Periodic Reporting for period 1 - SynLip (MCH/COX2-depend regulation of neuronal function and neuroinflammation in Alzheimer’s disease)
Periodo di rendicontazione: 2023-03-01 al 2025-02-28
During the development of this project, I optimized several tools and generated different datasets to accomplish the goals of the proposed project. Not all WP were accomplished as the project is terminated earlier. Nevertheless, I consider that this project provided me the opportunity to undergo major advances at the technical level, which will allow me to complete the proposed WP beyond the time covered by this grant. Initially, I proposed to isolate both neurons and microglia from CA1 region of the hippocampus, from adult animals. However, it became clear that isolating healthy neurons from adult brains is challenging and unsuccessful in our hands. On the other hand, we optimized the technical approach to isolate healthy microglia for lipidomics and scRNA sequencing. Moreover, we further optimized the proposed approach for the analysis of mRNA expression in microglia and we are currently performing scRNA sequencing. Moreover, we master all technical approaches to manipulate the MCH neurons under different mice backgrounds.
1.2.1 Work Package 1 - analysis of the impact of MCH treatment on lipid metabolism in hippocampal primary cultures using unbiased and targeted LC-MS
• Progress: fully completed
• We treated primary hippocampal cultures with MCH peptide for 4 hours
• We did not observe major changes in the lipidome of these neurons. Thus, we will perform additional experiments with longer treatments of MCH peptide.
• partly achieved
1.2.2 Work Package 2 - in vivo MCH-neurons activation (DREADDs) on Pmchcre and analysis of Ptgs2 mRNA levels and lipid profiles from CA1 pyramidal neurons.
• Progress: satisfactory progress
• In addition to the proposed analysis on Pmchcre, which is currently undergoing, we have reproduced the presented results in the initial proposed in a different group of animals: the App knock-in human mouse when comparing with the AppNL-G-F.
• We have acquired all the brain tissue essential to perform this WP.
• partly achieved
1.2.3 Work Package 3 - in vivo MCH-neurons activation (DREADDs) on AppNL-G-F.Pmchcre mice, a model that shows increased Ptgs2 mRNA in this region, to explore alterations on lipid metabolism on CA1 pyramidal neurons.
• Progress: not started
• We are currently performing these experiments
• Work in progress
• not achieved
1.2.4 Work Package 4 - Measurement of the mRNA levels of players involved in “On/Off” signals of CA1 pyramidal neurons from Pmchcre and AppNL-G-F.Pmchcre mice upon MCH-neurons activation
• Progress: satisfactory progress.
• Isolation of healthy neurons from adult animals is a challenging approach. Thus, we will perform mRNA measurements on total lysates of CA1 hippocampal regions dissected from these animals.
• Work in progress.
• not achieved
1.2.5 Work Package 5 - Analysis of the mRNA levels of players involved in lipid metabolism and lipid signature of hippocampal microglia from Pmchcre and AppNL-G-F.Pmchcre mice upon MCH-neurons activation
• limited progress
• As mentioned above, isolation of healthy neurons from adult animals is a challenging approach. Thus, we are restricting this analysis to hippocampal microglia. During the last months we successfully implemented the technical procedure to isolate healthy and pure suspension of microglia cells isolated from adult hippocampi, upon manipulation of the MCH neurons. We will ship these samples for lipidomics in the coming months, beyond the time of the project.
• Work in progress.
• partly achieved
1.2.6 Work Package 6- Analysis of the mRNA levels of the genes that characterize cellular states of hippocampal microglia from Pmchcre and AppNL-G-F.Pmchcre mice upon MCH-neurons activation (animals from Aim 1).
• Progress: satisfactory progress
• Initially, I proposed to perform mRNA analysis from bulk samples. However, we now propose to perform scRNA sequencing analysis on microglia isolated from these samples. As mentioned above, during the last months we successfully implemented the technical procedure to isolate healthy and pure suspension of microglia cells isolated from adult hippocampi, upon manipulation of the MCH neurons. We shipped the first samples for sequencing and are currently analysing data.
• Outcome and results.
• Achievement of scientific deliverables and milestones (fully/partly achieved, not achieved).
