Periodic Reporting for period 1 - SynERMCSs (Nanoscale organization and dynamics of ER-mitochondria contact sites upon induction of synaptic plasticity)
Periodo di rendicontazione: 2023-01-12 al 2025-01-11
The endoplasmic reticulum (ER) is an essential cellular organelle responsible for regulating intracellular calcium levels, translocating and secreting proteins, and coordinating organelle dynamics, among other functions. ER extends throughout the cell as a network of flattened sacs and tube-like structures, rearranging its functional domains according to cellular demands and establishing contact sites with other organelles to promote signalling and transfer resources. ER-mitochondria communication has been of particular interest, as it coordinates the mitochondrial calcium uptake (essential for energy production and cellular signalling), mitochondrial dynamics and lipid exchange. Disruption of normal ER function or ER-mitochondrial contacts has been associated with neurological disorders.
Neurons are highly polarised cells capable of quickly processing and transmitting information through electrical and chemical signals at specific sites - synapses. They have three main compartments: dendrites - branch-like processes that receive incoming signals in postsynaptic terminals and pass them to the cell body; cell body - responsible for processing information and maintaining the essential cellular functions; and axon - long extension with pre-synaptic terminals that send signals to other neurons further on the neuronal network. The communication occurring at synapses is not static; these connections can strengthen or weaken based on experience, a process called synaptic plasticity that is the basis for learning and memory. The dynamic nature of cellular organelles and synaptic proteins is essential to promote and support functional and structural adaptations underlying synaptic plasticity.
In dendrites, ER presents mainly a tubular morphology that may extend into dendritic spines (small protrusions where most excitatory synaptic transmission occurs), determining their responsiveness to synaptic activity. Activity-mediated dynamics of ER and mitochondria are necessary to uphold synaptic plasticity, and neuronal activity also increases ER-mitochondria contacts. However, studying these processes is challenging because synapses are small and tightly packed structures. Traditional imaging techniques have limitations in capturing the fine details of synaptic structures. Recent advancements in super-resolution microscopy have revolutionised our ability to visualise dynamic changes in living neurons. These techniques can offer unprecedented insight into the organisation and dynamics of dendritic ER and mitochondria upon synaptic activity and plasticity.
Hence, through the application of super-resolution microscopy techniques, this project aims to study how neuronal activity affects the organisation and dynamics of ER and its mitochondria contact sites, and to assess its functional significance for mechanisms of synaptic activity and plasticity.
Neurons are highly polarised cells capable of quickly processing and transmitting information through electrical and chemical signals at specific sites - synapses. They have three main compartments: dendrites - branch-like processes that receive incoming signals in postsynaptic terminals and pass them to the cell body; cell body - responsible for processing information and maintaining the essential cellular functions; and axon - long extension with pre-synaptic terminals that send signals to other neurons further on the neuronal network. The communication occurring at synapses is not static; these connections can strengthen or weaken based on experience, a process called synaptic plasticity that is the basis for learning and memory. The dynamic nature of cellular organelles and synaptic proteins is essential to promote and support functional and structural adaptations underlying synaptic plasticity.
In dendrites, ER presents mainly a tubular morphology that may extend into dendritic spines (small protrusions where most excitatory synaptic transmission occurs), determining their responsiveness to synaptic activity. Activity-mediated dynamics of ER and mitochondria are necessary to uphold synaptic plasticity, and neuronal activity also increases ER-mitochondria contacts. However, studying these processes is challenging because synapses are small and tightly packed structures. Traditional imaging techniques have limitations in capturing the fine details of synaptic structures. Recent advancements in super-resolution microscopy have revolutionised our ability to visualise dynamic changes in living neurons. These techniques can offer unprecedented insight into the organisation and dynamics of dendritic ER and mitochondria upon synaptic activity and plasticity.
Hence, through the application of super-resolution microscopy techniques, this project aims to study how neuronal activity affects the organisation and dynamics of ER and its mitochondria contact sites, and to assess its functional significance for mechanisms of synaptic activity and plasticity.
Advanced labelling techniques use small fluorescent molecules to label specific cellular structures or proteins of interest. The combination of these techniques with STimulated Emission Depletion (STED) microscopy (super-resolution method that improves resolution by selectively de-exciting fluorophores around a focal point) has unveiled the nanoscale organisation of the ER in dendritic spines of hippocampal neurons. The morphological diversity of structures can be categorised into single tubules, multi-tubular structures and dense structures. Live cell imaging studies revealed that these structures can be highly dynamic, rearranging in seconds. To understand how neuronal activity impacts ER dynamics in dendritic spines, genetically encoded calcium indicators targeting the synapse were combined with event-triggered STED. This STED implementation performs real-time analysis to detect calcium spikes and initiate STED imaging at active synapses, allowing selective imaging and analysis of ER present in active synapses.
While the resolution obtained by STED microscopy is sufficient to study ER’s structure and dynamics, its resolution is not enough to fully resolve ER-mitochondria interaction. This requires applying specific tools, like ER-mitochondria contact sensors that emit fluorescence when these contacts occur or studying proteins previously associated with these sites. Both strategies were used to study ER-mitochondria contacts in hippocampal neurons.
While the resolution obtained by STED microscopy is sufficient to study ER’s structure and dynamics, its resolution is not enough to fully resolve ER-mitochondria interaction. This requires applying specific tools, like ER-mitochondria contact sensors that emit fluorescence when these contacts occur or studying proteins previously associated with these sites. Both strategies were used to study ER-mitochondria contacts in hippocampal neurons.
By providing a detailed, high-resolution view on the neuronal ER and its contacts with mitochondria, this project contributes to a better understanding of how neurons adapt in response to neuronal activity. Further research should explore how these fundamental cellular dynamics contribute to brain health and disease.