Nikolaou Group – New LSI Group September 2024 Reasearch Interests Neurons are specialised cells in our body that have long extensions allowing them to form connections (called synapses) with other neurons, leading to the establishment of neural circuits. The main function of neural circuits is to conduct signals that coordinate our bodily functions, thoughts, sensations, and perceptions of the world. Overall, neural circuit function and ultimately behaviour depend on the precise formation of synaptic connections. Hence, changes in the way neurons are wired during development can lead to neurodevelopmental disorders such as autism spectrum disorder, intellectual disability, and Schizophrenia. Moreover, failure to properly maintain synapses throughout life often results in neurodegenerative conditions such as Alzheimer’s and motor neuron disease. Using zebrafish as a vertebrate genetic model system, the overarching aim of research in the Nikolaou group is to elucidate how functional neural connections are formed in the brain. We strive to understand the molecular and cellular mechanisms that regulate such important decisions, how connections are maintained throughout life and how deviations from the normal wiring program can lead to dysfunctions in the brain. Current projects available in our group include: (i) Linking neuronal structure with function. (ii) regulation of local RNA processing in neurons. (ii) generating zebrafish models for neurological diseases. Linking neuronal structure with function Neural circuit function and ultimately animal behaviour depend on the precise formation of synaptic connections in the brain. One of the main aims of research in our group is focused on understanding how neuronal structure relates to neuronal function. Previous research has shown that the laminar organisation of synaptic connections in the optic tectum (which are affected in a zebrafish robo2 null allele) is dispensable for the correct wiring of visual circuits, however it is crucial for the rapid assembly of neural networks. This suggests that neuronal structure is key to brain function. Several single-cell RNA sequencing results published by other groups revealed several genes that are uniquely expressed by neuronal subtypes. We are currently using this knowledge and through molecular genetic approaches widely used in our group (e.g., transgenesis, CRISPR/Cas9 knock-in) we label identifiable classes of neurons and determine their structure, connectivity patterns, neurotransmitter phenotype, functional response properties, and contribution to behaviour. Regulation of local RNA processing in neurons In neurons, RNAs localise to axons, dendrites and synapses (collectively known as neurites), where they facilitate rapid responses to local needs, such as axon growth/extension, branching, synapse formation, and synaptic plasticity. Recent studies have uncovered a diverse range of coding and non-coding RNAs localised within neuronal projections and shown that alterations in their abundance and/or metabolism can exert an influence in local decisions. RNA binding proteins (RBPs) mediate the vast majority of RNA trafficking and processing both in the nucleus and the cytoplasm. Whereas the protein-protein and RNA-protein interactions of RBPs in the nucleus are well-characterised, the function of analogous interactions in neurites remains elusive. We have recently shown that U1-70K/SNRNP70, a major RNA splicing regulator, localises to ribonucleoprotein complexes inside axons and regulates the establishment of neuromuscular synaptogenesis. We are hypothesising that the cytoplasmic/axonal pool of SNRNP70 modulates the axonal transcriptome through one or more of the following mechanisms: RNA trafficking, stability and degradation, translational repression, and local processing. Using imaging and molecular genetic techniques in zebrafish, our group has recently established knock-in and transgenic lines, which are enabling us to image the intricate relations between SNRNP70 and its axonal RNA targets in vivo. We currently have funding from the Academy of Medical Sciences to determine the protein-protein interactions mediating the axonal functions of SNRNP70. Moreover, we recently obtain a BBSRC grant (starts in early 2025) to explore the molecular mechanisms by which SNRNP70 regulates the axonal transcriptome. Many RBPs, including RNA splicing regulators, have been shown to form insoluble cytoplasmic aggregates, which interfere with the function of the neuron eventually leading to synapse loss and degeneration. We and other groups have shown that many splicing regulators localise to the nucleus and neurites in a bimodal fashion and many of these RBPs have been found to aggregate in neurodegenerative diseases e.g., Alzheimer’s disease and Amyotrophic Lateral Sclerosis (ALS). Another line of research in our group focuses on understanding how these disease-causing aggregates interfere with the function of these proteins not only in the nucleus but also locally within neurites, and what role they play in the breakdown of neurons. Understanding and treating neurological diseases Many genes linked to neurological diseases such as neurodevelopmental disorders and neurodegeneration have been shown to be important for neuronal development. As a first step in understanding human disease, we determine the physiological function of disease-related genes in the nervous system. Subsequent studies are focused on known