Periodic Reporting for period 1 - MemCode (Cracking the Synaptic Memory Code)
Berichtszeitraum: 2023-03-01 bis 2025-08-31
Despite their role in long-term information storage, synapses are highly dynamic and composed of rather short-lived components. In the adult mouse brain, it takes a couple of days for half dendritic spines to be replaced. Similarly at the molecular level most synaptic proteins have half-lives in the order of a week meaning they constantly need to be replaced by freshly produced ones. Thus, understanding how long-term memory can arise from unstable elements is one of today’s neuroscience’s greatest challenges. Overturning an old dogma, I discovered using in vivo and in vitro approaches that most synapses produce their own proteins locally at both the pre- and postsynaptic sites. Interestingly, classic plasticity paradigms produce unique patterns of rapid pre- and/or postsynaptic translation. This finding is driving a paradigm shift in our understanding of synaptic function. It is now possible to decode pre- and postsynaptic memory traces formed during learning.
I am now in the unique position to combine omics, cytometry, super-resolution and live-imaging techniques, and behavioral learning tasks to unravel how local production of new proteins contributes to information storage at synapses. Firstly, using live-imaging, I want to understand how and when protein synthesis is recruited in excitatory boutons. Secondly, using next generation sequencing and imaging, I will investigate how mRNA find their way to presynapses. Finally, using a genetically encoded neuronal activation tracker, I will follow the molecular changes and thus uncover the synaptic memory traces in the hippocampus and cortex after learning. Altogether, these experiments will tackle from molecules to neural networks the unresolved question of memory encoding in the brain. With an unprecedented resolution, we will gain critical insights into how memories are stored at synapses. Such a fundamental understanding of brain function is needed to provide new avenues against neurodegenerative diseases.
I am now in the unique position to combine omics, cytometry, super-resolution and live-imaging techniques, and behavioral learning tasks to unravel how local production of new proteins contributes to information storage at synapses. Firstly, using live-imaging, I want to understand how and when protein synthesis is recruited in excitatory boutons. Secondly, using next generation sequencing and imaging, I will investigate how mRNA find their way to presynapses. Finally, using a genetically encoded neuronal activation tracker, I will follow the molecular changes and thus uncover the synaptic memory traces in the hippocampus and cortex after learning. Altogether, these experiments will tackle from molecules to neural networks the unresolved question of memory encoding in the brain. With an unprecedented resolution, we will gain critical insights into how memories are stored at synapses. Such a fundamental understanding of brain function is needed to provide new avenues against neurodegenerative diseases.
MemCode explores how neurons use synaptic proteome remodelling at synapses -particularly at presynaptic boutons- to store information into neuronal networks over long periods of time. The project is divided into three aims as follows:
During the two first years of the project, I was able to recruit one PhD student and two Postdocs who are each working on one work package of the project under my supervision. We were able to establish key methods and gather preliminary results. In addition, while setting up experiments for Aim 1/WP1, we stumbled upon interesting observations that we decided to investigate further in parallel.
Precisely, we have established the following methods in the lab for the three work packages of the proposal:
- WP1: neuronal cell cultures in micro-fluidics devices, single synapse live imaging, metabolic labelling;
- WP2: fluorescence activated synapse sorting (FASS) for vGLUT1-GFP+ (excitatory synapses), long-read sequencing, RNA isoform identification;
- WP3 : Morris water maze memory task for mice, FASS, iDISCO.
For each WP, experiments are now running, and we are in the process of collecting and analysing data. Below I describe in more details our progress.
WP1: role of protein synthesis on presynaptic boutons
We performed a series of experiments investigating under which conditions protein synthesis is recruited in presynaptic boutons. Surprisingly, we found that the inhibitor of -secretase DAPT, after only 4h of treatment dramatically increases excitatory presynaptic boutons size and protein synthesis. This increase in protein synthesis is concomitant to an increase in neuronal network activity measured using gCaMP6 calcium imaging. Using SynaptoRed to measure single synapse release, we observed an increase in spontaneous (asynchronous) vesicle release. We performed additional experiments and found that this effect is mediate by the accumulation of a proteolytic product of APP (amyloid precursor protein) and precursor of Aβ (known for accumulating in Alzheimer’s disease) a peptide called APP-CTFβ. We believe that accumulation of APP-CTFβ could be one of the triggers of Alzheimer’s disease onset. A manuscript is currently under review describing this findings.
WP2: What is the origin of the transcripts in presynaptic terminals?
