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Mitochondrial Dynamics and Local Protein Synthesis in Dendrites

Periodic Reporting for period 1 - NEUROMITO (Mitochondrial Dynamics and Local Protein Synthesis in Dendrites)

Reporting period: 2016-01-01 to 2017-12-31

The human brain consumes 20% of the total energy in the body while it constitutes only 2% of the body weight. Particularly, proteostasis and modifying synaptic proteomes represent a large energy demand during synaptic plasticity and little is known on how the energy demands are met locally at dendrites and spines. Mitochondria, the powerhouses of cells, are found in neuronal compartments, but their molecular regulation in response to neuronal energy demands and their role in fueling local neuronal function remains largely unaddressed. During my Marie Curie Individual postdoctoral fellowship, I investigated the significance of mitochondria in local translation in the Schuman group, employing state-of-the-art imaging, proteomics and transcriptomic methodologies developed in the lab.

The overall goal of the project was to elucidate the significance of mitochondria during high-energy demands of local protein synthesis with the following objectives:
i) investigate mitochondrial compartmentalization and its activity-dependence in local dendritic translation; and
ii) analyse mitochondrial proteomics and transcriptomics during synaptic plasticity.

By refining and pushing the limits of conventional and super-resolution microscopy to image live dendritic mitochondria, experimentally manipulate local mitochondrial function in dendrites, label and visualize newly synthesized proteins in response to synaptic stimulation, I have demonstrated that: mitochondria exist in spatially stable compartments in dendrites and serve as local energy reserves to fuel synaptic protein synthesis during synaptic activity. These findings have revealed that in addition to the presence of localized translational machinery in dendrites, local compartments of energy exist, thereby opening up new unexplored questions on synaptic plasticity and metabolism.

In order to further investigate how the mitochondrial proteome is modulated during synaptic plasticity, I employed a previously reported strategy for labeling and isolation of the sub mitochondrial proteome, characterized and established it as a tool to examine the neuronal mitochondrial proteome and its regulation during neuronal activity.
"I investigated the significance of dendritic mitochondria in fueling local dendritic protein translation using conventional and super resolution fluorescence microscopy. Mitochondria were found to exist in spatial compartments of 30 μm that were stable for a period of 60 min in dendrites, but not in axons. Super resolution imaging of the measured mitochondrial compartments revealed that they were comprised of single or multiple mitochondrial filaments. On local perturbation of the function of these mitochondrial compartments, synaptic plasticity-induced protein translation was affected in spines. However, local protein translation during basal neuronal activity remained unaffected. These results suggest that local mitochondrial compartments fuel local synaptic protein translation in an activity-dependent manner (Figure 1, Rangaraju V et al, in review).

I developed and characterized a novel methodology to selectively label and isolate the mitochondrial proteome from sub mitochondrial compartments –mitochondrial matrix and outer mitochondrial membrane– in neuronal cultures. I am currently exploiting this methodology to study the regulation of mitochondrial proteome during neuronal activity (Figure 2, Rangaraju V et al. manuscript in preparation). I am also investigating the mitochondrial proteome in sub neuronal compartments –soma and neurites– using a novel platform to grow neurons in physically separated chambers.

In summary, I have demonstrated that in addition to the presence of localized translational machinery in dendrites, local compartments of energy exist to fuel local translation in an activity-dependent manner. Further, I have implemented a novel methodology to study the sub mitochondrial proteome in neurons to investigate the modulation of the mitochondrial proteome during neuronal activity.

These findings have been presented at various international conferences – Gordon Research Conference Cell Biology of the Neuron 2016; Gordon Research Conference Dendrites: Molecular Structure & Function 2017; EMBO Fellows’ meeting 2017; and at science seminars open to the public (Bar of Science of MPIBR 2017, Ivy Circle and Cornell alumni club of Frankfurt).

This project has resulted in 3 manuscripts:
1. Vidhya Rangaraju*#, Susanne tom Dieck and Erin M Schuman, Local translation in neuronal compartments: how local is local? EMBO reports 18, 693 (2017).
Number of citations: 16. # Corresponding author.
2. Vidhya Rangaraju*, Marcel Lauterbach, Erin M Schuman, Spatially stable mitochondrial compartments fuel activity-dependent local translation, in review.
3. Vidhya Rangaraju*, Christina Thum, Fiona Rupprecht, Julian Langer, Erin M Schuman, Local mitochondrial proteome remodeling during synaptic activity, in preparation.
Previous measurements of mitochondrial motility in dendrites have been limited to shorter durations of 3-10 min or much longer periods across development. Here, I have characterized and measured mitochondrial compartments and its dynamics in dendrites for a long duration of 60 min and have pushed the limits of the state-of-the-art to demonstrate that these mitochondrial compartments are comprised of single or multiple mitochondrial filaments in live neurons –previous observations of multiple mitochondrial filaments were only reported via static electron microscopic images. In order to investigate the functional significance of these mitochondrial compartments I characterized a novel methodology to locally perturb mitochondrial function by light in dendrites. Further, I have combined this methodology with the first time measurements of spine-specific modulation of the synaptic proteome following a synaptic plasticity-inducing stimulus that has made a significant contribution to the state-of-the-art in the field.

So far, mitochondrial proteomics has only been possible with purified mitochondrial samples, which are often associated with other organellar contaminations. The novel methodology I have developed in neurons to label and detect the sub mitochondrial proteome, facilitates better detection of the sub mitochondrial proteome with fewer contaminations and a wider dynamic range compared to a mitochondrial proteome dataset obtained from whole neuronal cultures or purified whole mitochondria. Since the mitochondrial proteome labeling is done in live neurons in situ, this methodology also allows the identification of novel neuronal mitochondrial proteins that were otherwise unidentified by conventional mitochondrial purification and proteomic strategies.

I strongly believe that elucidating the role of mitochondria in local translation sheds light on the novel molecules involved in the energetics of the process and the molecular mechanisms of synaptic plasticity and memory formation. Particularly, the findings of this research project will not only aid in better understanding of mitochondrial biology in the context of neuronal function, but also in neurological disorders –Alzheimer’s and Parkinson’s– that are associated with mitochondrial dysfunction aiding the development of novel therapeutic interventions in the future.
Figure 2 Sub-mitochondrial proteome labeling in neurons
Figure 1 Mitochondrial compartments fuel activity-dependent protein translation in spines.