BoTENT

Project: Trafficking of botulinum toxins in enteric nervous system / Prijenos botulinum toksina u enteričkom živčanom sustavu

Project leader: Ivica Matak, PhD, Research associate

Project collaborators: assist. prof. Ornella Rossetto, PhD; Marco Pirazzini, PhD, Research associate; Federico Fabris, M.Biotechnol / PhD candidate

Project duration: 01/10/2019 – 01/12/2019

This project is supported by Unity Through Knowledge Fund (UKF) Project call: “Gaining experience (2019), and the research group of assist. prof. Ornella Rossetto, Department of Biomedical Sciences, University of Padua, Italy

The aim of project visit is to establish collaboration and to share experimental experience between the visitor (Ivica Matak, Laboratory of Molecular Neuropharmacology, Department of Pharmacology, University of Zagreb School of Medicine), and the Neuroparalysis and Neuroregeneration Lab, at the Department of Biomedical Sciences at the University of Padua. Ivica Matak’s visit, lasting for two months, will be supervised by prof. Ornella Rossetto, one of the pioneering scientists in the investigation of basic mechanisms of action of botulinum toxin type A (BoNT/A) and other clostridial neurotoxins which characterized toxin entrance into neurons and translocation into neuronal cytosol and action on synaptic proteins, and characterization of the effects on the neuromuscular junction (Pirrazzini et al., 2017; Pirazzini et a., 2014; Schiavo et al., 1992).

During the proposed visit, Ivica Matak will join the ongoing research of the group in Padua involving investigation of botulinum toxins’ action on enteric nervous system. Different immunological serotypes of botulinum toxins, produced by anaerobic bacterium Clostridium botulinum are the most potent toxins known (Matak and Lacković, 2014; Pirazzini et al., 2017). The most common pathway of intoxication is ingestion of Clostridium-intoxicated food prepared in anaerobic conditions, and toxin absorption into the bloodstream through the intestinal gut lining (Arnon et al., 2001; Maxymowich et al, 1999)

Figure 1 Pharmacokinetics of botulinum neurotoxin type A (BoNT/A). Ingested or inhaled BoNT/A complex reaches the lumen of intestines or lung alveoles, and the neurotoxic part enters the bloodstream by transcytosis across epithelial layers. This is followed by systemic distribution into the extracellular fluid of peripheral tissues, such as muscles (auxiliary proteins indicated by dashed circle line dissociate from 150 kDa neurotoxin). Systemically distributed or intramuscularly (i.m.) injected BoNT/A then enters neuromuscular junctions and paralyses the muscle. Circulating BoNT/A cannot penetrate the blood-brain barrier, however, a portion of BoNT/A is retrogradely transported within peripheral nerves towards the CNS. Favorable pharmacokinetic properties for high BoNT/A toxicity are: the ability to survive the harsh conditions of GI tract and cross the epithelial layers to reach the bloodstream, sufficient half-life in the systemic bloodstream and specific recognition of neuronal terminals to promptly enter the cytosol of neuronal terminals (Ivica Matak, PhD thesis 2015).

The relevance of research theme is supported by clinical experience in botulism intoxication, as well as off label therapeutic use in treatment of gastrointestinal disorders (Brisinda et al., 2015; Vittal et al., 2006). Present knowledge suggests that botulinum toxins possess the effect in the enteric nervous system. However, most aspects of the botulinum toxin action, such as cell types targeted, distribution to distant body regions from the gut, possible interaction with the resident local immune system cells, as well as toxin distribution into CNS after toxin distribution from the gut, have never been characterized (Rossetto et al., 2015). It is of particular interest to study these actions since they are pathophysiologically relevant for the management of botulism, as well as potential therapeutic implications for the treatment of gastrointestinal disorders along different part of the gut axis, such as dysphagia, achalasia, dyspepsia, different intestinal hypermotility disorders, inflammatory bowel disorders, sphincter disorders etc.

The costs of travel, accommodation and monthly allowance are covered by Unity Through knowledge Fund (UKF), while the costs of research are covered by the Ornella Rossetto group, University of Padua. Total amount of funds = 24.500,00 HRK (Unity Through Knowledge Fund 20.000,00 HRK, University of Padua 4.500,00 HRK)

Objectives, significance and added value of the visit

Objectives of the research:

In model of natural botulism, the action of the toxins will be assessed at different time points by staining of the respective cleaved synaptic SNARE proteins at the enteric nervous system (myenteric and submucosal plexus), at the neuromuscular junction and at different sites of the central nervous system (spinal cord, and brain stem). Beside the imaging of whole mount and cryosection of these tissue preparations, primary culture of enteric neurons is going to be set up to test if BoNTs also alter intestinal homeostasis and immune response via blockade of neurons interacting with innate lymphoid cells and macrophages in the intestine.

