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Endoplasmic reticulum structure and synaptic function in Drosophila

Periodic Reporting for period 1 - SynapseER (Endoplasmic reticulum structure and synaptic function in Drosophila)

Reporting period: 2018-01-01 to 2019-12-31

Animal and human movement depends on the ability of nerve cells to carry signals along narrow projections known as axons, which in humans can extend as much as a metre from the centre of the cell, the cell body. Maintaining the structure and function of long axons to ensure good communication requires a lot of engineering.

In neurons, endoplasmic reticulum (ER) organelle shows physical continuity between dendrites, cell body and axonal presynaptic terminals, and has been termed “a neuron within a neuron” (Figure 1). The importance of ER in axons is suggested by the fact that mutations of ER-shaping proteins result in hereditary spastic paraplegia (HSP), a motor axon degeneration disease that preferentially affects the ends of axons furthest from the cell body on longer motor neurons, producing spasticity, paralysis and/or lack of sensation.

ER is present in presynapses, and mutations of ER-shaping proteins disrupt synaptic morphology or function. However, the physiological roles of ER distribution in this context are largely unknown. We address this question, examining the distribution and role of ER at presynaptic level, and mechanisms of dysfunction that are relevant for human neurodegenerative diseases.
The model organism used in this work was the fruitfly Drosophila, because of its powerful genetics, and methods to study the synapse where motor axons stimulate muscles (the neuromuscular junction; Figures 2 and 3). We generated mutant animals lacking the HSP ER-shaping protein (Reticulon), and studied neuromuscular junctions in wild-type and mutant animals. This has helped us to understand the roles of this protein and the consequences of altered ER distribution for local trafficking and organelle function.

We found that Reticulon loss of function causes structural defects in presynaptic ER distribution, and that these defects associate with altered synaptic morphology and signalling between muscles and neurons. Furthermore, we observed abnormal presynaptic calcium signaling, which it is known to be essential for neuronal function and whose dysregulation associate with neurodegeneration. These results will be published in open access in a scientific article that is in preparation.

In addition, we found that animals mutated for Reticulon and other two ER-shaping proteins (ReepA and ReepB) affects presynaptic ER calcium handling. We published this data, together with genetic tools we generated to visualize calcium signaling (Figure 2), in an open access repository (Oliva et al., 2020, bioRxiv, DOI: 10.1101/2020.02.20.957696) and it is under revision in a peer-reviewed scientific journal.

During this project, we also identified and characterized genetic tools to allow the identification and manipulation of specific types of motor neurons (Figure 3). This work has been published in an open access preprint server (Perez-Moreno and O'Kane, 2019, bioRxiv DOI: 10.1101/445577) and in an open-access peer-reviewed scholarly journal (Perez-Moreno and O’Kane, 2019, G3 DOI: doi.org/10.1534/g3.118.200809).

Our findings have been disseminated in our host department (Department of Genetics, University of Cambridge: Annual Departmental meeting 2018 and Internal Seminar Series 2019), in specialized scientific conferences (Tom Wahlig Symposium 2018, European Drosophila Neurobiology Conference 2018, and Cambridge Neuroscience Seminar 2019), as well as in public outreach events (European Commission Researchers Night 2018 and Cambridge Science Festival 2019). Furthermore, we have published a review article about axonal ER and neurodegeneration in a peer-reviewed journal (Öztürk et al., 2020, Front Neurosci, DOI: doi.org/10.3389/fnins.2020.00048).
This work has examined the distribution and role of ER at presynaptic level, and mechanisms of dysfunction that are relevant for human neurodegenerative diseases. In particular, we have studied the cellular consequences at presynaptic level of the loss of function of ER-shaping proteins, whose mutation in humans can cause HSP disease.

We found that proper presynaptic ER distribution is required for synaptic morphology and function. Our data may help to understand not only the biological function of presynaptic ER distribution, but also its relation with human disease. This knowledge is a prerequisite for finding therapeutic targets for HSP and similar axon degeneration diseases.

We have also presented genetics tools to study calcium signalling and specific types of motor neurons. Both set of tools may be of wide interest for the Drosophila neurobiology community, whose work has contributed for decades to our understanding of both fundamental neuroscience and neurological disorders.
1. Diagram of a neuron, where ER network is represented in blue
3. Posterior part of the axon and the muscle innervation (presynaptic terminals) by a single neuron
2. Presynaptic ER together with genetically encoded ER (top) or cytoplasmic (bottom) calcium sensors