Themistoklis Tsarouchas

All Publications

• The LncRNA MYRACL Regulates Human Oligodendrocyte Maturation and Myelination. Molecular therapy : the journal of the American Society of Gene Therapy Tsarouchas, T. M., Vacante, F., Kazakou, N. L., Wagstaff, L., Bennett, M., Zoupi, L., Gibson, E. M., Baker, A. H., Williams, A. 2025

  Abstract

  Recent studies have described disease-associated expression patterns of long non-coding RNAs (lncRNA) associated with neurodevelopment and neurodegeneration, highlighting their potential as regulators of function and therefore potential therapeutic targets. Oligodendrocyte dysfunction drives central nervous system (CNS) myelin disruption in neurological disorders, but the mechanisms underlying impaired myelin patterns are still poorly understood. In this study, we uncover a role for the long noncoding RNA (lncRNA) MYRACL (MYelination RegulAting oligodendroCyte associated LncRNA), as regulator of functional maturation and oligodendrocyte myelination. Analysis of RNA-sequencing data performed in human post-mortem brain tissue revealed MYRACL among the top enriched genes expressed in the oligodendrocyte (OL) population compared to the oligodendrocyte precursor cell (OPC) cluster. We validated this finding in an embryonic stem cell (ESC)-derived oligodendroglia cell culture model. Analysis of evolutionary conservation and protein coding potential showed that MYRACL is non-coding and may exhibit conserved regions across mammalian species. Further co-expression analysis of lncRNAs-mRNAs suggested that expression of MYRACL positively correlates with genes known to be involved in driving oligodendroglia differentiation. GapmeR-mediated knockdown of nuclear MYRACL disrupted OL maturation in vitro, while lentivirus-mediated overexpression promoted OL differentiation with enhancement of myelin formation in-vitro. Our findings highlight MYRACL as a novel regulatory mechanism in human OL maturation and myelination. By providing a human, translationally relevant platform, this work advances our ability to model human myelination in vitro and paves the way for precision medicine approaches targeting lncRNA-mediated dysregulation in neurodevelopmental and neurodegenerative diseases.

  View details for DOI 10.1016/j.ymthe.2025.08.011

  View details for PubMedID 40783783

• Protocol for assessing myelination by human iPSC-derived oligodendrocytes in Shiverer mouse ex vivo brain slice cultures. STAR protocols Tsarouchas, T. M., Zoupi, L., Williams, A., Gibson, E. M. 2025; 6 (1): 103609

  Abstract

  Human induced pluripotent stem cell (iPSC)-derived oligodendrocytes are a powerful tool for studying aberrant myelination in neurodegenerative and neurodevelopmental disorders; however, they often fail to myelinate in vitro. Here, we present a protocol for axonal ensheathment and perinodal segmentation using an ex vivo model. We describe steps for preparing Shiverer mouse brain slice cultures, oligodendrocyte transplantation, visualization, and analysis. This approach suits multiple culture formats, highlighting the potential for the screening of myelin-modulating drugs and compounds in a cost- and time-effective manner, while reducing animal use.

  View details for DOI 10.1016/j.xpro.2025.103609

  View details for PubMedID 39888721

• Effective Transcriptional Induction of the CARMN/miR-143/145 Complex Locus in Smooth Muscle Cells Using CRISPR Activation. Arteriosclerosis, thrombosis, and vascular biology Vacante, F., Sanchez-Esteban, S., Tsarouchas, T., Rodor, J., Bennett, M., Carroll, M. S., Beqqali, A., Baker, A. H. 2025

  View details for DOI 10.1161/ATVBAHA.124.322353

  View details for PubMedID 40740134

• C9ORF72 deficiency results in neurodegeneration in the zebrafish retina. The Journal of neuroscience : the official journal of the Society for Neuroscience Jaroszynska, N., Salzinger, A., Tsarouchas, T. M., Becker, C. G., Becker, T., Lyons, D. A., MacDonald, R. B., Keatinge, M. 2024