Patophysiological relevance of proposed research is providing explanation of the gut symptoms of the natural botulism, such as disturbances related to peristalsis. In addition, proposed research will assess the possibility if botulinum toxin might be therapeutically useful in treatment of many gastrointestinal spastic and inflammatory syndromes affecting different parts of the gut system. Additional aim addressed at the same time is to study the botulinum toxin spread away from the gut by observing its effects in distant body regions and CNS with behavioral and immunohistochemical techniques.

Added value to the visit is sharing the experimental experience related to research techniques between the visitor and co-applicant’s group. The visit is expected to provide results that will be published in international journals, and result in planning additional research activities in Zagreb and Padua, leading to application to funding grants from Science foundations in their respective countries, European Structural funds or Horizon programmes.

Expected measurable results of the visit and their potential users

Scientific results and potential use:

Characterization of the neuronal cell types (sensory, inhibitory, cholinergic) in plexus submucosus and plexus myentericus targeted by botulinum toxins will lead to better explanation of the gastrointestinal symptoms of botulism, as well as to potential mechanisms for treatment of gastrointestinal disorders.

-Establishment of primary intestinal cell cultures to study if botulinum toxin alters intestinal homeostasis and if immune response via blockade of neurons interacting with innate cells of immune system occurs in the intestine. This might contribute to study potential novel mechanisms of actions and uses in different gastrointestinal disorders.

– Characterization of the effect of botulinum toxins in distant regions of the body (peripheral neuromuscular junctions of skeletal muscles), and distant regions of the CNS, to study the pharmacokinetic properties and distribution of botulinum neurotoxins.

Applicant’s professional training:

– Ivica Matak will be fully acquainted with experimental tehniques used in proposed research, such as isolation of primary neuronal cultures from the intestine in our cell culture facility, recording of excitatory postsynaptic potentials and contraction imaging in ex vivo-isolated smooth muscle, as well as to study the morphology and functuion of peripheral neromuscular junction (ex vivo hemidiaphragm preparation).

-The experimental experience obtained will contribute to the introduction of novel experimental techniques to Department of Pharmacology in Zagreb, as well as to improve the potential for independent or common collaborative future projects with co-applicant and other researchers in the field.

Relevance and potential benefit of the visit

The research visit would result in common publications and improved scientific recognition od applicant and Administering institution. Funding by future projects resulting from this collaboration might result in strengthened scientific and infrastructural research potential of applicant’s Laboratory and increased scientific reputation of its parent institution (University of Zagreb School of Medicine), as well as doctoral training of young scientists in Croatia.

Plans for future collaborations, project applications and publications with host organization or other scientific groups in the world or private sector

During the visit, and depending on the results obtained, the applicant and co-applicant will discuss and propose the outline of future larger project application which will take place at both institutions. Ivica Matak will plan future project applications to Croatian funds: Croatian Science Foundation, UKF, as well as European structural funds, in collaboration with Ornella Rossetto’s group from Padua. Ivica Matak has already established collaboration with group of Matteo Caleo from CNR Neuroscience institute in Pisa, who is also a collaborator with Ornella Rossetto. Common project applications to different funding sources in Croatia, Italy, and European grants is expected, including possible collaboration with pharmaceutical industry. Since there are different aspects of the proposed research themes involving different fields of research (Toxinology, Toxicology, Pharmacology, Neuroscience), the result of visit, as well as future research activities which will take at both institutions following the visit, are expected to result in scientific publications in international jounrnals belonging to different areas of research.

Proposed communication and outreach of results

During the research visit, a public lecture by Ivica Matak will be organized on his experience research on botulinum toxin type A, nociception and muscle hyperactivity in Zagreb, as well as the introduction to the aims of research visit. This will enable promotion of the purposes of research visit, as well as to promote the research done in Zagreb and to establish contact among other scientists in Padua. Upon return from Padua, Ivica Matak will organize a public lecture at the Department of Pharmacology at the University of Zagreb, and inform the colleagues and interested scientists on the research visit. This lecture will be promoted among the Croatian Pharmacological Society and Croatian Microbiological Society members. Since there are different aspects of the proposed research themes involving different fields of research (toxinology, toxicology, pharmacology, neuroscience), the research done during the visit are expected to be part of several scientific publications in international neuroscience, pharmacology, and/or toxicology journals.

Management and organization of the visit

The administrational issues related to the project will be lead by project applicant and applicant’s institution. At the disposition to the project are following Departments of the university of Zagreb School of Medicine: 1.Financial department, which will be involved in the procurement of travel and accommodation in Padua, as well as financial reporting. Department for Science will be involved in reporting of project results and promotion of public lecture at the School of Medicine. The host institution in Padua, the Department of Biomedical Science will provide co-financing in the form of consumables and chemical reagents needed for research, as well as organization of the lecture at the Department. Ornella Rossetto and her staff will be at the disposition regarding professional training of the applicant (learning experimental techniques, technical support in performing experiments), as well as the procurement of chemical reagents needed for research.

