Periodic Reporting for period 4 - BrainNanoFlow (Nanoscale dynamics in the extracellular space of the brain in vivo)
Período documentado: 2023-06-01 hasta 2024-11-30
Evidence suggests that aggregates of proteins such as amyloid-beta play a key role in neurodegeneration, directly inducing toxic effects and acting as seeds to propagate the disease in the brain. A large fraction of these aggregates circulate the space in between brain cells and need to be constantly cleared from this narrow and complex space. The clearance mechanisms by which this extracellular "waste" is removed from the brain are not fully understood, as much of the dynamics happens at very small spatial scales and changes between wake and sleep. The project aim was to develop tools to study the extracellular clearance of protein aggregates using a combination of nanotechnology tools and high-resolution microscopy. This will have a deep impact in our understanding of clearance mechanisms in the brain and the mechanisms by which protein aggregates induce toxicity in the brain. In turn, the understanding of these mechanisms will give us better tools to fight against dementia.
The project made use of high-resolution microscopy to study how protein aggregates are cleared from the brain. We studied this with different levels of biological complexity, ranging from single-molecule imaging of individual protein aggregates to in vivo clearance experiments where we quantified cerebrospinal fluid flow and the drainage of protein aggregates from tissue into cerebrospinal fluid. We studied the dynamics of aquaporin-4 arrays, which are involved in cerebrospinal fluid circulation, and found that the array size and mobility is tuned by adrenergic signalling and osmotic changes. We also studied how aggregates with different structural features induce toxicity in cells, and we found striking differences in toxicity pathways arising from structural changes in the protein aggregate. Among those toxicity pathways, we particularly focused on the interactions of amyloid-beta with two receptors: TLR4 and PirB. We found that TLR4 inhibition can rescue most of the toxic effects of amyloid-beta aggregates. To study the clearance of extracellular solutes from the brain, we developed a method to trace flow of cereborspinal fluid in the brain using nanoparticles. This allowed us to quantify properties of perivascular flow and the circulation of cerebrospinal fluid in the brain parenchyma.
The most important achievements of the project that go significantly beyond the state of the art are:
- Successful tracking of individual nanoparticles in the brain of living anaesthetised animals.
- Characterisation of aquaporin-4 array changes upon stimuli related to sleep/wake cycles.
- Dissentanglement of complex toxicity pathways arising from protein aggregates in brain slices.
- Quantitative characterisation of the clearance routes of protein aggregates from the hippocampus.
- Discovery that the mobility of PirB changes with synaptic plasticity and is dysregulated by amyloid-beta aggregates.
- Successful tracking of individual nanoparticles in the brain of living anaesthetised animals.
- Characterisation of aquaporin-4 array changes upon stimuli related to sleep/wake cycles.
- Dissentanglement of complex toxicity pathways arising from protein aggregates in brain slices.
- Quantitative characterisation of the clearance routes of protein aggregates from the hippocampus.
- Discovery that the mobility of PirB changes with synaptic plasticity and is dysregulated by amyloid-beta aggregates.