Centre for Reconstructive Neuroscience - Institute of Experimental Medicine of the Czech Academy of Sciences

People

Deputy Head

Assoc. Prof. Pavla Jendelová, PhD

Researchers

Slaven Erceg, PhD

Karolína Turnovcová, MD, PhD

Kristýna Kárová, PhD

Lucia Machová Urdzíková, MD, PhD

Jana Svobodová Burianová, PhD

Jana Turečková, PhD

Assoc. Prof. Lýdia Vargová, MD, PhD

PhD Students

Anda Cimpean, PhD

Noelia Martinez-Varea, MSc

Barbora Smejkalová, PhD

Ingrid Vargová, MSc

Laboratory Technicians

Hana Vargošková

Karel Třešňák

Important Results

Why axons do not regenerate?

Mammalian neurons lose the ability to regenerate their central nervous system axons as they mature during embryonic or early postnatal development. Neuronal maturation requires a transformation from a situation in which neuronal components grow and assemble to one in which these components are fixed and involved in the machinery for effective information transmission and computation. To regenerate after injury, neurons need to overcome this fixed state to reactivate their growth programme. A variety of intracellular processes involved in initiating or sustaining neuronal maturation, including the regulation of gene expression, cytoskeletal restructuring and shifts in intracellular trafficking, have been shown to prevent axon regeneration. Understanding these processes, described in this review, will contribute to the identification of targets to promote repair after injury or disease.

Genetic differences between immature and mature neurons affect axon regeneration. In immature neurons, chromatin at promoters of regeneration-associated genes (RAGs) is open euchromatin, with acetylation marks. RAGs encode TFs STAT3, SOX11, KLF7, MYC and ATF3, enabling a robust transcriptional injury response and axon regrowth. Simultaneously, EZH2-mediated heterochromatin formation represses growth-suppressing genes. In mature neurons, RAG promoters are packed into methylated heterochromatin, limiting their post-injury expression. Instead, mature cells express genes for ion transport, excitability and synaptic function, plus receptors like NgR1 and PTPσ that mediate inhibition by chondroitin sulfate proteoglycans and CNS myelin, thus blocking regeneration.

Publication:

Hilton BJ, Griffin JM, Fawcett JW, Bradke F. Neuronal maturation and axon regeneration: unfixing circuitry to enable repair. Nat Rev Neurosci. 2024 Oct;25(10):649-667. doi: 10.1038/s41583-024-00849-3.

Integrin-Driven Axon Regeneration in the Spinal Cord Activates a Distinctive CNS Regeneration Programme

Reconstruction of nerve pathways after injury has not yet been possible, but a method of stimulating long-distance regeneration of sensory fibres in rodents has recently been developed. The study identifies the mechanisms that neurons must activate in order for their nerve fibres to regenerate. By profiling messenger RNAs in regenerating sensory neurons, the study finds that regenerating neurons initiate a novel CNS regeneration program that involves molecular transport, autophagy, ubiquitination, and modulation of the endoplasmic reticulum.

DRG axon growth and regeneration in the presence of inhibitors. The inhibitors used were: 3MA (Class III PI3K autophagy inhibitor), NSC697923 (K63-ubiquitination inhibitor), D4476 (casein kinase 1 inhibitor), MLN492 (NEDD8-mediated ubiquitination inhibitor), and MG132 (proteasome inhibitor). (A) Representative images of axotomized axons immediately after the cut and 2 hours post-injury. (B) Quantification of axon regeneration in the presence of inhibitors. (C) Continuous axon growth of uncut axons over a 2-hour interval. (D) Quantification of axon growth in the presence of inhibitors.

Publication:

Cheah M, Cheng Y, Petrova V, Cimpean A, Jendelova P, Swarup V, Woolf CJ, Geschwind DH, Fawcett JW. Integrin-Driven Axon Regeneration in the Spinal Cord Activates a Distinctive CNS Regeneration Program. J Neurosci. 2023 Jun 28;43(26):4775-4794. doi: 10.1523/JNEUROSCI.2076-22.2023.

The role of perineural networks in-memory storage

Perineuronal networks (PNNs) play a key role in the maturation and plasticity of neurons and synapses. PNNs affect the formation, retention, and extinction of memory in various animal models. According to a recently published study, the eyes and cavities in the PNN contain synapses and can therefore function as a memory repository that is even stable in conditions during which synapse retraction occurs, such as anoxia or hibernation. We studied this theory in mice put into a state resembling hibernation, in which there is a retraction of synapses and, at the same time, we monitored the spatial memory of these animals before and after hibernation. We used normal animals and animals that had an absence of nets in the hippocampus area; either due to cleavage by the enzyme chondroitinase ABC or genetically removed aggrecan, which is an important part of PNN. In our model, synapse retraction during hibernation caused memory deficits, but not to the level of naive animals. Both groups with enzymatically cleaved networks (hibernating and non-hibernating) had changes in the level of synaptic proteins located on parvalbumin neurons in the hippocampus during and after hibernation, which resulted in faster re-learning in the Morris water maze compared to mice in hibernation and the retraction of synapses preserved by PNN. Chondroitinase did not alter the memory deficit, while the long-term absence of PNN in the aggrecan knockout group led to partial memory retention, but there was no improvement in re-learning the task in the water maze.

