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The WNT_IMMUNE project started with the goal of studying how the increased or decreased levels of the immune-secreted Wnt3a could induce or not neuroprotection against stroke and/or recovery after the injury and to what extent, hippocampal adult neurogenesis process modulated by this immune-secreted Wnt subtype could contribute to recovery after ischemic injury. In order to address all these questions, we planned to generate a chimeric mouse model resulted from the injection of genetically modified bone marrow (BM)-derived cells. The generation of this model required the completion of the following steps:

1) Isolation of BM-derived cells from femur and/or tibia of donor mice expressing
macrophage/microgliadirected Green Fluorescence Protein (GFP) reporter gene.
2) Ex vivo correction of isolated BM-derived cells with inducible lentiviral constructions
overexpressing Wnt3a or a shRNA against this to abolish its expression.
3) Chimeric mice generation by reconstitution of appropriate recipient mice with the genetically
modified BM-derived cells isolated from donors
4) Study of the beneficial/detrimental outcomes derived from Wnt3a
overexpression/repression for the progression of the ischemic pathology.
5) Test how Wnt3a-regulated hippocampal adult neurogenesis could influence the recovery after
ischemic injury.

A limiting factor of the project was the availability of good genetic tools in order to start the ex vivo transduction of the BM derived cells. Our initial proposed constructs available in the Host laboratory (Dr. Stefano Pluchino’s laboratory) were tested and resulted unsuccessful for our scientific purposes. Consequently, we started the search of new appropriate genetic tools helped by the group of Dr. Alessandra Biffi who has recently moved from the HSR-TIGET Institute in Milan (Italy) to Boston Children’s Hospital/Dana Farber Cancer Institute and Harvard Medical School (USA). This process greatly delayed the completion of the first objectives of the proposal according to the time line provided in the original proposal.

This new beginning, and thanks to our collaborators, allowed us to successfully generate two different Doxycycline inducible systems to overexpress Wnt3a and three different shRNA candidates against this protein in another Doxycycline inducible system; tools that we, ourselves, designed and cloned. So far, Wnt3a overexpressing systems have been validated in 3T3 mouse embryonic fibroblasts in terms of not only expression but also in terms of secretion of the ligand and its ability to trigger Wnt signalling pathway in receptors cells by performing different analysis including western blot, qPCR, immunofluorescence staining, ELISA and luciferase assays. In this period, we have also generated lentiviral particles with the Wnt3a overexpressing system. Our efforts have been addressed to validate these particles in BM-derived primary macrophages. Our trials have demonstrated that these primary cells are receptive to be transduced with this lentiviral system. Further analysis about the MOI (multiplicity of infection), time of expression, protein levels and reporter gene expression are required in order to fully validate the construct in these cells.
Our project, however, required the validation of the genetic tools in BM precursor cells; a particular population of progenitors previously identified and described by the group of Dr.Alessandra Biffi as the main responsible cells generating long-term microglia/macrophages in the brain when transplanted. To this aim, and while further experiments were planned to be developed to identify our potential shRNA candidate against Wnt3a, we planned a short stay in the laboratory of our collaborators at Harvard University that would also allowed us to start the generation of the in vivo model. Two fellowships were awarded to this respect (EMBO short term fellowships and Travel grants sponsored by the Journal of Cell Science). The stay was scheduled from October 2016 to December 2016. As a result, we anticipated the completion of the 3 main points stated in the WNT_IMMUNE project by March 2017. Regrettably, due to financial issues the continuation of the Marie Curie IEF holder in the Host laboratory was not ensured for 2017, which made not possible the stay. However, we do believe that the generation of these constructs constitutes a major achievement, since it may set the basis for future experiments and collaborations in which Wnt signaling is the core of the investigations. Indeed, our tools, although specifically addressed to regulate the expression of Wnt3a, are perfectly valid to ensure the expression of any Wnt candidate in mind, broadening the spectrum and increasing the chances of successful collaborations. As it will be detailed in section 3, we also developed a contingency plan where promising results were obtained and whose potential impact in terms of publications and generation of valuable scientific knowledge is higher and more likely.

Considering the need of a contingency plan in case the proposed project was not giving the expected results or was considerably delayed (as in fact it happened), we decided to carry out a new/complementary project in parallel. Giving our interest in the stroke pathology and the putative contribution of Wnt signaling to it, we decided to study the role of Wnt3a in primary macrophages upon oxygen and glucose deprivation in an in vitro hypoxia model. Our idea was to better define the role of Wnt3a in the macrophage response regarding their ability to be activated, to acquire a pro or anti inflammatory phenotype and in the end, to modulate neurons response under detrimental conditions. We managed to successfully establish the hypoxia model and to generate interesting data regarding the role of Wnt3a in M1/M2 macrophage phenotype modulation. On the other hand, and in line with these preliminary results, we focused our attention on the angiogenesis process (formation of new blood vessels). A correlation between angiogenesis and improved functional outcome after ischemic stroke has been observed in both animal models and in human stroke patients. This “proangiogenic state” induced in response to an ischemic insult has been proposed to have multiple purposes in the hours to weeks after the injury, including the survival of the endothelial, glial and neuronal cell types in the penumbral area thanks to the growth factors whose expression is increased, the remove of damaged tissue by the neovascularization as well as the creation of a “vascular niche” in which neural stem cells are generated and allowed to migrate. Consequently, emerging therapeutic strategies for recovery implying the induction of angiogenesis will stimulate endogenous recovery mechanisms including neurogenesis, synaptogenesis, and neuronal and synaptic plasticity. To this regard, we tried to elucidate the basic mechanisms underlying a possible Wnt3a-induced pro-angiogenic effect in macrophages in an angiogenesis 3D In vitro model under hypoxia conditions. Our results arising from this part allowed us to define a positive effect on the formation of new tubular structures in anoxia (by using the tubular formation assay as an in vitro model to study angiogenesis) mediated by Wnt3a-primed BM-derived macrophages. Final data need to be fully analyzed but allow us to anticipate the publication of a brief paper reporting our findings to this respect.

With an annual incidence of approximately 250–400 in 100.000 inhabitants, about one million people in the EU suffer a stroke each year, many of them being struck by persistent long-term handicaps. Recent studies demonstrate that stroke may be by now the second cause of mortality in the world and, by 2020, its mortality will be increased by almost 50% of Ischemic stroke can affect individuals of any age, although incidence and prevalence of this condition sharply increases with age. On the other hand, population aging is occurring much faster. Thus, the number of people affected by stroke is expected to significantly grow. Despite fundamental insights have been made in understanding the complex pathophysiology of this disorder, nowadays the only efficacious treatment approved for ischemic stroke is thrombolysis. Unfortunately, only 5–10% of ischemic stroke patients can be elected to this treatment, because of its limited time window and its potentially dangerous side effects. Our results addressing the pro-angiogenic role of macrophages when stimulated by Wnt3a in vitro as a potential endogenous repairing mechanism that may work also in vivo, constitutes a step forward both towards the comprehension of the contribution of angiogenic mechanisms and of macrophages to the recovery from the disease and towards possible future therapeutic strategies targeting this process via modulation of Wnt signaling pathway. The potential results and future outcomes that will arise from it, in the form of scientific knowledge, high impact publications (in preparation), development of new research and diagnostic tools, presentations and collaborations, will be hopefully of high scientific and social impact, and will thus increase international visibility and be attractive for worldwide researchers whose research interest and activities are focused on neuroimmunology and neurological disorders (NDs), and who are interested in carrying on their scientific efforts on the investigation of dysfunctional molecular mechanisms in stroke.