1.2.7 Work Package 7 – (Progress): progress monitoring individual meetings to discuss the advances of the project with Prof. Tiago Gil Oliveira
• Progress: fully completed
• This has been done over the course of the project
• These meetings had a beneficial impact on the project development
• fully achieved
1.2.8 Work Package 8 - (Lab meetings): lab results presentation (slides) with Prof. Tiago Gil Oliveira and lab members.
• Progress: fully completed
• The results were presented in the labmeetings
• These discussions had a beneficial impact on the project development
• fully achieved
1.2.9 Work Package 9 - a) Supervision of students and b) lecturing in the post-graduate course in “Fundamentals in Neuroscience” at ICVS.
• Progress: fully completed
• a) over the project I supervised 3 MD students and 2 master students; b) I lectured twice in the Fundamentals in Neuroscience advanced course and in the immunobiology advanced course at ICVS.
• n/A
• fully achieved
1.2.10 Work Package 10 - a) advanced course the Advanced Course “Sleep, Brain Development and Neurodegeneration” from the The Neuroscience School of Advanced Studies. b) EMBO course on “Self-leadership for women scientist”
• Progress: satisfactory progress
• a) I attended the advanced course on “Sleep, Brain Development and Neurodegeneration” from the The Neuroscience School of Advanced Studies. b) EMBO course on “Self-leadership for women scientist Outcome and results – as a winner of the EMBO IG of this year I will be attending this course under this programme.
• partly achieved
1.2.11 Work Package 11 - WP11 (Dissemination): a) weekly labmeetings and journal clubs, b) oral presentations at the host institution, c) international conferences (AD/PD, GRC), d) submission of an article to bioRxv, e) internal events “ICVS Open day” and “The brain week”.
WP12 (Exploitation of results): a) meetings with Dr. Diederik Moechars (Janssen Pharmaceutica) and b) B.ACIS Center for Health Innovation
• Progress: good progress.
• a) weekly labmeetings and journal clubs were done; b) severaloral presentations at the host institution including the ICVS retreat was done; c) attended the ADPD conference, the FENS regional meetings, were I was selected for an oral presentation; d) the submission of an article to bioRxv was not done yet, as the project was terminated earlier; eparticipated in the open day at ICVS, the brain week, attended an interview in the RTP2 to talk about sleep, and was invited to speak to 6th grade students at my local school to teach about our brain.
• fully achieved
1.2.12 Work Package 12 - WP12 (Exploitation of results): a) meetings with Dr. Diederik Moechars (Janssen Pharmaceutica) and b) B.ACIS Center for Health Innovation
• Progress: good progress.
• WP12 (Exploitation of results): a) I regularly met with Dr. Diederik Moechars (Janssen Pharmaceutica)
• fully achieved
• Progress: fully completed
• We treated primary hippocampal cultures with MCH peptide for 4 hours
• We did not observe major changes in the lipidome of these neurons. Thus, we will perform additional experiments with longer treatments of MCH peptide.
• partly achieved
1.2.2 Work Package 2 - in vivo MCH-neurons activation (DREADDs) on Pmchcre and analysis of Ptgs2 mRNA levels and lipid profiles from CA1 pyramidal neurons.
• Progress: satisfactory progress
• In addition to the proposed analysis on Pmchcre, which is currently undergoing, we have reproduced the presented results in the initial proposed in a different group of animals: the App knock-in human mouse when comparing with the AppNL-G-F.
• We have acquired all the brain tissue essential to perform this WP.
• partly achieved
1.2.3 Work Package 3 - in vivo MCH-neurons activation (DREADDs) on AppNL-G-F.Pmchcre mice, a model that shows increased Ptgs2 mRNA in this region, to explore alterations on lipid metabolism on CA1 pyramidal neurons.
• Progress: not started
• We are currently performing these experiments
• Work in progress
• not achieved
1.2.4 Work Package 4 - Measurement of the mRNA levels of players involved in “On/Off” signals of CA1 pyramidal neurons from Pmchcre and AppNL-G-F.Pmchcre mice upon MCH-neurons activation
• Progress: satisfactory progress.
• Isolation of healthy neurons from adult animals is a challenging approach. Thus, we will perform mRNA measurements on total lysates of CA1 hippocampal regions dissected from these animals.
