Publicação
Unravelling mitochondrial dynamics and turnover in the synapse
| datacite.subject.fos | Ciências Médicas::Medicina Básica | pt_PT |
| dc.contributor.advisor | Epifânio, Vanessa Alexandra dos Santos Morais | |
| dc.contributor.author | Cardanho-Ramos, Carlos | |
| dc.date.accessioned | 2025-02-24T18:38:44Z | |
| dc.date.available | 2025-02-24T18:38:44Z | |
| dc.date.issued | 2024-04 | |
| dc.date.submitted | 2024-01 | |
| dc.description.abstract | Neurons rely on mitochondria for ATP production and Ca2+ homeostasis, particularly at the synapse. As subcompartmentalized cells, they have different pools of mitochondria in each compartment. Achieving a non-uniform distribution requires mitochondria not only to be transported, but also to be retained in regions with high energy demands and high levels of Ca2+ . One of the goals of this thesis was to understand what makes mitochondria travel back and forward in a neuron and which mechanisms were activated to retain this organelle at the synapse. To address this, mouse primary neuron cultures, with fluorescent labelled mitochondria, were used to study the impact of the actomyosin cytoskeleton in mitochondrial transport and anchoring at the synapse. Taking advantage of a proteomic screen comparing synaptic with non-synaptic mitochondria, we selected two candidate proteins related with organelle movement: cell division control protein 42 (cdc-42), involved in actin polymerization; and myosin-VI, capable of anchoring mitochondria to actin cables. In the course of this project, a paper was published addressing all our questions, forcing us to focus on a different topic. In the other project of this thesis, we focused on understanding how mitochondria replicate in neurons. It is assumed that mitochondria are generated in the cell body and travel to the synapse to exert their functions. However, considering the rate of mitochondrial transport in neurons, the time it would take for a single mitochondrion to travel from the cell body to the synapse exceeds the half-life of most mitochondrial proteins. Our goal was to understand whether mitochondrial replication occurred in the periphery of neurons and which mechanisms were involved. We developed a technique to assess mitochondrial replication in mouse primary neuron cultures using 5-bromo-2′- deoxyuridine (BrdU)- and 5-ethynyl-2´-deoxyuridine (EdU)-labelling, two thymidine analogues that are incorporated into mtDNA upon replication. Most BrdU/EdU staining was observed in the cell body, but staining was also present to a lower extent in the periphery. Using microfluidic devices, where axons can be isolated from the cell body, we were able to add EdU only to the axonal side, without interfering with the cell body. In these conditions, EdU staining was only present in axons, confirming that mitochondrial replication in neurons can occur away from the cell body. We hypothesized that mRNA and local translation must be at place in the periphery of neurons in order to provide all the necessary machinery for mitochondria to replicate. To test this, we assessed mitochondrial replication upon inhibition of both nuclear-encoded and mitochondrial-encoded protein translation. Mitochondrial replication in neurons decreased when nuclear-encoded protein translation was inhibited. However, no differences were observed upon inhibition of mitochondrial-encoded protein translation. Taking advantage of the proteomic screen comparing synaptic with non-synaptic mitochondria, two candidate proteins related with protein translation were found upregulated in the synaptic fraction – eukaryotic elongation factor 1 alpha 1 (eEF1a1), involved in nuclear-encoded protein translation; and mitochondrial translation elongation factor Tu (TUFM), involved in mitochondrial-encoded protein translation. We performed loss and gain of function assays with our candidate proteins and assessed their impact on mitochondrial replication. When eEF1A1 was downregulated, we observed a decrease in mitochondrial replication in the periphery of neurons. This effect was rescued by reintroducing eEF1A1. Regarding TUFM, no differences were observed. Our results confirm that mitochondrial replication can occur in the periphery of neurons, and that this process requires nuclear-encoded protein translation, mediated by eEF1A1. Understanding how mitochondrial replication occurs in neurons, particularly at the level of the synapse, provides novel lines of research to tackle the pathophysiological mechanisms underlying neurodegenerative diseases. | pt_PT |
| dc.identifier.tid | 101603886 | pt_PT |
| dc.identifier.uri | http://hdl.handle.net/10400.5/98699 | |
| dc.language.iso | eng | pt_PT |
| dc.relation | Unravelling mitochondrial dynamics under turnover in the synapse | |
| dc.relation | Quality Control and Maintenance of Synaptic Mitochondria | |
| dc.subject | biogénese mitocondrial | pt_PT |
| dc.subject | neurónio | pt_PT |
| dc.subject | fator de elongação da tradução | pt_PT |
| dc.subject | eEF1A1 | pt_PT |
| dc.subject | síntese proteica | pt_PT |
| dc.subject | mitochondrial biogenesis | pt_PT |
| dc.subject | neuron | pt_PT |
| dc.subject | translation elongation factor | pt_PT |
| dc.subject | protein synthesis. | pt_PT |
| dc.title | Unravelling mitochondrial dynamics and turnover in the synapse | pt_PT |
| dc.type | doctoral thesis | |
| dspace.entity.type | Publication | |
| oaire.awardTitle | Unravelling mitochondrial dynamics under turnover in the synapse | |
| oaire.awardTitle | Quality Control and Maintenance of Synaptic Mitochondria | |
| oaire.awardURI | info:eu-repo/grantAgreement/FCT//PD%2FBD%2F135521%2F2018/PT | |
| oaire.awardURI | info:eu-repo/grantAgreement/FCT/OE/COVID%2FBD%2F152520%2F2022/PT | |
| oaire.awardURI | info:eu-repo/grantAgreement/EC/H2020/679168/EU | |
| oaire.fundingStream | OE | |
| oaire.fundingStream | H2020 | |
| person.familyName | Cardanho-Ramos | |
| person.givenName | Carlos | |
| person.identifier.orcid | 0000-0001-6178-1654 | |
| project.funder.identifier | http://doi.org/10.13039/501100001871 | |
| project.funder.identifier | http://doi.org/10.13039/501100001871 | |
| project.funder.identifier | http://doi.org/10.13039/501100008530 | |
| project.funder.name | Fundação para a Ciência e a Tecnologia | |
| project.funder.name | Fundação para a Ciência e a Tecnologia | |
| project.funder.name | European Commission | |
| rcaap.rights | openAccess | pt_PT |
| rcaap.type | doctoralThesis | pt_PT |
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| thesis.degree.name | Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2024 | pt_PT |