Skip to main content

Extracellular brain proteolysis in neuronal plasticity and neuropsychiatric disorders

Final Report Summary - EXTRABRAIN (Extracellular brain proteolysis in neuronal plasticity and neuropsychiatric disorders)

"Extracellular brain proteolysis in neuronal plasticity and neuropsychiatric disorders" (EXTRABRAIN) was an EU FP7 Marie Curie-funded Initial Training Network coordinated by Prof. Leszek Kaczmarek from the Nencki Institute of Experimental Biology of the Polish Academy of Sciences, Warsaw, Poland. The Project Consortium consisted of 9 full Beneficiaries (1 from Poland, 3 from Germany, 2 from UK, 1 from Switzerland, 1 from France and 1 from Portugal), including 2 SMEs, and 1 Associated Partner (private company, non-SME).

The Project’s paramount ambition was to provide exceptional, multidisciplinary and intersectorial training in brain plasticity from the extracellular matrix perspective. The main focus of the training program was on expanding knowledge and on developing new treatments to the most costly brain disorders. The program focused on anxiety disorders, schizophrenia, mental retardation and Alzheimer’s. A unifying neurobiological concept in the consorted effort to tackle these conditions was the involvement of abnormal synaptic plasticity.

The fellows enrolled in EXTRABRAIN (11 Early Stage and 2 Experienced Researchers) benefited from a high-quality research training while being exposed to a cutting-edge research environment and facilities. The research projects were supervised by senior researchers from their home institution and from collaborative teams. This mutual effort resulted in developing a vibrant research community that benefited from collaborations between the teams.

The Project was divided into four workpackages:

WP1: Training

EXTRABRAIN delivered skills in three core training domains: task-specific research skills (delivered locally and through secondments), generic research skills (delivered locally, by the Associated Partner and at network meetings) and complementary skills (delivered locally, by the Associated Partner and at network meetings).

Seven successful training events were organized by the Consortium: 4 Practical Workshops (Paris, Warsaw, Magdeburg, Gottingen) and 3 Advanced Training Courses (Basel, Munich, Santorini). During the four Practical Workshops cutting-edge methodologies were shared among network partners offering a unique, multidisciplinary training to ESRs and ERs. These events were not open to researchers from outside to allow time for in-depth tutoring and hands-on practice with speakers recruited from within the network. Practical workshops were hosted by EXTRABRAIN partners specializing in particular key methodologies and having state-of-the-art facilities to ensure the programme was successfully delivered. The Workshops consisted of lectures, demos and hands-on-training sessions. Last day of each Practical Workshop was devoted to developing generic skills through lectures and practical training delivered the non-academic partners. To keep ESRs and ERs up to date with recent advances in theoretical aspects of brain plasticity in the ECM context, three Advanced Training Courses were organised. Lectures covered such key topics as Physiology of neurotransmission and synaptic microenvironment, Neuropsychiatric and neurodegenerative disorders and Experimental and computational methods for the analysis of microscopy imaging data. These courses were open to early career external participants, mainly from the hosting institutions.

