Living Systems Institute

Nikolaou Group

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.

Image Nikolas Nikolaou Fish vision

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 including xenotransplantations

– Transcriptome profiling of cells

– Live imaging of cell behaviour and function in vivo

– Behaviour analysis to study circuit function

– Primary neuron cultures

– Biochemical assays to study protein-protein interactions  

Willing to supervise doctoral students

We are always open to students interested in neuronal development and neural connectivity. PhD funded projects in the lab will appear below when become available.

PhD projects currently available to apply:

Project 1

SNRNP70 is a major spliceosome protein whose function as part of the spliceosome is well described. Surprisingly, SNRNP70 also localises to axons and synapses and this non-nuclear pool of the protein is important for neuromuscular connectivity, likely acting to form and maintain such contacts. At the molecular level, non-nuclear SNRNP70 was found to interact with several RNA binding proteins (RBP), some of which are linked to neurodegeneration e.g., TDP-43, and modulate the local transcriptome. Our evidence suggests that SNRNP70 binds to mRNAs outside the nucleus to regulate their axonal transport and stability. However, the precise role of extra-nuclear SNRNP70 is still elusive. What repertoire of global RBP-RNA interactions is modulated by cytoplasmic SNRNP70? Within what regions of transcripts are these binding sites clustered? More importantly, is the role of extra-nuclear SNRNP70 perturbed during neurodegeneration? Here, we will address these questions using zebrafish embryos/larvae and human iPSCs together with bioinformatic analyses.

Year 1:

  • Mutations in the TDP-43 (TARDBP) gene are a cause of familial ALS (Amyotrophic Lateral Sclerosis) and frontotemporal lobar degeneration (FTLD) with many mutations located in the C-terminal domain, affecting protein-protein interactions and TDP-43’s function. To determine whether the glycine-rich C-terminus domain of TDP-43 mediates interactions with SNRNP70, we will generate a construct containing this C-terminal domain, overexpress this in cells and perform co-IP experiments.
  • If SNRNP70-TDP-43 interactions remain, we will focus on familial ALS variants e.g., G348C, M337V, and N345K found in the C-terminus of TDP-43. We will induce these mutations and confirm whether any of these perturb SNRNP70-TDP-43 interactions.
  • Transgenic lines of zebrafish overexpressing DTP-43 ALS mutations that prevent such protein-protein interactions will be established.

Year 2:

  • Develop the methodology required to perform ePRINT on subcellular fractions (e.g., nucleus vs cytoplasm) of primary neurons or iPSCs. Confirmation that the methodology works by knockdown of key RBPs in the nucleus or cytoplasm e.g., FUS vs FMR1.
  • Zebrafish lines (snrnp70 KO, snrnp703xNLS-eGFP KI) will be used to generate primary neuron cultures and identify/characterise global changes in RBP-RNA sites when SNRNP70 is depleted or absent from cytoplasmic regions. The findings will be analysed to resolve potential mRNA binding signatures of cytoplasmic SNRNP70.

Year 3:

  • To establish links between neurodegeneration and extra-nuclear SNRNP70 function, the transgenic zebrafish generated during year 1 will be utilised. The consequence of mutant TDP-43 overexpression on the cytoplasmic SNRNP70 function will be determined including cytosolic SNRNP70 protein levels, changes in the abundance and mRNA transport of transcripts previously identified to be regulated by the extra-nuclear pool of SNRNP70 (e.g., rab1bb), and changes in the global RNA binding profiles linked to cytoplasmic SNRNP70. RBP-RNA binding alterations will also be investigated in human iPSC carrying the same TDP-43 mutations.

This project will advance our understanding of the molecular function of SNRNP70 during development of neuronal connections but also provide valuable insights into how SNRNP70 function might be perturbed during neurodegeneration. This project will suit a student who is keen on working at the interface between biology and computational analysis of transcriptomic datasets and is enthusiastic about optimising existing methodologies to identify and characterise protein-RNA interactions in subcellular compartments. This project will allow the student to learn a range of valuable skills for their future career in molecular/cell biology, imaging, transcriptomic data analysis, and working with cell and animal models for neurodegeneration.

Project 2

SNRNP70 is a major spliceosome protein whose function as part of the spliceosome is well characterised. Surprisingly, it also localises to extranuclear regions of neurons (axons, dendrites and synapses) and we have recently shown that this non-nuclear pool is essential for neuromuscular junctions (NMJs). At the molecular level, SNRNP70 regulates the local transcriptome. Our results also show that SNRNP70 co-localises with mRNAs outside the nucleus and regulates their axonal transport and stability in zebrafish. These finding suggest that cytoplasmic SNRNP70 utilises its RNA recognition motif (RRM) to bind to mRNAs. However, the RNA binding ability has not been directly demonstrated (e.g., functionally), and it’s still elusive whether SNRNP70 localises and regulates mRNA processing at NMJs. Here, we will address these questions using a combination of molecular genetics and synthetic peptide approaches.

Year 1:

  • To test the requirement for RNA recognition, a cytosolic SNRNP70 lacking its RRM will be expressed in zebrafish embryos. Additionally, to interfere with SNRNP70-RNA binding, the RRM sequence will be fused to a small epitope tag and ectopically expressed in cells. The effects these manipulations have on NMJs will be quantified to evaluate whether RRM is important for the extra-nuclear function of SNRNP70.
  • In parallel, a cell co-culture system for NMJs will be established by culturing human motor neurons with muscle fibres and demonstrating the presence of appositions between ChaT+/SV2+ axon terminals and alpha-BTX+ clusters. This co-culture system will be valuable for visualising NMJs later in the project.

Year 2:

  • To selectively tag SNRNP70 and monitor its localisation in cells, bioluminescent/fluorescent peptide binders that target a functionally inert SNRNP70 epitope will be designed. These peptide binders will be expressed in the co-culture system for analysing the localisation of SNRNP70 in cells.
  • To block the RNA binding ability of SNRNP70, photoactivatable peptide binders will be designed to target its RRM. The efficacy of these peptides in blocking RNA binding either throughout the cell (to test the functionality of the peptide) or in subcellular compartments (by fusion to soma, axon, synapse restricted proteins) will be tested. These experiments will enable us to determine whether the RNA binding of SNRNP70 is important for the formation, maintenance of NMJs or both by photoactivating the peptide binder before or after contacts have been made, respectively, and will be done in both live zebrafish and co-cultures for comparison.

Year 3:

  • Analogous RRM peptide binders for ALS-linked RNA-binding proteins e.g., TDP-43, SFPQ will also be designed and generated. Their efficacy will then be tested in both live zebrafish and co-culture systems.

This project will advance our understanding of the molecular function of SNRNP70 during development and maintenance of neuronal connections. This project will suit a student who is keen on working at the interface between biology and peptide chemistry and is enthusiastic about developing new methodologies to study protein-RNA interactions in cells. This project will allow the student to learn a range of valuable skills for their future career in molecular/cell biology, imaging, peptide design, and working with animal models.

Research staff positions

Highly 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.

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