Uncovering the machinery for the sorting of newly synthesized proteins into the axon

Project Information

NEUROSORTER

Grant agreement ID: 950617

DOI

10.3030/950617

Project closed

EC signature date 27 October 2020

Start date 1 November 2020

End date 31 October 2025

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 500 000,00

EU contribution

€ 1 500 000,00

Coordinated by

UNIVERSITEIT UTRECHT
Netherlands

Periodic Reporting for period 4 - NEUROSORTER (Uncovering the machinery for the sorting of newly synthesized proteins into the axon)

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

Summary of the context and overall objectives of the project

Neuronal development and function rely on the polarized distribution of organelles and transmembrane proteins (cargoes) across their somatodendritic and axonal domains. However, it is unknown how organelle organization regulates the polarized sorting of transmembrane proteins to ensure proper neuronal function. The classical model for sorting of newly synthesized transmembrane proteins to the plasma membrane (PM) follows the biosynthetic pathway via the rough endoplasmic reticulum (ER) and Golgi, which are restricted to the somatodendritic domain in neurons. It is unclear whether this classical secretion pathway is the main route for cargo sorting into the axon or whether alternative unconventional routes are used for axonal cargoes. A broad spectrum of human diseases is associated to cargo missorting. Neurons offer a unique advantage in spatial resolution to characterize unconventional routes, which could play a key role in human health and disease.
Within this NEUROSORTER project we aimed to: 1) identify the sorting routes for newly synthesized axonal proteins, 2) unravel the machinery required for Golgi-dependent and independent cargo sorting into the axon, and 3) elucidate its impact on neuronal development and function. By using high spatio-temporal resolution imaging and mass-spectrometry combined with novel strategies to control and track cargo secretion, we have deepened our understanding on how proteins are sorted to the axon.

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

Within this NEUROSORTER project, we have found that axonal ER tubules are required for local axonal translation. In particular, we have found that ribosomes interact with the axonal ER, and this interaction is mediated by the axonally distributed ER protein P180, which binds ribosomes in a mRNA-dependent manner. Axonally-enriched P180 mainly interacts with mRNAs encoding for transmembrane and luminal proteins. In addition, external neuronal cues regulate ER-ribosome interaction and axonal translation (Koppers et al., Dev Cell, 2024). Notably, we have identified an unconventional secretion mechanism for these locally translated transmembrane proteins, involving axonal ERES. Removal of axonal ERES components prevents unconventional secretion in the axon, and surprisingly also impairs local translation, suggesting an interplay between local translation and secretion. We discovered that axonal ERES interacts with the mRNA binding protein HDLBP1 (an interactor of P180) and with the tethering proteins Sec22B and NRZ complex for local cargo translation and secretion to the axonal plasma membrane (Nguyen et al., BioRxiv, 2025; unpublished data). The discovered mechanisms involved in local axonal translation and secretion play a key role in neuronal development (Koppers et al., Dev Cell, 2024; Nguyen et al., BioRxiv, 2025). Lastly, we have developed a novel spatiotemporal proteomics tool: Protein Origin, Trafficking And Targeting to Organelle Mapping (POTATOMap) to capture, for the first time, the dynamic interactome of newly synthesized secretory proteins from origin to destination. We have demonstrated the power of POTATOMap by studying the trafficking routes and sorting mechanisms for newly synthesized lysosomal membrane proteins, revealing the role of biosynthetic LAMP-positive compartments in the replenishment of axonal lysosomes, and in the delivery of synaptic proteins and RNA granules to the axon (Nat Commun., 2024).
These findings have been extensively shared within the scientific community over more than 20 presentations in international conferences.

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)

For long time has been questioned whether axonal translation existed, as most of the translation occurs in the soma of neurons. Cumulative evidence has now further supported the model of axonal translation, which has a key role in axon development and function. However, most of these studies have investigated translation of cytosolic proteins. We have now identified a mechanism for the translation of transmembrane proteins along the axon, which could explain why mRNA encoding for transmembrane proteins have been found in transcriptomics studies in isolated axons. This is the first time that it has been shown that ER tubules play a key role in local translation in mammalian cells. How do these locally translated proteins can exit the axonal ER in the absence of Golgi-like structures? We have found that axonally translated proteins exit the axonal ER via ERES in a Golgi function-independent manner. These ERES are in close proximity to newly identified translation and secretion machinery key players, which are required for cargo delivery to the axonal plasma membrane. These new discovered mechanisms for cargo translation and secretion at the axon may ensure fast responses to fulfil local demands for proper axon development and function. Our novel POTATOMap tool will help to identify additional routes and key players involved in the delivery of different cargoes and study trafficking disruption in human diseases with high spatio-temporal precision.

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