Literature references

Arnon, S. M., Schechter, R., Inglesby, T. V., Henderson, D. A., Bartlet, D. J., Michael, S.,Ascher, M. S., Eitzen, E., Fine, A. D., Hauer, J., Layton, M., Lillibridge, S.,Osterholm, M. T., O’Toole, T., Parker, G., Perl, T. M., Philip, K., Russell, F. K.,Swerdlow, D. L., Tonat, K., Working Group on Civilian Biodefense. 2001 Botulinumtoxin as a biological weapon: medical and public health management. JAMA 285,1059-1070.

Brisinda G, Sivestrini N, Bianco G, Maria G. Treatment of gastrointestinal sphincters spasms with botulinum toxin A.Toxins (Basel). 2015 May 29;7(6):1882-916. doi: 10.3390/toxins7061882.

Maksymowych, A.B., Reinhard, M., Malizio, C.J., Goodnough, M.C., Johnson, E.A.,Simpson, LL. 1999 Pure botulinum neurotoxin is absorbed from the stomach and small intestine and produces peripheral neuromuscular blockade. Infect. Immun. 67, 4708-4712.

Matak, I., Lacković, Z. 2014 Botulinum toxin A, brain and pain. Prog. Neurobiol. 119-120,39-59

Pirazzini M, Rossetto O, Eleopra R, Montecucco C. (2017). Botulinum Neurotoxins: Biology, Pharmacology, and Toxicology. Pharmacol Rev. 69(2):200-235.

Pirazzini M, Azarnia Tehran D, Zanetti G, Megighian A, Scorzeto M, Fillo S, Shone CC, Binz T, Rossetto O, Lista F, Montecucco C. (2014). Thioredoxin and its reductase are present on synaptic vesicles, and their inhibition prevents the paralysis induced by botulinum neurotoxins. Cell Rep. 8(6):1870-1878.

Rossetto O, Pirazzini M, Montecucco C. (2015). Current gaps in basic science knowledge of botulinum neurotoxin biological actions. Toxicon. 107(Pt A):59-63.

Schiavo G, Benfenati F, Poulain B, Rossetto O, Polverino de Laureto P, DasGupta BR, Montecucco C. Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature. 1992 Oct 29;359(6398):832-5

Vittal H, Pasricha PF. Botulinum toxin for gastrointestinal disorders: therapy and mechanisms. Neurotox Res. 2006 Apr;9(2-3):149-59.

Results of the projects

Experimental results: In a model of natural botulism evoked by oral gavage of clostridial toxins, we assessed the occurrence of enzymatic products of botulinum toxin type A (BoNT/A) in the plexus myentericus of the duodenum, spinal cord thoracal and lumbar regions, as well as brainstem. We found the toxin action in cholinergic and catecholamninergic neurons of duodenum. In the central regions, we observed the occurrence of truncated SNAP-25 products in sensory and motor regions of the thoracal, lumbar spinal cord and brainstem. In the sensory and motor regions of the CNS, the BoNT/A enzymatic activity occurred after short incubation period, suggesting possible entrance of the toxin via peripheral sensory and motor nerve endings, which is in line with known pharmacokinetic action of the toxin in peripheral tissues induced by toxin distribution to peripheral tissue via the bloodstream. Further research will be aimed at examining potential other types of intrinsic neurons within the intestine, e.g. peptidergic, immune cells such as intrinsic macrophages, as well as sensory primary afferents. The neuronal processes involved appear not to be of cholinergic origin, thus, demonstrating that the toxin activity both in the intestine and spinal cord is not selective for cholinergic neurons. Further stainings are needed to confirm the exact indentity of neuronal populations affected by the toxin activity, as well as to do and develop functional assays to assess the functional effects of the toxin in sensory and motor regions. The primary culture of enteric neurons from adult mice has been established, but further research is needed to obtain higher number of viable cells.

Project dissemination at the University of Padua: The project leader held a Powerpoint presentation on his research on botulinum toxin type A, nociception and muscle hyperactivity in Zagreb, as well as the introduction to the aims of research visit at the Department of Biomedical sciences, titled “Gli effetti centrali di BoNT/A” on 27th of October, 2019. The lecture was attended by professors Ornella Rossetto, Cesare Montecucco, Michela Rigoni, and assist. prof. Marco Pirazzini from the host laboratory, professors Aram Megighian and Matteo Caleo from other labs a the Department, as well as other PhD students and post-docs.

Partnership: The cooperation with the host institution (Department of Biomedical Sciences, University of Padua) went in perfect order according to the plan. The collaboration between the laboratories on the proposed research theme will continue further with regarding the immunohistochemical characterization of BoNT/A enzymatic activity in different peripheral and central regions. In addition, we started additional lines of research related to other serotypes of botulinum toxin in chemical pain behavioral parameters and evoked nociceptive neuronal activation. . Analyses of molecular markers of neuronal activation in brain tissue are currently underway in Zagreb. This will be the basis of a collaborative joint project proposal for International Association for the Study of Pain, together with assist prof. Marco Pirazzini.