, using Drop-seq for cell separation into oil vesicles. Despite a maternity leave pause, the deliverables for scRNA-seq libraries were successfully achieved. This package represents a crucial step, due that the rest of the project depended on the successful sequencing and on the quality of the data. The datasets obtained will be deposited for broader accessibility.
WP3 focused on analyzing scRNA-seq data from WP2, identifying mRNA candidates pivotal in neurogenesis and AD. The analysis revealed significant changes in STAU2 content at earlier stages of neurogenesis than previously described. Deliverables involving the analysis of RNA-seq datasets were achieved, providing a list of candidate genes associated with neurogenesis upon STAU2 knockout and in AD cells.
WP4 aimed to perform all the the experimental and functional analysis of selected targets identified in WP3. We optimized the transwell experiments to analyze changes in mRNA localization upon STAU2 KO and the APEX system to analyze the RNA-STAU2 protein complexes formed in each stage of neurogenesis. This work will continue even after the MSCA fellowship (ongoing collaboration between the Fellow and the Host lab).
WP5 and WP6 were about management, training and development. During the work on research projects, the Fellow also supervised a Erasmus+ Exchange Technician from the Gouti Lab at MDC (Berlin, Germany) and a Erasmus+ master student from Gratz University; and co-tutorized a Master Student from the University of Barcelona (UB). She has received training in single-cell library preparation and bioinformatics, hiPSC generation, maintenance, and characterization, and successful knock-outs by using CRISPR-Cas techniques (via collaboration during project in the Host lab). Furthermore, the Fellow got additional training in optical microscopy, flow cytometry analysis and how to give powerful presentations through IDIBELL Areté Program and Leadership and managerial skills through Marie Curie Alumni Association (MCAA) courses through Coursera. Management skills were further promoted collaborating closely with the post-award unit at IDIBELL, with constant communication with relevant departments, and utilization of the intranet for project details. Results of our research have been reflected in a scientific review (already accepted at Transcription Journal) and two forecoming papers (manuscripts in preparation). This work has been presented at three conferences (two via oral communication and one via poster communication). Moreover, this work was presented regularly at Department seminars at P-CMRC Department in IDIBELL and in the institutional fellows seminar. Moreover, the Fellow actively participated in science dissemination programs like INSPIRASTEAM and contributed to the MCAA newsletter, enhancing visibility in the scientific community Supervision and Training.
WP1 included deleting STAU2 from hiPSC lines and investigating the impact of Stau2 depletion on neuron's development. The genome editing CRISPR/Cas9 system was employed for precise STAU2 knockout, resulting in a stable hiPSC cell line. The differentiation potential of these cells was assessed, revealing accelerated neurogenesis in STAU2 knockout conditions. This work represents a scientific milestone, providing a robust platform for exploring Stau2's role in neurogenesis.
WP2 comprised the single-cell library formation and sequencing, using Drop-seq for cell separation into oil vesicles. Despite a maternity leave pause, the deliverables for scRNA-seq libraries were successfully achieved. This package represents a crucial step, due that the rest of the project depended on the successful sequencing and on the quality of the data. The datasets obtained will be deposited for broader accessibility.
WP3 focused on analyzing scRNA-seq data from WP2, identifying mRNA candidates pivotal in neurogenesis and AD. The analysis revealed significant changes in STAU2 content at earlier stages of neurogenesis than previously described. Deliverables involving the analysis of RNA-seq datasets were achieved, providing a list of candidate genes associated with neurogenesis upon STAU2 knockout and in AD cells.
WP4 aimed to perform all the the experimental and functional analysis of selected targets identified in WP3. We optimized the transwell experiments to analyze changes in mRNA localization upon STAU2 KO and the APEX system to analyze the RNA-STAU2 protein complexes formed in each stage of neurogenesis. This work will continue even after the MSCA fellowship (ongoing collaboration between the Fellow and the Host lab).
WP5 and WP6 were about management, training and development. During the work on research projects, the Fellow also supervised a Erasmus+ Exchange Technician from the Gouti Lab at MDC (Berlin, Germany) and a Erasmus+ master student from Gratz University; and co-tutorized a Master Student from the University of Barcelona (UB). She has received training in single-cell library preparation and bioinformatics, hiPSC generation, maintenance, and characterization, and successful knock-outs by using CRISPR-Cas techniques (via collaboration during project in the Host lab). Furthermore, the Fellow got additional training in optical microscopy, flow cytometry analysis and how to give powerful presentations through IDIBELL Areté Program and Leadership and managerial skills through Marie Curie Alumni Association (MCAA) courses through Coursera. Management skills were further promoted collaborating closely with the post-award unit at IDIBELL, with constant communication with relevant departments, and utilization of the intranet for project details. Results of our research have been reflected in a scientific review (already accepted at Transcription Journal) and two forecoming papers (manuscripts in preparation). This work has been presented at three conferences (two via oral communication and one via poster communication). Moreover, this work was presented regularly at Department seminars at P-CMRC Department in IDIBELL and in the institutional fellows seminar. Moreover, the Fellow actively participated in science dissemination programs like INSPIRASTEAM and contributed to the MCAA newsletter, enhancing visibility in the scientific community Supervision and Training.
This MSCA project aimed to perform multidisciplinary research for better understanding the role of Stau2 in neurogenesis and AD. Innovative aspects included: a) using cutting-edge techniques like single-cell RNA sequencing (scRNA-seq) together with hiPSC differentiation towards neuronal and glial cultures; b) implementation of RNA-protein pulldowns using APEX system and mass spectrometry for studying multicomponent protein-DNA complexes and optimization of transwell cultures of neuron-derived hiPSCs for proper separation of soma and neurites and analysis of STAU2 dependent changes in mRNA localization.
NEUROSTAD has had an impact through the scientific community. On AD field, we have identified pivotal transcriptomic changes and candidate genes associated with neurogenesis and this disease, surpassing current knowledge boundaries. Furthermore it has had a technical impact, since the project proved that single-cell sequencing should be considered as the “first-choice technique” for studying complex biological changes at different populations at the same time and at different stages. Our results also showed that the combination between such experiments and stem cell differentiated cultures provide an important research tool to study human developmental processes and neurodegenerative diseases (such as AD) which are difficult to study in vivo.
Moreover, the project's impact extends beyond scientific milestones; it holds significant socio-economic potential. The exploitation of these results, including scRNA-seq datasets and candidate genes, may pave the way for innovative therapeutic interventions (not only for AD but also for other neurodegenerative diseases). This project could also allow enhanced prevention and earlier detection. This way, patients could be given a better and wider life, and the costs of healthcare system could be reduced.
NEUROSTAD has had an impact through the scientific community. On AD field, we have identified pivotal transcriptomic changes and candidate genes associated with neurogenesis and this disease, surpassing current knowledge boundaries. Furthermore it has had a technical impact, since the project proved that single-cell sequencing should be considered as the “first-choice technique” for studying complex biological changes at different populations at the same time and at different stages. Our results also showed that the combination between such experiments and stem cell differentiated cultures provide an important research tool to study human developmental processes and neurodegenerative diseases (such as AD) which are difficult to study in vivo.
Moreover, the project's impact extends beyond scientific milestones; it holds significant socio-economic potential. The exploitation of these results, including scRNA-seq datasets and candidate genes, may pave the way for innovative therapeutic interventions (not only for AD but also for other neurodegenerative diseases). This project could also allow enhanced prevention and earlier detection. This way, patients could be given a better and wider life, and the costs of healthcare system could be reduced.