  Abstract

  Hexanucleotide repeat expansions within the gene C9ORF72 are the most common cause of the neurodegenerative diseases Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal dementia (FTD). This disease-causing expansion leads to a reduction in C9ORF72 expression levels in patients, suggesting loss of C9ORF72 function could contribute to disease. To further understand the consequences of C9ORF72 deficiency in vivo, we generated a c9orf72 mutant zebrafish line. Analysis of the adult female spinal cords revealed no appreciable neurodegenerative pathology such as loss of motor neurons, or increased levels of neuroinflammation. However, detailed examination of adult female c9orf72-/- retinas showed prominent neurodegenerative features, including a decrease in retinal thickness, gliosis, and an overall reduction in neurons of all subtypes. Analysis of rod and cone cells within the photoreceptor layer showed a disturbance in their outer segment structure and rhodopsin mis-localisation from rod outer segments to their cell bodies and synaptic terminals. Thus, C9ORF72 may play a previously unappreciated role in retinal homeostasis and suggests C9ORF72 deficiency can induce tissue specific neuronal loss.Significance statement Hexanucleotide expansions in the gene C9ORF72 are the most common cause of the Amyotrophic lateral sclerosis (ALS)/ Frontotemporal dementia (FTD) disease spectrum. The expansion reduces expression of C9ORF72 and so may play a role in neuronal loss. However, C9ORF72 loss of function has been comparatively understudied in vivo. Using the zebrafish as a model of C9ORF72 deficiency, we demonstrate that loss of C9ORF72 results in marked inflammation and neuronal loss in the aged adult zebrafish retina. Development of the retina is unaffected regardless of C9ORF72 status. This demonstrates that C9ORF72 loss of function can cause spontaneous neurodegeneration in vivo and highlights a novel role of C9ORF72 in retinal homeostasis.

  View details for DOI 10.1523/JNEUROSCI.2128-23.2024

  View details for PubMedID 38658168

• Rapid Testing of Gene Function in Axonal Regeneration After Spinal Cord Injury Using Larval Zebrafish. Methods in molecular biology (Clifton, N.J.) Drake, L. K., Keatinge, M., Tsarouchas, T. M., Becker, C. G., Lyons, D. A., Becker, T. 2023; 2636: 263-277

  Abstract

  Larval zebrafish show axonal regrowth over a complex spinal injury site and recovery of function within days after injury. Here we describe a simple protocol to disrupt gene function in this model using acute injections of highly active synthetic gRNAs to rapidly detect loss-of-function phenotypes without the need for breeding.

  View details for DOI 10.1007/978-1-0716-3012-9_15

  View details for PubMedID 36881306

  View details for PubMedCentralID 6729920

• CRISPR gRNA phenotypic screening in zebrafish reveals pro-regenerative genes in spinal cord injury. PLoS genetics Keatinge, M., Tsarouchas, T. M., Munir, T., Porter, N. J., Larraz, J., Gianni, D., Tsai, H. H., Becker, C. G., Lyons, D. A., Becker, T. 2021; 17 (4): e1009515

  Abstract

  Zebrafish exhibit robust regeneration following spinal cord injury, promoted by macrophages that control post-injury inflammation. However, the mechanistic basis of how macrophages regulate regeneration is poorly understood. To address this gap in understanding, we conducted a rapid in vivo phenotypic screen for macrophage-related genes that promote regeneration after spinal injury. We used acute injection of synthetic RNA Oligo CRISPR guide RNAs (sCrRNAs) that were pre-screened for high activity in vivo. Pre-screening of over 350 sCrRNAs allowed us to rapidly identify highly active sCrRNAs (up to half, abbreviated as haCRs) and to effectively target 30 potentially macrophage-related genes. Disruption of 10 of these genes impaired axonal regeneration following spinal cord injury. We selected 5 genes for further analysis and generated stable mutants using haCRs. Four of these mutants (tgfb1a, tgfb3, tnfa, sparc) retained the acute haCR phenotype, validating the approach. Mechanistically, tgfb1a haCR-injected and stable mutant zebrafish fail to resolve post-injury inflammation, indicated by prolonged presence of neutrophils and increased levels of il1b expression. Inhibition of Il-1β rescues the impaired axon regeneration in the tgfb1a mutant. Hence, our rapid and scalable screening approach has identified functional regulators of spinal cord regeneration, but can be applied to any biological function of interest.

