AIM 1: To investigate the potential actions of sIFNAR2 “in vitro” in modulating IFNβ signalling on neural functions. Lack of IFNβ/IFNAR1 in primary neurons and Neuro2A cell line affects survival, autophagy, neurite outgrowth, mitochondrial status. The recombinant sIFNAR2 protein (rsIFNAR2) has immunomodulatory actions per se in cell culture and different to IFNβ actions, but are unknown the pathways/genes that rsIFNAR2 induces. rsIFNAR2 could modulate the IFNβ-signalling, and the membrane receptors IFNAR1/2 could also have a role in this regulatory mechanism, so it is essential to use depleted cells for these proteins to study actions of sIFNAR2.
-Jak-Stat pathway evaluation after the IFNβ or IFNAR2 treatment on N2A Ifnar1-/- , Ifnar2-/- and IFNβ-/- cells generated by CRISPR/Cas9 editing. No activation was observed when cells were treated with rsIFNAR2, so also proved that the actions of srIFNAR2 are independent of the JAK-STAT signalling and its mechanism actions are induced through another pathway or mechanism.
-Effect of rsIFNAR2 in mitochondria homeostasis. A live cell analysis
using N2A cells showed that rsIFNAR2 treatment increases significantly both the mitochondrial mass and the mitochondrial membrane potential in the WT N2A but also in the depleted cells ones compared to the untreated one, considered as the negative control. This effect was independent of IFNβ signaling and confirmed by Western Blot of HSP60 and by IF of TOM20.
-Effect of rsIFNAR2 on autophagy. N2A-Wt and Ifnar1-/- cells showed a clear tendency to increase autophagy flux after treatment with rsIFNAR2 by the assessing p62, LC3B and Gabarapl1 expression.
-Effect of rsIFNAR2 on neurite outgrowth. Under neuronal differentiation and by a by a live cell analysis, N2A-Wt cells and knock out for Ifnar1-/- or for IFNβ-/- did not shown a tendency to increase the neurite outgrowth upon the rsIFNAR2 treatment.
Aim2:To investigate "in vivo" the potential actions of rsIFNAR2 in regulating the IFNβ-signalling in the PD pathology and the neuroinflammatory context of the PD-model (IFNβ/IFNAR1KO). Rationale: The Ifnb–/– or Ifnar1–/– mice exhibit spontaneous motor and cognitive impairments as well as like-PD neurodegeneration and rIFNβ rescues these defects. The sIFNAR2 can modulate the IFNβ signaling, but if rsIFNAR2 has positive effects on modulating the IFNβ signaling in these models are unknown.
-Recombinant sIFNAR2 as a treatment in a PD-like mouse model evaluation of effects on motor and cognitive functions.
Intracerebral rsIFNAR2 administration led to a clear improvement in memory, cognition, and motor coordination in IFNAR1-/- but not in IFNβ-/- mice, indicating a possible mediation of the inner IFNβ.
-The brain tissue analysis showed a significant decrease in TH+ neurons were observed in mice lacking IFN-β and a tendency in IFNAR1-KO at baseline (treated with PBS), compared to their control WT group. However, in vivo treatment with rsIFNAR2 was not able to prevent the degeneration of dopaminergic neurons or restore them in the SN. But for a more detailed insight into the degeneration of dopaminergic neurons, quantification of TH+ cells and neurite network in the striatum by IHC or IF would be essential.
Furthermore, neuronal activity as c-Fos positive cells was found significantly increased in the hippocampus in Wt and IFNAR1-/- mice treated with rsIFNAR2, which was consistent with the behavior improvements observed. The quantification of cell proliferation (PCNA marker) showed a significantly reduced level of PCNA-positive cells in IFNAR1-/- and IFNβ-/- mice, compared to WT mice treated with PBS. No impact was observed as consequence of the rsIFNAR2 treatment.
*Dissemination of the research results. I have done seminars for the entire audience of the department as well as specifically for the neuroscience groups. The data from this project are available in the internal group server, and some data is being prepared for update in open repositories. A research article is being prepared for publication in open access, and an abstract will be submitted to the next FENS Forum. I have mentored or supervised of a Bachelor Final Project and a Master student from the University of Copenhagen. I also organized a dissemination online activity because of the International Day of Women and Girls in Science with pupils from a primary school (29002150) in Málaga (Spain).