Electron beam electron microscopy was used to examine the number of active synapses in the CA1 region before, immediately and 24 hours after hibernation. We evaluated the number of active synapses at 37 ° C before and after cooling (16-18 ° C) (A). Only fully active synapses were taken for analysis, which newly appeared on the analyzed section (A1, detail A2). Both mice with intact and enzymatically cleaved perineural networks had a significantly reduced number of synapses immediately after hibernation, which resumed 24 hours later. However, in chondroitinase-treated animals, the number of synapses only returned to the level of the control animals and not to their own initial values. Also, the amount of the presynaptic marker Bassoon on parvalbumin-positive neurons temporarily decreased during hibernation and returned after 24 hours. HLS - a state resembling hibernation, CHABC chondroitinase, AGGKO aggrecan knockout.

The reduction of perineural networks by a hyaluronan synthase inhibitor improves the cognitive memory in mice

Hyaluronan (HA) is the basic molecule of the extracellular matrix in the central nervous system that forms the perineuronal networks (PNNs) that surround the subpopulations of neurons. PNNs control synaptic stabilization both during development and in the adult brain, and PNN disruption has shown the reactivation of nerve plasticity. We investigated the possibility of memory prolongation by reducing PNN production by an inhibitor of HA synthesis. Adult mice were fed a diet containing a 5% HA synthesis inhibitor for 6 months. This application reduced the level of glycosaminoglycans in the brain to 72% compared to the controls. The memory was tested using the New-Object Recognition Test (NOR) and the Spontaneous Alternation Test (SA). NOR tests were performed at 2, 3, 6 and 7 months with a delay of 3 h and 24 h after exposure to the subjects. A significant improvement in the NOR score was found in the animals treated with 4-MU at 3 and 6 months after a 24-hour delay, compared to the control group. However, 1 month after the end of treatment, the effect of the HA synthesis inhibitor on memory increase did not persist. Immunohistochemistry confirmed a decrease in PNN by lower intensity staining of Wisteria floribunda around neurons. In addition, a shorter and less arborization of aggrecan networks was observed around dendrites in the hippocampus after 6 months of HA synthesis inhibitor treatment.

Representative immunofluorescence staining of WFA in the hippocampal region of CA1-CA3 in the control group, the group treated for 6 months with an HA synthesis inhibitor and after 1 month of elution. (B) WFA staining intensity and a total number of WFA-positive cells in the hippocampal region. A significant decrease in WFA intensity staining was observed after 6 months of HA synthesis inhibitor administration and persisted after 1 month of washout compared to control. WOE - washing effect. Scale: 50 μm.

Current Information

2023

The cooperation with Prof. Britta Eickholt from Charite University (Berlin) resulted in a joint Mobility Grant awarded by the CAS and DAAD, within the frame of which students from both departments are exchanged.
Collaboration with Joost Verhaagen from Amsterdam has enabled additional preparation of viral vectors in his laboratory also in 2023 (Dr. Kristýna Kárová and Lydia Knight).
In March 2023, B. Niuvenhuis, R. Eva and E. Bradbury visited the Centre and discussed the results of the joint projects and further collaborations planned for the sustainability period.

2022

A new grant “Hyperactive PI3 kinase and activated integrin for corticospinal regeneration” started (supported by the Swiss International Foundation for Research in Paraplegia)
Several foreign internships took place – Institute of Biochemistry, Charité-Universitätsmedizin Berlin, University King’s College London
Presentation of project results at the FENS 2022 congress in Paris, the Miami axon repair workshop, the 7th Neurological Disorders Summit in San Francisco, the AMBRA conference in Wrocław, the TERMIS EU European Congress in Krakow, the conference of the American Neuroscience Society
“Regeneration meeting” was organized in May 2022 with the participation of members of the laboratory of prof. Elizabeth Bradbury (King’s College London)
Minisymposium “Regeneration and plasticity of the central nervous system” took place with the participation of several foreign speakers
Dr Kwok gave a plenary lecture “The Role of Extracellular Matrix in Nervous System Regeneration” at the Biocev Days conference
Team members participated in popularization events Brain Awareness Week, The Week of the Czech Academy of Sciences, Researchers’ Night.

