Engineering vasoactive probes for brain-wide imaging of molecular signaling

Project Information

AVATar

Grant agreement ID: 101040901

DOI

10.3030/101040901

EC signature date 8 March 2022

Start date 1 May 2023

End date 30 April 2028

Funded under

• European Research Council (ERC)

Total cost

€ 1 492 968,00

EU contribution

€ 1 492 968,00

Coordinated by

MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV
Germany

Periodic Reporting for period 1 - AVATar (Engineering vasoactive probes for brain-wide imaging of molecular signaling)

Reporting period: 2023-05-01 to 2025-10-31

Summary of the context and overall objectives of the project

Brain function depends on spatiotemporally defined brain-wide signalling via molecules such as neurotransmitters. No current technology can measure signalling molecules throughout the brain with sufficient spatial and temporal resolution in living mammals. This poses a major roadblock for understanding how molecular neuronal communication coordinates whole-brain function.

Magnetic resonance imaging (MRI) currently provides the highest brain-wide resolution. Dynamic imaging of blood flow and oxygenation in the finely arborized vasculature, so-called functional MRI (fMRI), can visualize whole-brain function in mammals and humans. However, MRI is inherently insensitive, which precludes it from accessing neurotransmitter signalling that occurs at (sub)micromolar concentrations and fMRI does not provide molecular information underlying measured hemodynamic signals.

This project aims develop protein-based vasoactive sensors, called AVATar, that convert neurotransmitter signalling into hemodynamic contrast to visualize brain-wide molecular signalling dynamics that shape healthy and pathological brain function. The project is structured along three objectives:

1) Engineering AVATars that convert neurotransmitter signalling into hemodynamic signals.
2) Brain delivery via non-invasive routes.
3) Application for fMRI of brain-wide neurotransmitter signalling in rodents.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Objective 1 aims to develop neurotransmitter-sensing AVATars along two different designs featuring competitive or allosteric functional mechanisms. We succeeded in generating an allosteric AVATar that acts at nanomolar concentrations and responds to physiological neurotransmitter concentrations and generated all key building blocks of the competitive AVATar design and produced all parts for a serotonin-sensing competitive AVATar.

In objective 2, we achieved genetic encoding and virus-mediated expression of AVATars, which facilitates wide-field, minimally invasive brain delivery. In situ expression circumvent two challenges of alternative delivery through the bloodstream, namely homogenous brain delivery and potential peripheral effects of AVATars outside the brain.

Progress towards objective 3 was largely hampered by delayed approvals of animal protocols. We are currently establishing the fMRI methodology for imaging AVATars in vivo.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The AVATar technology is still at an early development stage, so assessing its impact is still speculative. That said, our achievements from the first 2-year reporting period represent major progress for the field of brain imaging and understanding of molecular brain function in several ways:

1) The allosteric neurotransmitter-sensing AVATar we developed shows an unprecedented combination of detectability at low doses and sensitivity towards its target neurotransmitter. This paves the way towards fMRI detection of neurotransmitter release at physiological levels, which has not been possible with current technologies. Upcoming in vivo experiments will assess the full potential of AVATars for such applications.

2) Our progress in making AVATars genetically encodable and express them in situ through adeno-associated viruses facilitates brain-wide targeting and functionality of these probes. With this, we circumvent a major roadblock of existing molecular MRI probes, namely delivering sufficient doses of the probe into the brain in a non-disruptive and homogenous manner. Genetic encoding has been the key advantage of fluorescent sensors and was the main driver behind the massive success of GFP-derived tools. Still, in situ expression of molecular MRI sensors has not been achieved so far.

Together, these achievements will, address a major challenge in neuroscience: enabling imaging of neurotransmitters throughout the entire mammalian brain will address long-standing research questions into the fundamental role of long-range molecular signalling patterns in brain function.