Periodic Reporting for period 2 - ENSEMBLE (Structure and functions of the brain extracellular space)
Reporting period: 2022-11-01 to 2024-04-30
Brain research has made tremendous progress over the last few decades in nearly all areas of investigation with the exception of one: the extracellular space (ECS). It is however a key compartment defined as the web-like space between brain cells, filled with a myriad of molecules that enable brain functions and homeostasis. How molecules navigate in the ECS is a very important, yet unsolved, challenge that precludes conceptual advance in brain science and innovation in therapeutics (e.g. immunotherapy). The lack of knowledge is mainly due to the absence of dedicated investigation strategies for such a complex and finely structured biological entity. Our ground-breaking project (ENSEMBLE) will shed light on the conceptual and methodological roadblocks that have prevented us from understanding the fine architecture of the ECS and how molecules navigate within it throughout the brain. We posit that molecular diffusion in the ECS is locally regulated by the properties of the ECS, which is essential for brain functions. Four world-class scientists, L. Groc (molecular neuroscience), E. Bezard (systems neuroscience), L. Cognet (optics & nanoscience), and U.V. Nägerl (neurophotonics), team up to develop and apply unconventional investigation approaches, based on original nano-imaging strategies (super-resolution microscopy and carbon nanotube/nanoparticle tracking), to the in vivo brain. Yet, to consider and achieve such an experimental and multidisciplinary tour de force a side-by-side and daily interactive effort is necessary. Thanks to our complementary expertise and geographical proximity, ENSEMBLE will provide a unique opportunity to unveil in vivo the structure and functions of this crucial brain compartment and will offer a new theoretical and experimental framework to manipulate molecule navigation. The ENSEMBLE project will also cross-fertilize the fields of nanoscience, optical imaging, organ pathophysiology and immunotherapy.
The ENSEMBLE project started in May 2021, i.e. during the COVID outbreak. Even though we faced (like everyone else) enormous difficulties, we are proud to claim massive progress in line with the planned objectives.
We have achieved the set-up of the ENSEMBLE lab with two dedicated systems, proprietary to the ENSEMBLE project, established within the dedicated 150 m2 open lab (refurbishment completed as per plan, most hired staff working into that project-dedicated environment). These two set-ups are due to merge at some point to enable the dual mode of operation for the in vivo imaging of both SUSHI and SWCNT tracking.
ENSEMBLE is about gaining knowledge of the extracellular space (ECS), the still terra incognita of the brain. A lot of effort has been put into technological advances such as (but not limited to) (i) the continuous search for informative reporter nanoparticles (variations around single carbon nanotubes -CNTs – for gaining different levels of information; diffusion properties of various Quantum Dots of different sizes), (ii) the development of several innovative strategies to achieve volumetric localization of single carbon nanotubes (CNTs) deep in live brain samples, (iii) the first ever in vivo (anesthetised mouse) SUSHI pivotal study that improves the axial resolution for STED microscopy in the deeply embedded hippocampus (Bancelin et al., Neurophotonics 2023) and (iv) the validation of the neuronal organoid technology based upon encapsulated inducible pluripotent stem cells as a system model of ECS.
This first period witnessed great technological advances and paved the way for ground-breaking discoveries. Among the several brain areas of interest to ENSEMBLE, we have achieved the first nanoscale map of the hippocampal ECS. We showed that the hippocampal ECS is highly heterogeneous and sensitive to neuronal activity (Grassi et al., Cell Reports, 2023). In addition, we provided the first description (Grassi et al., Cell Reports, 2023) of immunoglobulin (IgG) diffusion with the hippocampal ECS, emphasising the heterogeneity of the hippocampal maze. This study is emblematic of ENSEMBLE as it demonstrates that ECS rheology and diffusive properties differ from compartment to compartment and that such differences lead to dramatic changes in IgG accessibility. We began to investigate local synaptic nano environment in the brain ECS lying within 500 nm of postsynaptic densities using a correlative imaging approach (Paviolo et al. Nano Letters, 2022) in order to study the modulation of the molecular interaction between synaptic and extrasynaptic neurotransmitter receptors by the ECS and its constituent (ECM). Finally, we began tackling the pathophysiology of two neurodegenerative diseases, namely Parkinson’s disease and Alzheimer’s disease, characterised by the accumulation and aggregation of proteins, -synuclein and A-, respectively, which travel within ECS from one brain cell to another, making the ECS an essential compartment in regulating their propagation. We currently use both the CNT and Quantum dot tracking approaches to map ECS characteristics in the substantia nigra and the striatum to provide a protein-specific ECS atlas of the mouse brain locally infused with unlabeled (i) aggregated recombinant species-specific -synuclein (so-called preformed fibrils – PFFs) and (ii) Lewy Bodies extracted from the brain of PD patients.Finally combining SUSHI, CNT and quantum dot imaging, we unravelled the peculiar ECS organisation and behaviour within and around the amyloid plaques in an AD transgenic mouse model providing the first-ever study combining the three imaging modalities, illustrating in an exemplary manner, ENSEMBLE’smotto.
