Quality Control and Maintenance of Synaptic Mitochondria

Synaptic mitochondria compared to mitochondria in other cells, need to cope with increased calcium load, more oxidative stress, and high demands of energy generation during synaptic activity. My hypothesis is that synaptic mitochondria have acquired specific mechanisms to manage local stress and that disruption of these mechanisms contributes to neurodegeneration.
How mitochondria sense their dysfunction is unclear. Even more intriguing is the question how they decide whether their failure should lead to removal of the organelle or dismissal of the complete neuron via cell death. We anticipate that these decisions are not only operational during disease, but might constitute a fundamental mechanism relevant for maintenance of synaptic activity and establishment of new synapses.

SYNAPTICMITOCHONDRIA aims to uncover uncharacterized novel components of synaptic mitochondria and to scrutinize quality control mechanisms that synaptic mitochondria use to sense and maintain their intrinsic function in a healthy brain. Even though mitochondrial function has been implicated in synapses maintaining their robustness, little is known about the composition, maintenance and intrinsic properties of synaptic mitochondria. Here we propose to use two complementary approaches to address these questions. A proteomic approach will be used to identify the protein fingerprint of synaptic mitochondria and to compare them to mitochondria from other tissues. Additionally, the identification of key players of the proposed regulatory pathways involved in intrinsic mitochondria quality control will also be attained. In a complimentary approach, we will exploit our findings and use in vitro and in vivo experimental approaches to measure mitochondrial function of synaptic versus non-synaptic mitochondria and the relevance of those changes for synaptic function. Our work will unravel the specific properties of synaptic mitochondria and provide much needed insight in their hypothesized predominant role in neurodegenerative disorders.

Mitochondria at the synapse have a pivotal role in neurotransmitter release, but almost nothing is known about synaptic mitochondria composition or specific functions. Synaptic mitochondria compared to mitochondria in other cells, need to cope with increased calcium load, more oxidative stress, and high demands of energy generation during synaptic activity.
The first aim of this research project is to identify and characterize novel components of synaptic mitochondria. For this, we have determined the proteomic profile that discriminates synaptic mitochondria from non-synaptic mitochondria, and through Bioinformatics analysis we have identified our 10 top candidate proteins. At present, we have determined the protein fingerprint of synaptic mitochondria. By assessing several mitochondrial parameters, such as cristae organization, bioenergetic profile and overall mitochondrial capacity of synaptic mitochondria compared to non-synaptic mitochondria, we have determined that synaptic mitochondria have a unique electron transport chain organization that enables them to deal with the high energy demands present at synapse. Additionally, we have also unveiled that synaptic mitochondria have a higher flexibility for different fuel sources. Moreover, we have observed that mitochondrial biogenesis can occur at the level of the synapse, and that mitochondrial replication does not only occur at the soma, as previously described.
We have also developed a methodology that enables the determination of mitochondrial properties in vivo in a living mouse brain. For this, we have capitalized on a mitochondrial fluorescently tagged transgenic mouse and in combination with 2-Photon live imaging we can determine mitochondrial properties in a healthy and diseased mouse brain.
The second aim of this research project is to pinpoint mitochondrial quality control (MQC) mechanisms that synaptic mitochondria use to sense and maintain their intrinsic function. We have established reporter cell lines that will aid in understanding the cross-talk between PINK1/PARL/PGAM5. However, we concluded that this crossroad probably does not have a major contribution to regulating the quality control mechanisms of mitochondria. Nevertheless, we further pursued the role of PINK1 in dictating overall mitochondrial fate, and to understand the function of this mitochondrial gatekeeper protein PINK1 in different neural cell types. For this, we have developed a methodology that enables the isolation and culture of primary neurons, astrocytes and microglia cells from the same mouse brain. The understanding of the crosstalk mediated by PINK1 between this different neural cell types within the context of a healthy brain will determine how these cells go astray in a neurodegenerative scenario. Furthemore, we have also scrutinize the impact of several parkinson's disease related PINK1 clinical mutations on overall PINK1 kinase function.
Our integrative approach, based on using an unbiased approach and a candidate gene approach, is providing a unifying model revealing the mechanisms involved in maintaining a healthy pool of mitochondria at the synapse.

SYNAPTICMITOCHONDRIA will significantly advance the understanding of the role of synaptic mitochondria in maintaining synapse function.
This work will reveal a proteome specific to synaptic mitochondria and unravel the quality control mechanisms that regulate synaptic mitochondria in a healthy and damaged brain. Additionally, we will have developed a unique array of methodology to evaluate the function of mitochondria in synapses which will position us in a unique way to address the many speculative links made between mitochondria and neurodegenerative disorders. Finally, a successful outcome of this research project will enable the identification of molecular mechanisms that cause neurodegenerative disorders, opening the avenues to new therapeutic approaches and moreover, to a contribution of overall brain function.

Periodic Reporting for period 5 - SynapticMitochondria (Quality Control and Maintenance of Synaptic Mitochondria)

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