WP2: Research

The overall scientific objective of EXTRABRAIN was to understand the functional role of the ECM in major brain disorders such as schizophrenia, mental retardation, Alzheimer's disease and anxiety disorders. Specifically, this project aimed: (i) to understand how neuronal activity regulates extracellular enzymes such as proteases; (ii) to identify new ECM/CAM cleavage products that may act as signalling molecules, their receptors and downstream signalling in neurons and glia; and (iii) to develop novel tools and reagents aimed at modulation of neuronal activity-driven ECM enzymatic activities. Having achieved these objectives the Consortium would be able to provide multi-faceted, internationally-supported training to the young researchers participating in the project, to the highest standard available to date.
The scientific objective of EXTRABRAIN was achieved fully in the course of the Project the following scientific results were obtained:
ESR1 Behnam Vafadari (Partner#1 NENCKI) demonstrated that the combination of the altered levels of MMP-9 and psychosocial stress can lead to schizophrenia related behaviors in mice. He also showed the dendritic spines shape and density changes of these mice in different brain regions (Basolateral Amygdala, Hippocampus and Prefrontal cortex). ESR2 Ahmad Salamian (Partner#1 NENCKI) showed that pharmacological and genetic inhibition of MMP-9 activity affect cholinergic- induced synaptic plasticity in hippocampal neurons. ESR3 Gabriela Matuszko (Partner#2a DZNE Magdeburg) demonstrated a reduction of ECM and parvalbumin expression in the medial prefrontal cortex of ketamine-treated rats that served as a model schizophrenia-like phenotype. ESR4 Maura Ferrer Ferrer (Partner#2a DZNE Magdeburg) found that neurotrypsin deficiency reveals impairments of specific forms of long-term potentiation and behaviour as well as spine morphology in vivo. ESR5 Valentina Brambilla (Partner#3 UNEXE) found that stimulation of PAR-1 via thrombin receptor causes shrinkage of the dendritic tree. The student also found that an extracellular protease Htra1 is upregulated by restraint stress and can regulate stress-induced neuronal plasticity. ESR6 Alberto Labrador-Ramos (Partner#3 UNEXE) found that extracellular proteases that cleave PAR-1 (tPA and plasmin) also cleave membrane tyrosine kinase EphA4. ESR7 Matteo Tripodi (Partner#4 FMI) was making in utero injections experiments aiming at delivering tamoxifen locally in order to activate the CreER protein and thereby introducing a tool that would eventually allow him to have genetic control of either early- or late-born Parvalbumin neurons in adult mice. ESR8 Bárbara Oliveira (Partner#5 MPG) found associations of normal variants in the genes encoding the Fragile X protein family with severity of autistic phenotypes. Additionally, using human induced pluripotent stem cells (IPS) she found that circulating autoantibodies targeting the NR1 subunit of the NMDA receptor have a functional impact on the availability of the receptor at the cell membrane leading to internalization of the receptor. This effect was observed using human samples and samples from other mammals besides humans broadening the view on the presence of these autoantibodies across species and their role on autoimmunity. ESR9 Elena Montagna (Partner#2b DZNE Munich) investigated the activity of astrocytes in APP KO mice, by the use of 2-photon in in vivo microscopy. She found impairment in the kinetics and in the dynamics of calcium in astrocytes of mice lacking APP. This correlates with altered mitochondria morphology and mitochondria protein expression, observed in astrocytic culture. Additionally she investigated spine dynamics in several APP-KI lines. Outcomes revealed and confirmed that APP is necessary for the maintenance of spine density. Moreover in vivo studies attributed a crucial role to APP ectodomain in modulating spine plasticity as response to enriched environment. ER1 James Reynolds (Partner#6 UCL) has initiated studies on physiological responses to sensory and noxious stimuli to assess the role of ECM-astroglial crosstalk in functional responses in vivo. ESR10 Alexandra Nothnagel (Partner#7 ENS) showed that factors such as embryonic orthodenticle homeobox 2 protein (Otx2), known to modulate the timing of critical periods in this model, did regulate the formation of perineuronal nets (PNNs), the expression of Parvalbumin and the expression of a synaptic markers in vitro. She also showed that global activity of the network could interact with Otx2 effects, and that the mouse cortical culture treated with Otx2 could serve as an in vitro model for critical period synaptic plasticity. ESR11 Adrián Posado Fernández (Partner#8 SVIDA) characterized a monoclonal antibody for targeting amyloid beta from AD patients, and produced two new antibodies for targeting phosphorylated tau and acetylated tau respectively. ER2 Liesbeth Micholt (Partner#9 EVOTEC) established a high density neuronal culture to test different neuronal labeling methods for dendritic spines (diolistic labeling, AAV mediated GFP-infection, lipofectamine GFP transfection). The ER2 position was then taken over by Joana Pedro (Partner#9 EVOTEC) who optimized cell culture conditions and identified compounds capable of regulating synaptic number.

WP3: Management

EXTRABRAIN management structure was designed to ensure smooth functioning of the network through experienced leadership and clear demarcation of responsibilities. The Project Coordinator, was effectively supported by the Supervisory Board and Advisory Committee, experienced five-strong management office at NENCKI, EXTRABRAIN Project Manager and local committees organising individual events. The EXTRABRAIN Supervisory Board consisted of the representatives of each Project Partner and met during each of the Training Events and Network Meetings (every 6 to 8 months). The EXTRABRAIN International Advisory Board consisted of 3 members: Prof. Ron Stoop from the Faculty of Biology and Medicine, Universite de Lausanne, Switzerland, Dr. Jonathan Fadok from the Department of Psychology of the Tulane University, New Orleans, USA and Prof. Zoltan Nusser from the Institute of Experimental Medicine, Laboratory of Cellular Neurophysiology, The Institute of Experimental Medicine HAS in Budapest, Hungary. The Committee provided external judgment and advice on the activities of the network.