We have achieved the set-up of the ENSEMBLE lab with two dedicated systems, proprietary to the ENSEMBLE project, established within the dedicated 150 m2 open lab (refurbishment completed as per plan, most hired staff working into that project-dedicated environment). These two set-ups are due to merge at some point to enable the dual mode of operation for the in vivo imaging of both SUSHI and SWCNT tracking.
ENSEMBLE is about gaining knowledge of the extracellular space (ECS), the still terra incognita of the brain. A lot of effort has been put into technological advances such as (but not limited to) (i) the continuous search for informative reporter nanoparticles (variations around single carbon nanotubes -CNTs – for gaining different levels of information; diffusion properties of various Quantum Dots of different sizes), (ii) the development of several innovative strategies to achieve volumetric localization of single carbon nanotubes (CNTs) deep in live brain samples, (iii) the first ever in vivo (anesthetised mouse) SUSHI pivotal study that improves the axial resolution for STED microscopy in the deeply embedded hippocampus (Bancelin et al., Neurophotonics 2023) and (iv) the validation of the neuronal organoid technology based upon encapsulated inducible pluripotent stem cells as a system model of ECS.
This first period witnessed great technological advances and paved the way for ground-breaking discoveries. Among the several brain areas of interest to ENSEMBLE, we have achieved the first nanoscale map of the hippocampal ECS. We showed that the hippocampal ECS is highly heterogeneous and sensitive to neuronal activity (Grassi et al., Cell Reports, 2023). In addition, we provided the first description (Grassi et al., Cell Reports, 2023) of immunoglobulin (IgG) diffusion with the hippocampal ECS, emphasising the heterogeneity of the hippocampal maze. This study is emblematic of ENSEMBLE as it demonstrates that ECS rheology and diffusive properties differ from compartment to compartment and that such differences lead to dramatic changes in IgG accessibility. We began to investigate local synaptic nano environment in the brain ECS lying within 500 nm of postsynaptic densities using a correlative imaging approach (Paviolo et al. Nano Letters, 2022) in order to study the modulation of the molecular interaction between synaptic and extrasynaptic neurotransmitter receptors by the ECS and its constituent (ECM). Finally, we began tackling the pathophysiology of two neurodegenerative diseases, namely Parkinson’s disease and Alzheimer’s disease, characterised by the accumulation and aggregation of proteins, -synuclein and A-, respectively, which travel within ECS from one brain cell to another, making the ECS an essential compartment in regulating their propagation. We currently use both the CNT and Quantum dot tracking approaches to map ECS characteristics in the substantia nigra and the striatum to provide a protein-specific ECS atlas of the mouse brain locally infused with unlabeled (i) aggregated recombinant species-specific -synuclein (so-called preformed fibrils – PFFs) and (ii) Lewy Bodies extracted from the brain of PD patients.Finally combining SUSHI, CNT and quantum dot imaging, we unravelled the peculiar ECS organisation and behaviour within and around the amyloid plaques in an AD transgenic mouse model providing the first-ever study combining the three imaging modalities, illustrating in an exemplary manner, ENSEMBLE’smotto.
As detailed in the above section, the project has moved forward on several frontlines. The progresses are described above in details.
The expected results until the end of the project are the following:
i) Defining the ECS characteristics in brain slices (hippocampus, cortex, striatum...) from organotypic and acute preparations.
ii) Defining the ECS in vivo in the cortex of mice using both SPT and SUSHI-based methods
iii) Defining the movement of extracellular proteins, such as autoantibodies, in the ECS of brain areas
iv) Defining the ECS in models of neurodegenerative diseases.
The expected results until the end of the project are the following:
i) Defining the ECS characteristics in brain slices (hippocampus, cortex, striatum...) from organotypic and acute preparations.
ii) Defining the ECS in vivo in the cortex of mice using both SPT and SUSHI-based methods
iii) Defining the movement of extracellular proteins, such as autoantibodies, in the ECS of brain areas
iv) Defining the ECS in models of neurodegenerative diseases.