candidate risk human mutant variants. Our group has expertise in generating transgenic animals as well as CRISPR/Cas9 knock-out and knock-in lines. Phenotypic characterisations include assessments of amount of mRNA and/or protein levels and distribution of proteins within tissues at cellular and subcellular level. Changes in neuronal morphology and synaptogenesis are also explored. Genetically encoded calcium reporters and light-sheet microscopy are used to record neuronal activity in the entire larval brain and generate network activity maps to compare with control neural networks. In parallel, animal behaviour is investigated to examine the functional outputs of the nervous system. We currently have funding from the Royal Society to generate zebrafish epilepsy genetic models. It is estimated that more than half of childhood epilepsies are due to genetic aetiologies. Seizures are difficult to control, and medications e.g., anticonvulsant drugs are mostly focused on reducing their effect. This project aims to identify better treatments for childhood epilepsies. Currently, only few in vivo models for epilepsy are available and most of these are mouse genetic models, which are not suitable for high-throughput drug screening. We are using zebrafish (the least sentient vertebrate animal model with high level of conservation of anatomical and physiological brain connectivity that is translatable to humans) to establish genetic models of mutations known to cause childhood epilepsies. A major advantage of using zebrafish is that the system is amenable to large-scale small molecule screening, once a disease model is established. Broad technical expertise in our group – Genetic alterations using CRISPR/Cas9 and transgenesis techniques – Grafting/transplantation of cells or tissues – Transcriptome profiling of cells – Live imaging of cell behaviour and function in vivo – Behaviour analysis to study circuit function – Primary neuron cultures Willing to supervise doctoral students We are always open to students interested in neuronal development and connectivity. PhD funded projects in the lab will appear here when become available. PhD opportunities currently available in our group (click on the links below for more details on how to apply): GW4 BioMed MRC DTP Project More details about this project can be found by clicking the above link. LSI PhD Programme in Complex Living Systems Imaging the life of an endogenous RNA transcript from birth to death in an in vivo setting mRNAs are synthesised in the nucleus by RNA polymerases. However, this is only the first in a series of processing steps transcripts will undergo, which will determine the amount of protein being produced. For instance, an mRNA in the form of pre-mRNA transcript is capped, spliced and polyadenylated before being transported from the nucleus to the cytoplasm. Within the soma cytoplasm, the transcript undergoes quality control to ensure mRNA integrity and function, and eventually is translated into a protein by ribosomes. The mRNA is eventually degraded to regulate protein levels. Each of these steps is highly regulated, contributing to gene expression and protein synthesis within the cell. This is especially evident in neurons, where the soma harbouring the mRNA-producing nucleus can be millimetres and even centimetres away from their eventual destination. This underscores the importance of RNA transport, stability and localized translation at synapses, growth cones and dendritic and axonal branch points for normal neuronal development and function. The overall aim of this project is to monitor the life of an endogenous mRNA, from birth to death, using zebrafish as an in vivo model organism. This project will allow the student to learn a range of valuable skills for their future career in molecular/cell biology, imaging, mathematical modelling and working with animal models. In detail, the student will learn how to: generate targeting constructs for CRISPR/Cas9-mediate gene editing in zebrafish to insert specific sequences into candidate genes for monitoring their mRNA products. use imaging techniques to monitor the life of targeted mRNA transcripts within neuronal compartments. use mathematical modelling to examine mRNA dynamics, such as those occurring during nuclear export, axonal transport and stability/degradation. This project will suit a student who is keen on working at the interface between biology and mathematical modelling, and is enthusiastic about developing new tools and approaches to label and monitor individual mRNA molecules of interest within their natural environment. Research staff positions Motivated researchers interested in joining our group are always welcomed. If interested, please contact us well in advance for an informal discussion. Current positions will be advertised below when available. Postdoctoral Research Associate position (apply by 12/11/2024) Teaching interests Nikolas is a Senior Lecturer in the Department of Clinical and Biomedical Sciences (Exeter Medical School). His teaching roles span across both undergraduate (UG) and postgraduate (PG) degrees. He delivers lectures on the UG Neuroscience course programme. He supervises final year UG and offers MSc lab projects. He supervises on average 2 PhD students working in his lab. NEU1006 – Introduction to Neuroscience NEU2018 – Neural Circuits NEU3001 – UG research projects NEU3008 – Frontiers in Neuroscience NEUM006 – MSc Research dissertation projects