The Project was officially launched on September 1, 2013. Its Consortium Agreement was finalized and signed in April 2014. The EXTRABRAIN website www.extrabrainitn.eu was created and maintained, regularly updated throughout the Project lifetime. The international recruitment process was carried through a very specific procedure set up by the EXTRABRAIN Consortium that followed closely the EC guidelines. All Fellows received a proper employment contracts. The Project financial management was overseen by the Office of International Cooperation and Project Management at NENCKI. Individual partner institutions were responsible for the management of funds allocated to them in line with the contract, national legislation and their internal regulations.

WP4: Dissemination and exploitation

All ESR/ER projects were based on extensive interactions between the academic and industrial centres providing a framework for commercial exploitation of the outcomes of ER/ESR projects. Industry/academia partnerships provided access to the state-of-the-art technologies and developed an appropriate management structure and IPR policies to facilitate commercial exploitation.

The Project website (www.extrabrainitn.eu) has been online since 2013. It has been updated on regular basis. Information about the Project, as well as announcements concerning available positions within the Project, have been accessible to the scientists of the Network and to the Internet users from all over the world. The ESRs contributed to a special section of the EXTRABRAIN website devoted to public dissemination of their research projects (described their work in a language appropriate for non-specialist audience). The descriptions were posted on the website.

The EXTRABRAIN teams have produced 21 publications, some of them coming from fruitful collaborations between particular teams. All publications and presentations by members of the project consortium - including all funded fellows - acknowledged the EU financial support received.

The ESRs and ERs participated to numerous domestic and international meetings and conferences outside the network where the work from EXTRABRAIN was disseminated via talks or posters. The EXTRABRAIN findings were further disseminated to the public by talks given locally by partners and/or ERs/ESRs.

EXTRABRAIN Project did not result in any patent applications, trademarks or registered designs.

Socio-economic impact of the Project

The EXTRABRAIN project capitalized on capacity building in well-established European research institutions and combined expertise in the ECM and its proteolysis. Furthermore, EXTRABRAIN involved collaborative interactions that allowed to merge the unique and complementary expertise in the field. The Project provided a very complementary capacity building programme for the recruited ESRs and ERs with a combination of training on new techniques, specific training in the domain of brain disorders, as well as management / commercialisation / industrial liaison. They constitute a community of PhD and post-doctoral fellows trained to achieve a high and uniform standard of knowledge in basic science. We believe the novel data produced by EXTRABRAIN has a marked impact on science. Their novelty and originality, as certified by the quality of journals they have been published in, should be greatly appreciated by worldwide scientific community. The Project increased human resource capacity in critical neuroscience areas, study root causes of common mental and neurological disorders from the matrix biology perspective and raised awareness of the mental disease burden.

Gender equality

EXTRABRAIN Consortium gave much effort to ensure selection committees had a proper gender balance. All partner institutions followed equal opportunity policies and made sure female recruits were not disadvantaged. As a result, six out of the 11 recruited ESR fellows and one of the 2 recruited ER fellows were female (58%). The gender parity was reached, and there was substantial indication that recruitment was gender neutral, as monitored by the Project Equal Opportunities and Recruitment Lead. All the fellows were treated professionally by their co-workers and the Supervisors did their best to integrate them into their host institution. All partner institutions recognised rights for parental leave and favour working conditions enabling researchers to combine career development with family life.

Ethical issues

Research involving animal experimentation within the EXTRABRAIN network was undertaken with a clear scientific purpose. Animals were used quite extensively in this work and the choice of the species used in animal experiments was a complex decision and was based on a consideration of animal welfare, practicality and scientific rationale. All experiments were conducted in rats and mice. The 3R’s rule (Replace, Reduce, Refine) was intensively applied to obtain relevant data from the minimum number of animals possible. Each lab of the EXTRABRAIN network was regulated by its national policy and rules on animal experimentation. All the required ethical approvals by the competent local/national ethical/legal bodies, together with relevant authorizations for animal experiments were obtained.

Conclusions

EXTRABRAIN Project was carried out successfully and its deliverables were met. There were no outstanding problems. The Fellows cooperated as a highly interactive group both during the training events and during the intervals between them. All the students were successfully encouraged to attend relevant meetings outside their institutions. The close attention they were receiving both from their PIs and from other members of the Consortium paid off in terms of the production of a team of well-trained scientists, who will go on to successful careers. The research results the ESRs and ERs produced have found the way to major scientific publications that appeared in the top notch journals, which also shall greatly contribute to the scientific careers of the fellows trained within the EXTRABRAIN.