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Mechanisms of synaptic growth and plasticity

Periodic Reporting for period 1 - Neuronal Trafficking (Mechanisms of synaptic growth and plasticity)

Reporting period: 2016-03-01 to 2018-02-28

Understanding how neurons work is one of the biggest challenges of this century. Neuronal structure and connectivity are genetically determined but can be altered in response to changing levels of activity, a process known as plasticity. Until we understand the biology behind neuronal development and function, it will be difficult to develop informed clinical trials. Defects in synaptic morphology and activity-dependent plasticity are a hallmark of neurodegenerative disorders, which usually involve an initial phase of synapse loss and neuronal simplification, followed by death. A significant problem is that, by the time patients have manifestations of disease, major neuronal cell death has irreversibly occurred, precluding analysis of the processes that led to the loss of neurons - and this, poses a challenge for the development of treatments.

My strategy was to use the nervous system of Drosophila. Because 75% of all human disease genes have related sequences in Drosophila, we expect that these studies will contribute to the dissection of the mechanisms that, when disrupted, may lead to disease.
My by long-term goal is to understand how neurons grow and remodel. We added two aims to this proposal. Based on these alterations, our objectives are:
1. To dissect the role of Ral/exocyst in postsynaptic growth and activity-dependent plasticity.
2. Regulation of nerve bundle structure and function by Ral GTPase
3. In vivo and in real time mechanisms of synaptic growth
4. To uncover novel regulators of neuronal growth

We hope that knowing the basic mechanisms by which neurons acquire their shape and change it in response to activity, and the dissection of the genes that regulate these processes will allow the development of novel therapeutic strategies to delay the progression of neurodegenerative disorders.
In this project, we want to answer a long-standing question in basic neurobiology, which concerns how wired neurons grow and remodel. Below are the main discoveries made in each of the Aims proposed.

Aim 1.

We wanted to uncover the pathways downstream of postsynaptic Ral/exocyst activation and to characterize the genetic cascade that converts synaptic activity into postsynaptic membrane growth in a Ral/Exocyst-dependent manner. We focused on a novel pathway that regulates neuronal morphology in response to activity that required Ral and the Exocyst for the regulation of membrane growth at the synapse in response to neuronal activity. Rab proteins are associated with most of vesicular trafficking pathways and are considered traffic regulators. Additionally, Rabs can interact with motors and with tethering complexes, thereby orchestrating trafficking within cells. Since we know that Ral and Exocyst are required to recruit membrane vesicles that allow the postsynaptic SSR to grow, and knowing that Rab GTPases are central traffic regulators of many biological processes, we asked if and which Rab GTPases contribute to postsynaptic growth during development and in response to changes in synaptic activity.

From these studies, we concluded that no single RabCA over-expression could mimic RalCA. We characterized the distribution of all Drosophila Rabs at the NMJ in the several SSR size backgrounds, and now have a complete picture of the expression profiles of these central traffic regulators, which can guide our future studies. Finally, we identified 3 Rab-GTPases that are required for Ral-dependent Sec5 recruitment to the NMJ. We are now unveiling how these Rabs are interacting with Ral and testing if they contribute to different aspects of synaptic growth.

Aim 2.
The correct development of nerve bundles is an essential process that assures axonal fasciculation and insolation, which are critical factors for neuronal function. In vertebrates, myelinating and non-myelinating Schwann cells are responsible for ensheathing and supporting the axons within peripheral nerves. In invertebrates, such as Drosophila, larval peripheral nerves are also composed of several glial layers, which assure axonal wrapping and fasciculation

We observed that Ral mutants have abnormally thick intersegmental nerve bundles, and abnormal locomotion. While much is known about the factors that regulate neuronal pathfinding, much less is known regarding the contribution of glia for nerve bundle formation. We analyzed the morphology of each cell type present in nerve bundles and uncovered a novel role for Ral in glia. We dissected the pathway through which Ral is exerting its function, and are currently working on the mechanism by which Ral gives rise to these defects.

Aim 3.

The mechanisms by which activity-dependent bouton formation occurs are poorly understood. By doing live imaging of Drosophila larvae and by changing two critical parameters relative to other studies, we were able to observe in vivo bouton formation, in real time. This high temporal resolution time-lapse imaging of Drosophila NMJs revealed that the dynamics and morphological changes observed were distinct from growth cone-mediated bouton. We discovered that, in response to synaptic-activity, boutons form by a mechanism used by cells to migrate in 3D-environments. This aim uncovered a new, undescribed, mode of neuronal growth. Additionally, we found that the muscle may have a mechanical role during this process. We suggest that motor neuron and muscle have an intricate interplay of mechanical forces and biochemical signaling, which are integrated, and result in the regulation of the number of new synaptic structures that form upon intense muscle activity.

In the future, we hope to extend our studies to genetic models of neuromuscular degenerative disorders and to the vertebrate NMJ. We hope to open new avenues that will lead to the development of therapies based on neuronal remo
Our goal was to contribute for the understanding of the factors and mechanisms that regulate neuronal growth and plasticity. We used the Drosophila NMJ as a model synapse. I will pursue this research by exploring how biophysical and biomechanical properties of muscles and neurons influence synaptic growth. This area of neuroscience is largely unexplored and my discoveries can lead to a better understanding of how neurons and muscles interact from a biomechanical point of view. This relationship is critical during synapse formation and maintenance, and dysfunction in these processes is known to be the underlying cause of many neurodegenerative and neurodevelopmental disorders.

By contributing to the understanding of the molecular mechanisms that guide neuronal growth, this proposal and my future work can guide us towards the development of strategies that aim to translate this basic knowledge onto the treatment of neuromuscular or neurodegenerative diseases. A better understanding of the causes and mechanisms underlying these diseases will allow the creation of new approaches to prevent and treat them, bridging the gap between scientific knowledge and health services.
Summary of Aims 1-3