Role of Igr family members in AER renewal during limb bud development

Tese de mestrado. Biologia (Biologia Evolutiva e do Desenvolvimento). Universidade de Lisboa, Faculdade de Ciências, 2011During limb development, the proximal-distal outgrowth is controlled by a thickening of ectodermal cells at the distal tip of the limb, termed apical ectodermal ridge (AER). This transient embryonic structure is essential for the patterning and limb outgrowth, being a conserved feature in vertebrate development and it is also important to maintain proliferation in adjacent tissues before their differentiation. Despite AER induction and maintenance are orchestrated by complex interactions between the FGF, WNT/β-catenin and BMP signaling pathways, little is known about the molecules involved in the maintenance of the proliferation versus apoptosis and the renewal of its cells during development. In recent studies, it has been showed by our lab that one of these molecules, oct4, could be involved in the control of proliferative balance within the AER cells. In this thesis we present evidences of the involvement of two more molecules in this process, lgr5 and lgr6, which are known as adult stem cells markers. In here, we describe the expression pattern of lgr5 and lgr6 during limb bud development using the chicken embryo as a model and show that their expression patterns in the limb bud are consistent with the areas of cell proliferation within the AER. Moreover, we performed lgr5 gain-of-function experiments through in ovo electroporation and studied the relationship of lgr5 and lgr6 with different signaling pathways known to be involved in the AER induction and maintenance. The phenotypes obtained point to the involvement of lgr5 in the maintenance of a proliferative niche in the AER. We also present here, evidences showing that lgr6 is controlled by WNT signaling. Our results support a model in which lgr5 and lgr6 control the activation and maintenance of a niche of undifferentiated cells that are in continuous proliferation at the base of AER. This niche can be responsible for the renewal of AER until it disappears by massive programmed cell death

Targeting senescent cells improves functional recovery after spinal cord injury

© The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)Persistent senescent cells (SCs) are known to underlie aging-related chronic disorders, but it is now recognized that SCs may be at the center of tissue remodeling events, namely during development or organ repair. In this study, we show that two distinct senescence profiles are induced in the context of a spinal cord injury between the regenerative zebrafish and the scarring mouse. Whereas induced SCs in zebrafish are progressively cleared out, they accumulate over time in mice. Depletion of SCs in spinal-cord-injured mice, with different senolytic drugs, improves locomotor, sensory, and bladder functions. This functional recovery is associated with improved myelin sparing, reduced fibrotic scar, and attenuated inflammation, which correlate with a decreased secretion of pro-fibrotic and pro-inflammatory factors. Targeting SCs is a promising therapeutic strategy not only for spinal cord injuries but potentially for other organs that lack regenerative competence.D.P.d.C. was supported by a FCT PhD fellowship (PD/BD/105770/2014). I.M. was supported by a FCT post-doctoral fellowship (SFRH/BPD/118051/2016). A.M.C. was supported by a FCT fellowship (PTDC/BOM-MED/3295/2014). A.F.D. was supported by CONGENTO LISBOA-01-0145-FEDER-022170, co-financed by FCT (Portugal) and Lisboa2020, under the PORTUGAL2020 agreement (European Regional Development Fund). D.N.-S. was supported by a FCT PhD fellowship (SFRH/BD/138636/2018). D.C. was supported by a FCT PhD fellowship (PD/BD/114179/2016). L.S. was supported by a FCT IF contract. The project leading to these results has received funding from a FCT grant (PTDC/MED-NEU/30428/2017) and “la Caixa” Banking Foundation and FCT, I.P., under project code HR18-00187.info:eu-repo/semantics/publishedVersio

