Periodic Reporting for period 1 - ParaplegiaAxonsER (Functional interactions between endoplasmic reticulum and mitochondria in Drosophila axons)
Reporting period: 2017-03-01 to 2019-02-28
Hereditary Spastic Paraplegias (HSPs) are a group of disorders characterised by lower limb spasticity and weakness, caused by selective degeneration of motor neurons. Whilst there are many genes implicated in HSP, the most commonly mutated group of genes are those involved in shaping the membrane of a specialised intracellular compartment, the endoplasmic reticulum (ER). The ER is critical in maintaining homeostasis within the cell, constantly sensing and relaying information in every part of the neuron. One key feature of the ER is its continuity, it invades every part of the cell through a network of sheets and tubules, which is key for its communication. Using Drosophila (fruit fly) as a model organism we disrupted several HSP genes that have been identified in humans, and observed that the integrity and continuity of the ER was perturbed in the longest motor neurons, analogous to the human condition [1]. To understand the functional consequences of this disruption, and learn more about how these mutations might lead to neurodegenerative disease, we needed to develop tools to interrogate this. One of the main ways in which the ER communicates, within itself and with other organelles, is with calcium ions.
Therefore I aimed to develop fluorescent calcium sensors for use in Drosophila, targeted to the interior (lumen) of ER tubules, to monitor changes in the level of calcium. Together with sensors in other subcellular compartments (mitochondria, cytoplasm and post-synapse), I planned to interrogate changes to calcium handling in HSP-mutant flies.
Therefore I aimed to develop fluorescent calcium sensors for use in Drosophila, targeted to the interior (lumen) of ER tubules, to monitor changes in the level of calcium. Together with sensors in other subcellular compartments (mitochondria, cytoplasm and post-synapse), I planned to interrogate changes to calcium handling in HSP-mutant flies.
My first objective was to develop the ER lumenal calcium sensors to specifically interrogate the role of ER calcium in neurodegeneration, with a particular focus on genetic causes which affect ER integrity. I successfully adapted sensors that were developed and used in neurons in culture [2], for use in Drosophila, through adaptation of the plasmid for injection into Drosophila embryos, with eventual generation of transgenic animals. I confirmed the ER localisation of the sensors within the cell, and together with sensors outside of the ER lumen; in the cytoplasm, have observed fluxes across the ER membrane. I am preparing a publication on using these sensors as tools to learn about calcium handling of the ER, and how mutations that affect ER integrity may cause neurodegenerative disease.
In addition to the ER lumen it is also of interest to monitor calcium in the cytoplasm and in the mitochondria (the power generators of the cell that are regulated by their contacts with the ER), and at the muscle interface where neuronal innervation occurs. I have obtained (cytoplasmic, mitochondrial mitoGCaMP3, and post-synaptic), and created (mitochondrial CEPIA2mt, CEPIA3mt and CEPIA4mt) transgenic stocks that contain sensors in these compartments for a complete toolkit to monitor neuronal calcium. Monitoring calcium in these different compartments gives us a more complete picture of the movement of calcium, and how changes in calcium handling as a result of genetic disruption, results in changes in synaptic transmission to the postsynapse.
Using the cytoplasmic sensor I have observed changes in calcium handling in mutants that disrupt two ER-shaping HSP proteins. The first protein, Reticulon-like 1 (Rtnl1), is involved in curving of the ER membrane. It’s mutation results in decreased ER levels with occasional fragmentation[1, 3]. The cytoplasmic sensor showed that the presynaptic response of Rtnl- mutant flies (lacking Rtnl1 protein) was significantly decreased compared to control flies. Additionally the Rtnl1- mutants took longer to reach peak fluorescence, and longer to decay. This implies reduced excitability of the presynaptic terminal, and could implicate ER pathophysiology in releasing to and/or uptaking calcium from the cytoplasm. To verify these results I am now increasing the number of samples, in addition to examining changes in the other subcellular compartments.
The second protein, atlastin (Atl), has a different role to Rtnl within the ER membrane. It is localised to specific regions within the membrane where it joins and fuses membranes of apposing ER tubules, thereby making the ER more interconnected. Mutation of the atl gene in Drosophila results in an increased amount of ER tubules, with preliminary Fluorescence Recovery After Photobleaching (FRAP) and Electron Microscopy (EM) analyses suggesting that they are less connected. Preliminary results using the cytoplasmic sensor suggest that, conversely to Rtnl, there is an increased presynaptic response compared to control. Taken together these results are quite intriguing, as although preliminary, they suggest a correlation between the amount of ER, and the excitability of the neuron. More samples, and recordings in other subcellular compartments will assist in verifying this observation.
