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Cellular and molecular mechanisms of neuronal remodelling at the neuromuscular junction
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Nervous system function depends on the coordinated activity of populations
of neurons that are connected and communicate at junctions called synapses.
Synapses are first established during embryogenesis using a highly precise genetic
program from which neural circuits are defined, allowing processes such as
perception, learning, memory, or locomotion. However, circuit wiring can be
further remodeled using neuronal activity-dependent plasticity mechanisms.
Therefore, synaptogenesis is a long-developmental process that involves synapse
formation, maintenance, refinement, and elimination. The plasticity of neural
circuits is critical because it allows for information storage and environmental
adaptation in young individuals and throughout adulthood. Hence, it is not
surprising that defects in synaptic connectivity and plasticity are characteristic of
numerous neurodevelopmental and neurodegenerative diseases.
Synapses are typically found in round varicosities formed at neuronal axons,
called synaptic boutons, which are conserved structures from invertebrates to
humans. Although boutons are important synaptic compartments where
neurotransmission occurs, very little is known about the mechanism and dynamics
of bouton formation, especially during the postembryonic period. Initial circuit
formation is relatively well-characterized and requires intricate developmental
events prior to synaptogenesis, including cell fate specification, cell migration, axon
guidance, and synaptic target selection, all of which occur almost simultaneously
in billions of neurons during vertebrate nervous system development. During this
period, the mechanisms that give rise to synaptic boutons and synapses involve
the formation of labile and dynamic growth cones (GCs), comprised of filopodia
and lamellipodia, which guide axons towards their targets and differentiate into
round boutons upon arrival at their destination. Once formed, the circuit
architecture can change in response to developmental cues and external stimuli,
such as synaptic activity, allowing for more selective remodeling, including bouton
formation and elimination. However, how these and other structural changes are
coordinated between synaptic partners and the mechanisms used for neuronal
remodeling are still unknown.
The aim of this PhD thesis was to address a fundamental question in
neuroscience: How are synaptic boutons formed and integrated into wired neurons? The Drosophila neuromuscular junction (NMJ), a synapse formed
between motor neurons (MNs) and skeletal muscle fibers that is essential for
muscle contraction and movement, was adopted as a model to study this question.
In addition to clearly discernible boutons, NMJs show robust structural plasticity
during larval growth, with new boutons added to the neuronal arbor through
developmental and activity-dependent processes. The analysis was performed
mainly in 3rd instar larvae, in which developmental bouton addition is nearly
completed, after induction of structural plasticity using depolarization protocols
with a solution containing elevated K+ that mimics intense activity, and allows for
the rapid addition of new boutons to the NMJ. This stimulation method was used
and combined with live samples of Drosophila larvae to dissect the details of
bouton outgrowth in real-time.
Neuronal migration and growth are critical for proper synaptic wiring in the
nervous system. To date, the mechanisms described for bouton formation have
involved filopodia or lamellipodia structures. However, live imaging of
unanesthetized Drosophila larval NMJs with high temporal resolution (in secs)
sheds light on an unreported mechanism for bouton addition in wired neurons,
which can potentially be used for rewiring of mature neurons. In the Drosophila
larval NMJ, synaptic bouton addition in response to intense activity does not occur,
as in the embryonic stage, where a GC differentiates into round boutons. Instead,
new boutons emerge rapidly as round protrusions of the neuronal membrane
resembling blebbing, a pressure-driven mechanism used by other cells in 3D
migration and tissue invasion. Moreover, new boutons exhibit key characteristics
of blebbing: a reduction in filamentous actin (F-actin) during their growth and the
recruitment of non-muscle myosin II (MyoII), an essential regulator of blebbing.
Because the NMJ is deeply inserted into the muscle, for the MN to attach new
boutons to the muscle, it must further invade it, which is mechanistically similar to
migration across other tissues. It is possible that MNs have adopted a strategy in
which blebbing is combined with activity-dependent signaling pathways to
modulate the formation of new boutons during intense neuronal activity.
Interestingly, an association between muscle contraction and bouton formation
was observed in live imaging movies, suggesting a role of the muscle during this
process. Using several strategies, the muscle contraction potential was
manipulated during NMJ stimulation, which showed that this mechanism of bouton formation requires muscle contraction around the neuronal membrane.
One hypothesis is that the muscle plays a mechanical role in MN plasticity and
bouton remodeling, possibly by compressing the MN during contractility and
increasing its confinement, which is an important factor in promoting blebbing.
These findings suggest that in addition to biochemical signaling, a balance of
mechanical forces cooperates between MN and muscle cells to coordinate activitydependent
bouton formation at the Drosophila NMJ.
This research further expanded our understanding of the mechanisms that
control synaptic bouton assembly in neurons and identified a mechanism,
blebbing, by which wired neurons may form new boutons, allowing their structural
expansion and plasticity, using transsynaptic physical interactions as the main
driving force. Further investigation of this mechanism can contribute to a clear
understanding of the normal neuronal structure and function, which is essential
for studying responses to injury or disease.
Descrição
Palavras-chave
Synaptic Bouton Activity-Dependent Structural Plasticity Drosophila Neuromuscular Junction