  View details for DOI 10.1371/journal.pgen.1009515

  View details for PubMedID 33914736

  View details for PubMedCentralID PMC8084196

• Dynamic control of proinflammatory cytokines Il-1β and Tnf-α by macrophages in zebrafish spinal cord regeneration. Nature communications Tsarouchas, T. M., Wehner, D., Cavone, L., Munir, T., Keatinge, M., Lambertus, M., Underhill, A., Barrett, T., Kassapis, E., Ogryzko, N., Feng, Y., van Ham, T. J., Becker, T., Becker, C. G. 2018; 9 (1): 4670

  Abstract

  Spinal cord injury leads to a massive response of innate immune cells in non-regenerating mammals, but also in successfully regenerating zebrafish. However, the role of the immune response in successful regeneration is poorly defined. Here we show that inhibiting inflammation reduces and promoting it accelerates axonal regeneration in spinal-lesioned zebrafish larvae. Mutant analyses show that peripheral macrophages, but not neutrophils or microglia, are necessary for repair. Macrophage-less irf8 mutants show prolonged inflammation with elevated levels of Tnf-α and Il-1β. Inhibiting Tnf-α does not rescue axonal growth in irf8 mutants, but impairs it in wildtype animals, indicating a pro-regenerative role of Tnf-α. In contrast, decreasing Il-1β levels or number of Il-1β+ neutrophils rescue functional regeneration in irf8 mutants. However, during early regeneration, interference with Il-1β function impairs regeneration in irf8 and wildtype animals. Hence, inflammation is dynamically controlled by macrophages to promote functional spinal cord regeneration in zebrafish.

  View details for DOI 10.1038/s41467-018-07036-w

  View details for PubMedID 30405119

  View details for PubMedCentralID PMC6220182

• Wnt signaling controls pro-regenerative Collagen XII in functional spinal cord regeneration in zebrafish. Nature communications Wehner, D., Tsarouchas, T. M., Michael, A., Haase, C., Weidinger, G., Reimer, M. M., Becker, T., Becker, C. G. 2017; 8 (1): 126

  Abstract

  The inhibitory extracellular matrix in a spinal lesion site is a major impediment to axonal regeneration in mammals. In contrast, the extracellular matrix in zebrafish allows substantial axon re-growth, leading to recovery of movement. However, little is known about regulation and composition of the growth-promoting extracellular matrix. Here we demonstrate that activity of the Wnt/β-catenin pathway in fibroblast-like cells in the lesion site is pivotal for axon re-growth and functional recovery. Wnt/β-catenin signaling induces expression of col12a1a/b and deposition of Collagen XII, which is necessary for axons to actively navigate the non-neural lesion site environment. Overexpression of col12a1a rescues the effects of Wnt/β-catenin pathway inhibition and is sufficient to accelerate regeneration. We demonstrate that in a vertebrate of high regenerative capacity, Wnt/β-catenin signaling controls the composition of the lesion site extracellular matrix and we identify Collagen XII as a promoter of axonal regeneration. These findings imply that the Wnt/β-catenin pathway and Collagen XII may be targets for extracellular matrix manipulations in non-regenerating species.Following spinal injury in zebrafish, non-neural cells establish an extracellular matrix to promote axon re-growth but how this is regulated is unclear. Here, the authors show that Wnt/β-catenin signaling in fibroblast-like cells at a lesion activates axon re-growth via deposition of Collagen XII.

  View details for DOI 10.1038/s41467-017-00143-0

  View details for PubMedID 28743881

  View details for PubMedCentralID PMC5526933

• Spinal motor neurons are regenerated after mechanical lesion and genetic ablation in larval zebrafish. Development (Cambridge, England) Ohnmacht, J., Yang, Y., Maurer, G. W., Barreiro-Iglesias, A., Tsarouchas, T. M., Wehner, D., Sieger, D., Becker, C. G., Becker, T. 2016; 143 (9): 1464-74

  Abstract

  In adult zebrafish, relatively quiescent progenitor cells show lesion-induced generation of motor neurons. Developmental motor neuron generation from the spinal motor neuron progenitor domain (pMN) sharply declines at 48 hours post-fertilisation (hpf). After that, mostly oligodendrocytes are generated from the same domain. We demonstrate here that within 48 h of a spinal lesion or specific genetic ablation of motor neurons at 72 hpf, the pMN domain reverts to motor neuron generation at the expense of oligodendrogenesis. By contrast, generation of dorsal Pax2-positive interneurons was not altered. Larval motor neuron regeneration can be boosted by dopaminergic drugs, similar to adult regeneration. We use larval lesions to show that pharmacological suppression of the cellular response of the innate immune system inhibits motor neuron regeneration. Hence, we have established a rapid larval regeneration paradigm. Either mechanical lesions or motor neuron ablation is sufficient to reveal a high degree of developmental flexibility of pMN progenitor cells. In addition, we show an important influence of the immune system on motor neuron regeneration from these progenitor cells.

  View details for DOI 10.1242/dev.129155

  View details for PubMedID 26965370

  View details for PubMedCentralID PMC4986163