Activity-dependent turnover of the Perineural Extracellular Matrix
by Gabriel Ignacio Cassinelli Petersen
Date of Examination:2024-04-02
Date of issue:2024-04-05
Advisor:Prof. Dr. Silvio Rizzoli
Referee:Prof. Dr. Silvio Rizzoli
Referee:Prof. Dr. Michael Thumm
Referee:Prof. Dr. Ralf Dressel
Persistent Address:
http://dx.doi.org/10.53846/goediss-10429
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Abstract
English
The different molecules present in the extracellular space of the brain make-up the perineural extracellular matrix (ECM). One of the specializations of the perineural ECM, the perineural nets (PNNs), are lattice-like structures that ensheathes different neurons. The ECM surrounding synapses is termed the perisynaptic ECM which, just as PNNs, is primarily made up of hyaluronan, chondroitin-sulphate proteoglycans (CSPGs), and tenascins, which interact and cross-link. The proteins that make up the perisynaptic ECM have been shown to have extremely long half-lives, far exceeding those of most other neural proteins. The perisynaptic ECM has also been shown to severely restrict synaptic plasticity by impeding the mobility and morphological changes of synapses. At the same time, super-resolution microscopy experiments in the last decades have provided evidence that synapses are able to change in morphology and size more frequently than previously thought. This is difficult to accommodate with the notion of a stable and constricting perisynaptic ECM. Hence, the aim of this study was to investigate whether perineural ECM components were remodelled within short timeframes, and whether such a remodelling was dependent on the activity of synapses. To answer this question, an experiment based on imaging of immunocytochemically treated primary hippocampal cell culture was devised. All ECM epitopes available at timepoint zero were first blocked by non-fluorescently tagged antibodies. The cells were then incubated with or without bicuculline treatment, and finally labelled with new fluorophore-conjugated antibodies. Hence, any new fluorescence should reflect epitopes that emerged during the incubation period. The cells were then imaged using fluorescence, confocal, and STED microscopy. This experiment was performed on tenascin-R, neurocan, N- acetylgalactosamine moieties, and hyaluronan epitopes. For all targets, new epitopes emerged within a twelve-hour timeframe. The new epitope pool even reaching sometimes a size larger than 50 % of the initial pool. There was a significant association of bicuculline treatment with an increased pool of new epitopes for all studies epitopes, except for N- acetylgalactosamine moieties. A correlation analysis revealed for tenascin-R revealed a strong positive correlation between new epitopes and more active synapses. A significant negative correlation between both parameters could be measured for neurocan, although only one biological replica was sued in this analysis severely limiting its validity. Overall, the results of this study suggest that while the half-life of different perineural ECM components is very long, a significant level of remodelling takes place. For tenascin-R follow-up experiments performed in the laboratory revealed a novel recycling mechanism. Future experiments are necessary to elucidate the mechanism behind this remodelling for CSPGs and hyaluronan, and the relevance of this phenomenon in brain physiology and pathology.
Keywords: Perineural ECM; Tenascin; Neurocan; WFA; Hyaluronan; Neurophysiology