• Work in progress.
• not achieved
1.2.5 Work Package 5 - Analysis of the mRNA levels of players involved in lipid metabolism and lipid signature of hippocampal microglia from Pmchcre and AppNL-G-F.Pmchcre mice upon MCH-neurons activation
• limited progress
• As mentioned above, isolation of healthy neurons from adult animals is a challenging approach. Thus, we are restricting this analysis to hippocampal microglia. During the last months we successfully implemented the technical procedure to isolate healthy and pure suspension of microglia cells isolated from adult hippocampi, upon manipulation of the MCH neurons. We will ship these samples for lipidomics in the coming months, beyond the time of the project.
• Work in progress.
• partly achieved
1.2.6 Work Package 6- Analysis of the mRNA levels of the genes that characterize cellular states of hippocampal microglia from Pmchcre and AppNL-G-F.Pmchcre mice upon MCH-neurons activation (animals from Aim 1).
• Progress: satisfactory progress
• Initially, I proposed to perform mRNA analysis from bulk samples. However, we now propose to perform scRNA sequencing analysis on microglia isolated from these samples. As mentioned above, during the last months we successfully implemented the technical procedure to isolate healthy and pure suspension of microglia cells isolated from adult hippocampi, upon manipulation of the MCH neurons. We shipped the first samples for sequencing and are currently analysing data.
• Outcome and results.
• Achievement of scientific deliverables and milestones (fully/partly achieved, not achieved).
1.2.7 Work Package 7 – (Progress): progress monitoring individual meetings to discuss the advances of the project with Prof. Tiago Gil Oliveira
• Progress: fully completed
• This has been done over the course of the project
• These meetings had a beneficial impact on the project development
• fully achieved
1.2.8 Work Package 8 - (Lab meetings): lab results presentation (slides) with Prof. Tiago Gil Oliveira and lab members.
• Progress: fully completed
• The results were presented in the labmeetings
• These discussions had a beneficial impact on the project development
• fully achieved
1.2.9 Work Package 9 - a) Supervision of students and b) lecturing in the post-graduate course in “Fundamentals in Neuroscience” at ICVS.
• Progress: fully completed
• a) over the project I supervised 3 MD students and 2 master students; b) I lectured twice in the Fundamentals in Neuroscience advanced course and in the immunobiology advanced course at ICVS.
• n/A
• fully achieved
1.2.10 Work Package 10 - a) advanced course the Advanced Course “Sleep, Brain Development and Neurodegeneration” from the The Neuroscience School of Advanced Studies. b) EMBO course on “Self-leadership for women scientist”
• Progress: satisfactory progress
• a) I attended the advanced course on “Sleep, Brain Development and Neurodegeneration” from the The Neuroscience School of Advanced Studies. b) EMBO course on “Self-leadership for women scientist Outcome and results – as a winner of the EMBO IG of this year I will be attending this course under this programme.
• partly achieved
1.2.11 Work Package 11 - WP11 (Dissemination): a) weekly labmeetings and journal clubs, b) oral presentations at the host institution, c) international conferences (AD/PD, GRC), d) submission of an article to bioRxv, e) internal events “ICVS Open day” and “The brain week”.
WP12 (Exploitation of results): a) meetings with Dr. Diederik Moechars (Janssen Pharmaceutica) and b) B.ACIS Center for Health Innovation
• Progress: good progress.
• a) weekly labmeetings and journal clubs were done; b) severaloral presentations at the host institution including the ICVS retreat was done; c) attended the ADPD conference, the FENS regional meetings, were I was selected for an oral presentation; d) the submission of an article to bioRxv was not done yet, as the project was terminated earlier; eparticipated in the open day at ICVS, the brain week, attended an interview in the RTP2 to talk about sleep, and was invited to speak to 6th grade students at my local school to teach about our brain.
• fully achieved
1.2.12 Work Package 12 - WP12 (Exploitation of results): a) meetings with Dr. Diederik Moechars (Janssen Pharmaceutica) and b) B.ACIS Center for Health Innovation
• Progress: good progress.
• WP12 (Exploitation of results): a) I regularly met with Dr. Diederik Moechars (Janssen Pharmaceutica)
• fully achieved