Mouse TrueBlot® ULTRA: Anti-Mo

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The tyrosine phosphatase LAR acts as a receptor of the nidogen-tetanus toxin complex
Sunaina Surana1,2,3,*
, David Villarroel-Campos1,2, Chiara Panzi1,2,3, Sergey S. Novoselov1,2, Sandy
Richter1,4 Giuseppe Zanotti4 and Giampietro Schiavo1,2,3,*
1
Department of Neuromuscular Diseases, Queen Square Institute of Neurology, University College
London, London WC1N 3BG, United Kingdom.
2
UCL Queen Square Motor Neuron Disease Centre, University College London, London WC1N 3BG,
United Kingdom.
3
UK Dementia Research Institute, University College London, London WC1E 6BT, United Kingdom.
4
Department of Biomedical Sciences, University of Padova, Padova 35121, Italy.
*
Correspondence to: Sunaina Surana (s.surana@ucl.ac.uk) and Giampietro Schiavo
(giampietro.schiavo@ucl.ac.uk)
perpetuity. It is made available under aCC-BY 4.0 International license.
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
bioRxiv preprint doi: https://doi.org/10.1101/2023.02.03.526966; this version posted February 4, 2023. The copyright holder for this
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Abstract
Tetanus toxin is one of the most potent neurotoxins and is the causative agent of tetanus. This neurotoxin
binds to the neuromuscular junction and, after internalisation into motor neurons, undergoes long-distance
axonal transport and transcytosis into spinal cord inhibitory interneurons. Inside the cytoplasm of
interneurons, the catalytic chain of the toxin blocks neurotransmitter release, leading to spastic paralysis.
Whilst the effects of tetanus toxin intoxication have been extensively studied, the molecular composition
of its receptor complex is still poorly understood. We have previously shown that the extracellular matrix
proteins nidogens are essential for binding of the toxin to the neuromuscular junction. In this study, we
show that the tyrosine phosphatase LAR interacts with the nidogen-tetanus toxin complex and enables its
uptake into motor neurons. Binding of LAR to the toxin complex is mediated by its fibronectin III domains,
which we have harnessed to inhibit tetanus toxin entry into motor neurons. Surprisingly, this function of
LAR is independent of its role in regulating the neurotrophic activity of the TrkB receptor, which has
previously been shown to augment the axonal transport of signalling endosomes containing tetanus
neurotoxin. These findings identify a multi-subunit complex acting as a protein receptor for tetanus
neurotoxin, and demonstrate a novel endocytic trafficking route for extracellular matrix proteins in neurons.
Our study paves the way for dissecting the molecular mechanisms that control the recognition and uptake
of physiological ligands and pathological proteins at the neuronal plasma membrane, as well as their
targeting to the axonal retrograde pathway for long-distance transport within the nervous system.
Keywords: Extracellular matrix/LAR/Neuromuscular junction/Nidogen/Tetanus toxin
perpetuity. It is made available under aCC-BY 4.0 International license.
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
bioRxiv preprint doi: https://doi.org/10.1101/2023.02.03.526966; this version posted February 4, 2023. The copyright holder for this
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Introduction
Tetanus neurotoxin (TeNT) is one of the most toxic molecules identified to date. Produced by the
anaerobic, Gram-positive bacterium Clostridium tetani, TeNT causes tetanus, a neuroparalytic syndrome
characterised by lockjaw, opisthotonus, muscle stiffness and increasingly painful spasms, ultimately
leading to respiratory failure and death (Farrar et al, 2000). Despite the availability of an effective vaccine
and various antitoxin preparations, tetanus is a leading cause of mortality in neonates and unvaccinated
adults in developing countries due to limited resources, lack of enforcement of appropriate public
healthcare measures and scarce availability of treatments (Thwaites et al, 2015; Pirazzini et al, 2022).
TeNT is formed by three modular domains, each of which is essential for intoxication of the nervous
system. In the active toxin, these domains are arranged in a heavy chain (H chain) and a catalytic light
chain (L chain). Generated from a single polypeptide by proteolytic cleavage, both subunits remain
associated via non-covalent interactions and a conserved inter-chain disulphide bond. The H chain is
further subdivided into two regions: an amino terminal (HN) and a carboxy terminal (HC) domain, which are
responsible for membrane translocation and receptor binding, respectively (Schiavo et al, 2000; Surana et
al, 2018). After bacterial spore germination and TeNT production, the active toxin accumulates in the
synaptic space at the neuromuscular junction (NMJ) and binds to the plasma membrane of motor neurons
with sub-nanomolar affinity by virtue of its HC domain (Pirazzini et al, 2016). This results in rapid
internalisation of the neurotoxin, followed by its long-distance, retrograde transport towards the neuronal
cell body in the spinal cord (Salinas et al, 2010). TeNT then undergoes trans-synaptic transfer into
inhibitory interneurons, where the HN domain drives the pH-dependent translocation of the L chain from
the endocytic lumen of synaptic vesicles into the cytosol (Pirazzini et al, 2016). The L chain, which
possesses zinc protease activity, cleaves the synaptic vesicle protein synaptobrevin, leading to a cessation
of neurotransmitter release (Schiavo et al, 1992, 1994). This perturbs the balance of excitatory and
inhibitory inputs to motor neurons, leading to motor neuron hyperactivity and spastic paralysis (Schiavo et
al, 2000; Surana et al, 2018).
Given the high neuro-specificity and extreme toxicity of TeNT, several studies have endeavoured to
identify the presynaptic receptors responsible for its entry into motor neuron terminals. The C-terminal
region of the HC domain (HCC) was found to bind with high affinity to polysialogangliosides of the G1b
perpetuity. It is made available under aCC-BY 4.0 International license.
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
bioRxiv preprint doi: https://doi.org/10.1101/2023.02.03.526966; this version posted February 4, 2023. The copyright holder for this