Exploring the dendritic molecular machinery responsible for neuronal polarization

Project Information

SortAx

Grant agreement ID: 101031398

DOI

10.3030/101031398

Project closed

EC signature date 19 April 2021

Start date 1 September 2021

End date 30 March 2024

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 147 815,04

EU contribution

€ 147 815,04

Coordinated by

CENTRO DE NEUROCIENCIAS E BIOLOGIACELULAR ASSOCIACAO
Portugal

Periodic Reporting for period 1 - SortAx (Exploring the dendritic molecular machinery responsible for neuronal polarization)

Reporting period: 2021-09-01 to 2023-08-31

Summary of the context and overall objectives of the project

The high morphological and functional polarization of neuronal processes relies on exquisite mechanisms to accurately sort
and traffic proteins to their rightful location. It is therefore of no surprise that accurate formation and function of neuronal
circuits depends on these trafficking mechanisms, which are indeed often impaired in neurodegenerative disorders and
normal aging.

In this proposal, I hypothesize that dendritic endosomes are key sorting stations for polarization of axonal cargoes. I
therefore propose to elucidate the key axonal trafficking pathways of endogenous cargoes, the intracellular trafficking and
endosomal machinery involved and assess their importance for axonal development and connectivity. Importantly, I will
adopt an unbiased approach using cutting-edge technology to focus on the endogenous protein pool, which differs
considerably from prior studies in the field, in which axonal trafficking route has been followed for a limited number of
cargoes under overexpression conditions.

Given that abnormal axonal trafficking increases susceptibility to neurodegenerative diseases, including Alzheimer´s and
Parkinson, with this project I will also contribute to the design of new therapies to alleviate neurodegeneration by deciphering
the molecular mechanisms of axonal cargo trafficking.

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

The specific objectives of the present research plan were:

- To uncover key axonal trafficking pathways of endogenous cargoes (WP1.1 and WP1.2):

We first identified axonal proteins present in our in vitro model using microfluidic chambers combined with mass spectrometry (MS). Afterward, we selected several candidates that are poorly characterized in terms of axonal transport and function. Currently, we are studying the transport pathways used by these candidates through live-cell imaging and characterizing their subcellular distribution in dendrites and the axon by immunocytochemistry.

- To identify the dendritic protein machinery of intracellular trafficking and endosomal sorting involved (WP2.1 and WP2.2):

We performed proximity-dependent labeling using endosomal markers fused to TurboID, a biotin ligase. This allowed us to identify the axonal proteins that are transported along the dendrites and to identify endosomal proteins that are necessary for the transport of axonal cargoes.

- To assess their importance for axonal development and brain connectivity (WP3.1 and WP3.2):

We are currently studying the function of several proteins identified in previous tasks for axonal development and brain connectivity.

Additionally, I have been very actively involved in science communication, science outreach, teaching, and reviewing activities. I have also been involved in publishing manuscripts with collaborators and writing grants, with some of them being successful. This has tremendously boosted my skills as a scientist, ultimately allowing me to become an independent Principal Investigator. Some examples are:

Dissemination:

During the duration of this project, I presented the results obtained here orally:
- 2024: Talk at the EMBO workshop in Molecular Neurobiology (Heraklion, GR).
- 2023: Talk at the Department of Life Sciences, University of Coimbra (Coimbra, PT).
- 2023: Talk at the ESN-ISN 2023 Meeting (Porto, PT).
- 2023: Talk at the 2nd Surfaceome Meeting (Scanno, IT).
- 2022: Seminar at the Institute of Health Innovation and Research – i3S (Porto, PT).

And in the form of a poster:
- 2024: EMBO workshop in Molecular Neurobiology (Heraklion, GR).
- 2023: FENS Regional Meeting (Albufeira, PT).
- 2022: 8th European Synapse Meeting (Coimbra, PT).

Outreach:

- 2023: Participation in the project “33 Voices of CNC-UC”. The CNC-UC celebrated 33 years of history in November 2023. This project aimed to give voice to some of those who have made up or currently make up the CNC-UC and have been or are part of this history.

- 2023: Interview for Ciência Viva: I highlighted the impact of my summer internship experience at Ciência Viva, during high school, on my career as a researcher.

- 2022: CNC-UC welcome day: I introduced our lab and projects to the newcomers of CNC-UC.

- 2022: European Researchers’ night: in the context of the Green labs initiative at CIBB, we prepared an activity for the general public to illustrate the different type of plastics we can find in day-life products and how to proceed to recycle them. We also explain to the general public the measures we are taking in the lab to decrease the ecological footprint of our research activities.

- 2022: Observatório RUC: interview at the Radio of the University of Coimbra (RUC) during the Brain Awareness Week. We discussed our research at CNC-UC, and some of the challenges of a career in an academic research environment.

- 2021: Meet the Scientists: activity organized by the Brain Gain initiative of CNC-UC with the purpose of introducing the topic of research to high-school and university students and inspire them to follow a career as a researcher.

Teaching at PhD Programs and Other Courses:

- 2024: Lecturer at the Synapse Assembly, Function and Plasticity course (PhD Program in Experimental Biology and Biomedicine of the Center for Neuroscience and Cell Biology) (Coimbra, PT).

- 2024: Instructor for Experimental Work at the Synapse Assembly, Function and Plasticity course (PhD Program in Experimental Biology and Biomedicine of the Center for Neuroscience and Cell Biology) (Coimbra, PT).

- 2023: Lecturer at the Cellular and Molecular Neurobiology course (Master in Cellular and Molecular Biology of the Department of Life Sciences of the University of Coimbra) (Coimbra, PT).

- 2023: Instructor for Experimental Work at the Advanced Techniques for Synapse Biology Course (Cajal Advanced Neuroscience Training) (Bordeaux School of Neuroscience, FR).

- 2023: Instructor for Experimental Work at the Synapse Assembly, Function and Plasticity course (PhD Program in Experimental Biology and Biomedicine of the Center for Neuroscience and Cell Biology) (Coimbra, PT).

- 2022: Lecturer at the Synapse Biology course (PhD Program in Experimental Biology and Biomedicine of the Center for Neuroscience and Cell Biology) (Coimbra, PT).

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)

In this research plan, by taking advantage of recent technological advances such as CRISPR-Cas9-based endogenous protein tagging and proximity-dependent labeling, we helped understand how endogenous newly synthesized proteins are specifically sorted into the axon, thus shedding light on a long-standing controversy in the field. Namely, we identified some of the molecular protein machinery involved in Golgi-to-axon transport of neuronal membrane cargoes, which is ultimately responsible for nearly every aspect of neuronal function, from brain wiring to behavioral encoding, and shed light on the function of uncharacterized proteins in the brain. Finally, SortAx will also generate novel therapeutic targets because mutations in genes encoding key components of the axonal transport machinery, including motor proteins and motor adaptors, have been implicated in multiple neurological disorders.

In SortAx we will uncover the machinery of dendritic endosomes during neuronal polarization