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Molding the Brain: Drosophila Neurotrophins in Brain Plasticity and Neurodegeneration

Final Report Summary - NPN (Molding the Brain: Drosophila Neurotrophins in Brain Plasticity and Neurodegeneration)

The NPN project aimed to investigate the functions of Drosophila neurotrophins in the formation and plasticity of the adult fruit-fly brain.

An organism’s ability to respond to its environment and learn from experience is crucial for survival, and in the course of evolution, the interaction of organisms with their environment results in diverse brain types and behaviours. In the brain, adaptation at a cellular level is manifested as synaptic and structural plasticity, including changes in neuronal number, axonal and dendritic complexity, in neural circuits and in the volume of different brain domains. Structural plasticity has long been known to be dependent on neural activity in vertebrates. There is evidence that the brain of the fruit-fly Drosophila is also structurally plastic: enriched environments, sensory stimulation and increased neuronal input cause increased volume in specific brain regions, while deprivation of stimuli reduces brain size and neuronal complexity. Different brain domains respond differently to sensory deprivation, and flies with mutations in genes involved in learning and memory have decreased plasticity, revealing functional correlates of structural plasticity. Plasticity of neuronal complexity is also present in the locomotor system of Drosophila larvae. Conversely, failure of plastic mechanisms may underlie the neurodegeneration that occurs as we age and enhancing plasticity is protective against neurodegeneration. The understanding of how such structural changes come about, and whether they are a requirement for normal brain function, is scarce, and it is an important area of neuroscience.

The neurotrophins (NTs) are the main protein family linking structure and function in the mammalian brain. They have important functions in nervous system development, as they regulate cell proliferation and neuronal survival, axon targeting and connectivity, thus matching the appropriate number of neurons to the target to enable neuronal function and normal behaviour. NTs are also required for synaptogenesis, synaptic transmission, learning, memory and cognition. Most psychiatric and neurodegenerative diseases involve abnormal NT function. For their influence in nervous system structure and function, the NTs are also perhaps the most promising factors to induce nervous system regeneration after damage. For a long time the NTs were thought to be restricted to vertebrates. Whilst this provided a mechanistic explanation for the outstanding properties of the mammalian brain, it was also taken to imply that invertebrate and vertebrate brains might be formed and function in fundamentally different ways. However, over the recent decade, orthologues and homologues of the NTs have been found across the animals, throughout most invertebrates within the deuterostomes and protostomes, as well as the vertebrates. This means that the NTs most likely have fundamental functions in the formation and function of all brains.

The Host laboratory recently discovered a neurotrophin family in Drosophila (called Drosophila neurotrophins). They were shown to regulate neuronal survival and axon targeting, and more recently they have been shown to be required for synaptogenesis (Sutcliffe, Forero, Zhu, Robinson and Hidalgo 2013 PLoS One). The Host laboratory also invested a considerable effort to identify the DNT receptors, which were unknown when this project started. The discovery of the receptors was recently published by the host team in a publication in which the Researcher is also an author (McIlroy et al 2013 Nature Neuroscience).

This project aimed at investigating whether DNTs are involved in the formation of the adult brain and their potential involvement in the regulation of experience dependent structural brain plasticity. This will shed light towards understanding the mechanisms by which structure and function are linked in the brain. Ultimately, this will also provide insights into understanding how the brain works, and what fails during degeneration and upon damage.

The tasks for the project were to determine the circuits involving DNTs in the fly brain, investigate how the DNTs influence brain plasticity in normal development, and upon experience, and whether these mechanisms may also be investigated in the context of neurodegeneration.

To identify the circuits in which the DNTs are expressed and/or functional a variety of methods were used, including in situ hybridisations, generation of transgenic flies with DNT-specific GAL4-reporters, use of MiMIC-GFP gene trap insertions, and generation of custom made antibodies. The GAL4 approach revealed very interesting patterns in the adult brain. However, this was not always an ideal method, since it some cases it also revealed cells that did not coincide with those labelled by the antibodies. MiMIC-GFP revealed the endogenous pattern of some of the genes, but it did not work for all. The antibodies revealed interesting distributions of the endogenous proteins in the adult brain. This was on the whole the best method, but in some cases we were not able to demonstrate un-disputably that the antibody was specific only to one particular DNT and that there was no cross-hybridisation. Thanks to these major efforts, we have been able to establish a very detailed map of where the DNT ligands are expressed in the adult brain. In the course of this project, the Host lab identified the DNT receptors. Using the same multiple methods as above, the expression patterns of the receptors in the adult brain were also characterised by the Researcher. The characterisation of the expression patterns in the adult brain of ligands and receptors resulted in data that contributed to a publication in which the Researcher is an author (McIlroy, Foldi, Aurikko, Wentzell, Lim, Fenton, Gay and Hidalgo, 2013 Nature Neuroscience). The Researcher also provided in vivo co-IP evidence that the candidate receptors could physically bind the ligands in transgenic flies, and these data also form part of the paper in Nature Neuroscience. We obtained further abundant data on the distribution of the ligands and receptors that were not included in that paper and will form part of future publications.

To identify the cellular events regulated by the DNTs and their receptors, new null mutant alleles were generated. Cellular events and processes such as axonal patterns and cell number were analysed, and for which several defects were observed. The analysis of their influence of the DNTs in dendrite patterns is currently under way by a student in the Host lab supervised by the Researcher.

To investigate the involvement of the DNTs in structural experience dependent plasticity, flies have been bred in different conditions of constant darkness and compared to flies bred in constant light. This has been done in different genotypes in which the function of the DNTs and their receptors is altered. Data are currently being analysed. To this aim, the Researcher has extended her stay in the Host lab (using Host funds) for a further four months after the termination of the Marie Curie Fellowship.

The aim to investigate the functions of the DNTs in neurodegeneration was abandoned, due to the unforeseen delays in achieving the other aims. We encountered problems with: the incubator in which the flies were bred for these experiments broke down in multiple ways (first, the temperature went, another time the neon light bulb); the experiments were thus carried out in another incubator which was subsequently shown not to retain temperature constant, and thus the data resulting from those experiments were invalidated; and finally, the power station supplying energy to the University of Birmingham burnt down, which resulted in the complete loss of power for one week, and the breakdown of a confocal microscope (which the insurance refused to pay for, and as a result was never mended).

The project has so far resulted in one publication in the high profile journal Nature Neuroscience. We anticipate the remaining data will contribute to at least one more publication.