Innovative therapies for spinal cord injury : a zebrafish approach

Spinal cord injury (SCI) is a debilitating condition that affects millions of people worldwide. It leads to devastating motor and sensory dysfunctions and elicits cardiac and respiratory alterations, bladder and bowel dysfunction, as well as, sexual dysfunction and other complications. This complex neurological condition has a huge impact on the health, quality and life expectancy, thus constituting a high socioeconomic and psychological burden for patients and their families. After a traumatic SCI, current management approaches consist in the complete immobilization and constant monitorization of the patient (blood pressure, respiratory and cardiac alterations), administration of anti-inflammatory drugs, surgical decompression of the spinal cord to stabilize the spinal column and rehabilitative care. However, none of these efforts result in a cure for SCI patients. In fact, due to the complexity and multitude of SCI facets, there is still no effective treatment available on the market for this indication. The biphasic SCI pathophysiology comprises primary and secondary injuries which trigger a complex cascade of events that can lead to beneficial or detrimental responses. However, the reparative responses are limited and the fibroglial scar formation and the inhibitory microenvironment constitute the main obstacles for SCI repair in mammals. In contrast to mammalian models, zebrafish show robust spinal cord recovery without a fibroglial scar formation. Thus, in addition to having emerged as a valuable tool for drug discovery, zebrafish has also emerged as an attractive model for the study of SCI. In fact, despite having different outcomes, zebrafish and mammals share a considerable number of disease-associated targets and drug metabolic pathways. Therefore, the idea of developing a phenotypic-based in vivo screening using a larval zebrafish model of SCI to select bioactive chemical compounds with therapeutic potential to SCI, becomes particularly appealing. Thus, we designed a simple, fast and efficient drug screening platform using a zebrafish larval spinal cord transection model. This approach allows the selection of small molecules with locomotor rescue properties in this zebrafish larva model of SCI. We validated our screening platform by showing that Riluzole and Minocycline, two different pharmacologic classes of molecules that have entered in clinical trials for SCI indication, promote rescue of the motor function of the transected larvae. Further validation of the platform was demonstrated through the blind identification of D-Cycloserine, a molecule scheduled to enter phase IV clinical trials for SCI. Next, we used this larval zebrafish drug discovery platform to blindly screen 113 bioactive compounds from a FDA-approved small molecule library. As we used known FDA-approved drugs, we took advantage of previous studies and reports to further select three candidate compounds, Tranexamic acid, Pefloxacin mesylate and Eletriptan Hbr that showed significant motor rescue properties in the pro-regenerative SCI model. As we intended to validate the translational value of zebrafish larvae in the SCI context, we tested the therapeutic efficacy of the three candidate compounds in a rodent contusion (pro-fibrotic) model of SCI. We showed that Pefloxacin mesylate did not preserve its therapeutic effect on motor function in contused mice but exhibited a therapeutic effect on another important SCI sequel, the bladder dysfunction. Importantly, we showed that Tranexamic acid and Eletriptan Hbr molecules maintain their locomotor recovery properties in T9-contused mice. We showed that Tranexamic acid reduces the extension of the lesion and we proposed that its therapeutic effect on motor function could be explained by a possible role in limiting the toxic effect of the parenchymal haemorrhage, thereby controlling the extension of the lesion and improving the locomotor outcome of SCI animals. Besides proving that the therapeutic effect of Eletriptan Hbr was preserved in a mammalian SCI model, we also explore its therapeutic potential in a second rodent study. This second study aimed to determine if a longer therapeutic administration would result in greater improvement in the long term and allowed to further evaluate its therapeutic effect on the demyelination status, fibrotic scar and microglia/macrophages at a chronic phase of the injury. We suggested that perhaps the locomotor improvement properties of Eletriptan Hbr could be explain by a possible mediation by PDGFRβ+ cells. Additionally, we also demonstrated that Eletriptan Hbr led microglia to display a morphology that is more similar to that one characteristic of moderately activated microglia which suggests that maybe Eletriptan Hbr has the ability to modulate the inflammatory response. Overall, we demonstrated in this study the predictive value of zebrafish for phenotype-based drug screenings and presented a full proof-of-concept of transected spinal cord zebrafish larval model for the identification of new therapeutics for SCI. Notably, it was presented a simple, fast and automated phenotypic screening with proved efficacy in a mouse contusion model of SCI. Besides demonstrating the translational value of this zebrafish drug screening platform, we also showed the importance of testing the effectiveness of the new molecules identified on this platform, in a mammalian model before translation to clinical trials. Ultimately, this approach combined with drug repositioning allowed to identify a potential new use for two molecules (Tranexamic acid and Eletriptan Hbr) already in use in the clinic for other therapeutic indications. We believe that the strategy here presented, associated with combinatorial therapeutic approaches, promises to accelerate the discover and rapid translation of effective treatments for SCI in humans

Tapentadol prevents motor impairments in a mouse model of dyskinesia

© 2019 IBRO. Published by Elsevier Ltd. All rights reserved.The motor features in Parkinson's disease (PD) are associated with the degeneration of dopaminergic cells in the substantia nigra in the brain. Thus, the gold-standard in PD therapeutics still consists of dopamine replacement with levodopa. However, as the disease progresses, this therapeutic option becomes less effective and can be accompanied by levodopa-induced complications. On the other hand, several other neuronal pathways have been implicated in the pathological mechanisms of PD. In this context, the development of alternative therapeutic options that modulate non-dopaminergic targets is emerging as a major goal in the field. In a phenotypic-based screen in a zebrafish model of PD, we identified tapentadol as a candidate molecule for PD. The therapeutic potential of an agent that modulates the opioid and noradrenergic systems has not been explored, despite the implication of both neuronal pathways in parkinsonism. Therefore, we assessed the therapeutic properties of this µ-opioid receptor agonist and norepinephrine reuptake inhibitor in the 6-hydroxydopamine mouse model of parkinsonism. We further submitted 6-hydroxydopamine-lesioned mice to chronic treatment with levodopa and evaluated the effects of tapentadol during levodopa OFF states and on levodopa-induced dyskinesia. Importantly, we found that tapentadol halted the aggravation of dyskinesia and improved the motor impairments during levodopa OFF states. Altogether, our findings raise the hypothesis that concomitant modulation of µ-opioid receptor and norepinephrine transporter might constitute relevant intervention strategies in PD and that tapentadol holds therapeutic potential that may be translated into the clinical practice.This study was sponsored by TechnoPhage S.A. and Eurostars program (ES#5553) from EUREKA (a program run by the European Commission). Rita L. Vaz was supported by a grant (SFRH/BD/78077/2011) from Fundação para a Ciência e Tecnologia. Tiago F. Outeiro was supported by the DFG Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB).info:eu-repo/semantics/publishedVersio