Exploitation and Dissemination. I have presented these results in poster format at several scientific symposia (Tom Wahlig Stiftung 2017 and 2018, Federation of European Neuroscience Societies (FENS) Forum 2018, Neurofly 2018, Cambridge Neuroscience Symposium (CNS) 2019), as well as in an oral presentation at the Genetics Internal Seminar Series, Department of Genetics, University of Cambridge. I also gave an oral presentation to the wider research community at Newnham College, to which I am affiliated, in 2017. A scientific publication of the ER lumenal sensor development in is progress, with analysis of calcium handling in HSP-mutant flies to follow.
In addition to the ER lumen it is also of interest to monitor calcium in the cytoplasm and in the mitochondria (the power generators of the cell that are regulated by their contacts with the ER), and at the muscle interface where neuronal innervation occurs. I have obtained (cytoplasmic, mitochondrial mitoGCaMP3, and post-synaptic), and created (mitochondrial CEPIA2mt, CEPIA3mt and CEPIA4mt) transgenic stocks that contain sensors in these compartments for a complete toolkit to monitor neuronal calcium. Monitoring calcium in these different compartments gives us a more complete picture of the movement of calcium, and how changes in calcium handling as a result of genetic disruption, results in changes in synaptic transmission to the postsynapse.
Using the cytoplasmic sensor I have observed changes in calcium handling in mutants that disrupt two ER-shaping HSP proteins. The first protein, Reticulon-like 1 (Rtnl1), is involved in curving of the ER membrane. It’s mutation results in decreased ER levels with occasional fragmentation[1, 3]. The cytoplasmic sensor showed that the presynaptic response of Rtnl- mutant flies (lacking Rtnl1 protein) was significantly decreased compared to control flies. Additionally the Rtnl1- mutants took longer to reach peak fluorescence, and longer to decay. This implies reduced excitability of the presynaptic terminal, and could implicate ER pathophysiology in releasing to and/or uptaking calcium from the cytoplasm. To verify these results I am now increasing the number of samples, in addition to examining changes in the other subcellular compartments.
The second protein, atlastin (Atl), has a different role to Rtnl within the ER membrane. It is localised to specific regions within the membrane where it joins and fuses membranes of apposing ER tubules, thereby making the ER more interconnected. Mutation of the atl gene in Drosophila results in an increased amount of ER tubules, with preliminary Fluorescence Recovery After Photobleaching (FRAP) and Electron Microscopy (EM) analyses suggesting that they are less connected. Preliminary results using the cytoplasmic sensor suggest that, conversely to Rtnl, there is an increased presynaptic response compared to control. Taken together these results are quite intriguing, as although preliminary, they suggest a correlation between the amount of ER, and the excitability of the neuron. More samples, and recordings in other subcellular compartments will assist in verifying this observation.
Exploitation and Dissemination. I have presented these results in poster format at several scientific symposia (Tom Wahlig Stiftung 2017 and 2018, Federation of European Neuroscience Societies (FENS) Forum 2018, Neurofly 2018, Cambridge Neuroscience Symposium (CNS) 2019), as well as in an oral presentation at the Genetics Internal Seminar Series, Department of Genetics, University of Cambridge. I also gave an oral presentation to the wider research community at Newnham College, to which I am affiliated, in 2017. A scientific publication of the ER lumenal sensor development in is progress, with analysis of calcium handling in HSP-mutant flies to follow.
Development of the ER lumenal sensors gives us unparalleled insight into the functional aspects of the ER in relation to calcium handling. In my hands, this will enable understanding of how perturbations to ER integrity affect function.
More broadly, perturbations to calcium handling are widely considered as precursors to the molecular mechanisms underlying neurodegeneration, and these sensors will allow insight into the contribution of the ER in these pathways. While development of therapies is not a short-term goal of this work, it is a long-term one, and a better mechanistic understanding of the pathogenesis of HSP (and neurodegenerative disease more broadly), is the first step in this process.
More broadly, perturbations to calcium handling are widely considered as precursors to the molecular mechanisms underlying neurodegeneration, and these sensors will allow insight into the contribution of the ER in these pathways. While development of therapies is not a short-term goal of this work, it is a long-term one, and a better mechanistic understanding of the pathogenesis of HSP (and neurodegenerative disease more broadly), is the first step in this process.