2021

conference Regeneration I with the participation of partner organizations from Poland, Slovakia and Hungary (Massakowski Medical Research Centre Polish Academy of Sciences, Neuroimunologický ústav SAK, BioTalentum Ltd.), BIOCEV, Vestec
presentation of project results – The 13th Conference of the Czech Neuroscience Society, Prague
presentation of project results – The 19th Congress of the Czech and Slovak Spine Society, Špindlerův Mlýn

2020

lecture prof. Dr. Andreas Faissner (Department of Cell Morphology & Molecular Neurobiology , Ruhr-University, Bochum, Německo) – Structural and functional characterization of the CNS matrisome
new grants: Visegrad fund „V4RM – Bridging the gap between science, education and enterprise in regenerative medicine“ and EMBO short-term fellowship
presentation of results at the VIBes in Biosciences 2020 conference, Belgium

2019

lecture by prof. Mark H. Tuszynski, M.D., Ph.D. (Director, Center for Neural Repair, Department of Neurosciences, University of California – San Diego) – Neural Stem Cells for Spinal Cord Injury
lecture by prof. Martin E. Schwab (Institute for Regenerative Medicine, University of Zurich and Dept. of Health Sciences and Technology, ETH Zurich) – Neurobiological mechanisms of functional recovery after spinal cord injury or stroke; from the lab bench to the clinic with a neurite growth-enhancing therapy
presentation of project results at the TERMIS European Chapter Meeting 2019, Greece
presentation of results at conference XIV. European Meeting on Glial Cells in Health and Diseases, Portugal
study stay of a member of the implementation team at the University of Leeds, Great Britain – the study of the effect of ECM disruption in the spinal cord lesion on the regeneration of spinal cord tissue
lecture by prof. Catherina G. Becker, FRSB (Center for Discovery Brain Sciences, The University of Edinburgh) – The immune systems controls successful spinal cord repair in zebrafish
presentation of results at the SFN Neuroscience 2019 conference

2018

lecture by Dr. Elisabeth Bradbury (King’s College London) – Restoring function after spinal cord injury: targeting glial scar matrix and endogenous repair processes
lecture by Dr. Kristiana Franzeho (Department of Physiology, Development and Neuroscience University of Cambridge) – The mechanical regulation of neuronal development and regeneration
lecture by prof. Joosta Verhaagena (Netherlands Institute for Neuroscience) – Neuronal regeneration: from gene networks to gene therapy
lecture by prof. Britty Eickholt (Charité – Universitätsmedizin Berlin) – Injury induced drebrin controls astrogliosis and scar formation by regulating tubular endosomes and adhesion responses
Mgr. Neumann participated in a 3-day workshop on the application of viral vectors to spinal ganglia at the Netherlands Institute for Neuroscience, Amsterdam
three-month internship with Mgr. Hahn in the laboratory of Joao Relvas, Institute for Molecular and Cell Biology, University of Porto
Conference – Controlling neuronal plasticity – developmental disorders and repair in Villa Lanna, Prague

2017

conference – Regeneration, Plasticity, Protection (Prof. James Fawcett)
a two-day workshop of experts from the JOHN VAN GEEST CENTER FOR BRAIN REPAIR, which falls under the Department of Clinical Neuroscience at the University of Cambridge
DRG INJECTION WORKSHOP (Dr. Menghon Cheah)
Perineuronal net treatments for memory loss (Dr. Sujeong Yang)
training workshop on the topic of in situ isolation of DRG in the cervical and lumbar spine, accompanied by practical examples of how to open the correct vertebrae and identify the appropriate dorsal ganglia
established cooperation with Dr. Vincenzo de Paola of Imperial College London (in vivo imaging of organotypic sections) and Dr. Diego Peretti of the University of Cambridge (hibernation model)
three-month internship with Dr. Růžičky at the workplace of Dr. Vincenza di Paola
lecture by prof. Toshitaka Oohashi – The Hyaluronan and Proteoglycan Link Proteins: Organizers of the Brain ECM and Key Molecules for Neuronal Function and Plasticity
internship Mgr. Dubišová at the University of Cambridge
participation of project members at the TERMIS EU 2017 Congress of Tissue Engineering and Regenerative Medicine, organization of the symposium “New trends in CNS repair” within the congress, three lectures and two poster presentations
established cooperation with the group of Dr. Andras Lakatos from Cambridge, lecture on the topic: Regulatory networks controlling protective and detrimental astrocyte phenotypes in injury and ALS.
During the year, the Center’s workplace was gradually built and investment equipment, including a Lightsheet microscope, was purchased

Reconstructive Neuroscience

People

Deputy Head

Researchers

PhD Students

Laboratory Technicians

Important Results

Why axons do not regenerate?

Publication:

Integrin-Driven Axon Regeneration in the Spinal Cord Activates a Distinctive CNS Regeneration Programme

Publication:

The role of perineural networks in-memory storage

The reduction of perineural networks by a hyaluronan synthase inhibitor improves the cognitive memory in mice

Current Information

2023

2022

2021

2020

2019

2018

2017

Projects

Modulation of extracellular matrix to enhance neuroplasticity for the rescue of age-related memory deficits

Publications

Collaborators

iemcas