In this work package, we want to unravel the “code(s)” leading to the targeting of specific mRNAs into axons and presynaptic boutons. Previous work suggests that non-coding elements in the 3’untranslated regions (3’UTRs) of mRNA are key to this process. Thus, as a starting point for this project, we needed establish in our lab long read sequencing -the approach of choice to capture 3’UTR diversity in transcriptomes- and develop downstream analysis pipelines for mRNA isoform identification and classification. We have collected presynaptic terminals from two brain regions (cerebellum and forebrain) and successfully used long-read sequencing to identify mRNA isoforms. We are in the process of analysing these data and establishing the analysis pipelines.
WP3: What are the molecular traces associated with memory storage?
During this first phase of the project, we have established all the methods necessary to achieve our goal. Firstly, after obtaining our animal licence and authorization we started to breed our mouse lines to obtain the triple transgenic line: vGlut1-GFP+/+ / fosTRAP2+/+ / tdTomato+/+. Secondly, we optimized our learning paradigm to obtain robust memory in our mouse line for up to a week. Finally, we established iDisco tissue clearing and hemi-brain imaging in our lab. We are now capable to count the number of neurons activated during our memory task in different brain regions. We are currently wrapping-up this validation experiments and will in the next month’s start synapse isolation for proteomics and transcriptomics.]
During the two first years of the project, I was able to recruit one PhD student and two Postdocs who are each working on one work package of the project under my supervision. We were able to establish key methods and gather preliminary results. In addition, while setting up experiments for Aim 1/WP1, we stumbled upon interesting observations that we decided to investigate further in parallel.
Precisely, we have established the following methods in the lab for the three work packages of the proposal:
- WP1: neuronal cell cultures in micro-fluidics devices, single synapse live imaging, metabolic labelling;
- WP2: fluorescence activated synapse sorting (FASS) for vGLUT1-GFP+ (excitatory synapses), long-read sequencing, RNA isoform identification;
- WP3 : Morris water maze memory task for mice, FASS, iDISCO.
For each WP, experiments are now running, and we are in the process of collecting and analysing data. Below I describe in more details our progress.
WP1: role of protein synthesis on presynaptic boutons
We performed a series of experiments investigating under which conditions protein synthesis is recruited in presynaptic boutons. Surprisingly, we found that the inhibitor of -secretase DAPT, after only 4h of treatment dramatically increases excitatory presynaptic boutons size and protein synthesis. This increase in protein synthesis is concomitant to an increase in neuronal network activity measured using gCaMP6 calcium imaging. Using SynaptoRed to measure single synapse release, we observed an increase in spontaneous (asynchronous) vesicle release. We performed additional experiments and found that this effect is mediate by the accumulation of a proteolytic product of APP (amyloid precursor protein) and precursor of Aβ (known for accumulating in Alzheimer’s disease) a peptide called APP-CTFβ. We believe that accumulation of APP-CTFβ could be one of the triggers of Alzheimer’s disease onset. A manuscript is currently under review describing this findings.
WP2: What is the origin of the transcripts in presynaptic terminals?
In this work package, we want to unravel the “code(s)” leading to the targeting of specific mRNAs into axons and presynaptic boutons. Previous work suggests that non-coding elements in the 3’untranslated regions (3’UTRs) of mRNA are key to this process. Thus, as a starting point for this project, we needed establish in our lab long read sequencing -the approach of choice to capture 3’UTR diversity in transcriptomes- and develop downstream analysis pipelines for mRNA isoform identification and classification. We have collected presynaptic terminals from two brain regions (cerebellum and forebrain) and successfully used long-read sequencing to identify mRNA isoforms. We are in the process of analysing these data and establishing the analysis pipelines.
WP3: What are the molecular traces associated with memory storage?
During this first phase of the project, we have established all the methods necessary to achieve our goal. Firstly, after obtaining our animal licence and authorization we started to breed our mouse lines to obtain the triple transgenic line: vGlut1-GFP+/+ / fosTRAP2+/+ / tdTomato+/+. Secondly, we optimized our learning paradigm to obtain robust memory in our mouse line for up to a week. Finally, we established iDisco tissue clearing and hemi-brain imaging in our lab. We are now capable to count the number of neurons activated during our memory task in different brain regions. We are currently wrapping-up this validation experiments and will in the next month’s start synapse isolation for proteomics and transcriptomics.]
We identify a new toxic peptide that could trigger Alzheimer’s disease associate synaptic disfunction prior to Aβ accumulation. We believe this finding could significantly impact the Alzheimer’s research field and open new avenues for innovative therapeutic strategies. We are currently looking for industrial collaborators to develop blockers for this peptide and we would like to submit a patent.