Periodic Reporting for period 1 - ParkIFNAR (Soluble IFNAR2 in Parkinson's disease and its role in the regulation of IFNβ in a neuroinflammatory context.)
Reporting period: 2020-09-14 to 2022-09-13
Over 1.2 million people in Europe alone suffer from Parkinsonian disorders (PD), and it has a rising prevalence. This neurodegenerative condition led to decreased motor activity and other complications including cognitive impairment. Current medications for patients are focused on treating the symptoms, however, no curative treatment is available.
Evidence indicates that neuroinflammation is partly responsible for the observed PD pathology. The host lab reported a dysfunctional type I interferon signaling is associated with PD, but the role of their receptor in PD is not yet understood. Here, we aimed to investigate the role of type I interferon (IFN) receptors, specially its soluble IFNAR2 receptor (sIFNAR2), in Parkinson’s disease (PD) pathology. Of note, the host lab has identified a new mutation in IFNAR1 associated with a family of PD patients, and I found that PD patients present lower serum levels of sIFNAR2 than healthy controls indicating dysregulation in this pathway. So, we hypothesize that a defective IFNβ-IFNAR signalling is associated with PD pathology, and this could be partly mediated by the function of sIFNAR2 as this protein is able to modulate the endogenous IFNβ and interplay with membrane-expressed IFNAR1 or IFNAR2. Our objective was to investigate "in vivo" the potential actions of sIFNAR2 in regulating the IFNβ-signalling in the PD pathology and the neuroinflammatory context of the PD-model (IFNβ/IFNAR1KO).
Evidence indicates that neuroinflammation is partly responsible for the observed PD pathology. The host lab reported a dysfunctional type I interferon signaling is associated with PD, but the role of their receptor in PD is not yet understood. Here, we aimed to investigate the role of type I interferon (IFN) receptors, specially its soluble IFNAR2 receptor (sIFNAR2), in Parkinson’s disease (PD) pathology. Of note, the host lab has identified a new mutation in IFNAR1 associated with a family of PD patients, and I found that PD patients present lower serum levels of sIFNAR2 than healthy controls indicating dysregulation in this pathway. So, we hypothesize that a defective IFNβ-IFNAR signalling is associated with PD pathology, and this could be partly mediated by the function of sIFNAR2 as this protein is able to modulate the endogenous IFNβ and interplay with membrane-expressed IFNAR1 or IFNAR2. Our objective was to investigate "in vivo" the potential actions of sIFNAR2 in regulating the IFNβ-signalling in the PD pathology and the neuroinflammatory context of the PD-model (IFNβ/IFNAR1KO).
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).
-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).
In ParkIFNAR my aim was to investigate the potential of srIFNAR2 potentially targeting immunomodulatory processes, as a new pharmacological approach in PD mouse models. Interestingly, we found that mice treated with srIFNAR2 exhibited clear impairments in both memory and motor functions, corresponding to symptoms observed in PD patients. IFNAR1-/- but not IFNb-/- PD-mice treated with rsIFNAR2 exhibited improved memory, better learning, and improved motor function. The treatment even significantly improved the brain functions in WT mice. Parkinson’s Disease (PD), the most common Parkinsonian disorder, is the fastest growing in terms of prevalence, disability and mortality, and it has an estimated cost of €13.9 billion annually in Europe. Nevertheless, the etiology of PD remains largely unknown, slowing the development of successful therapeutic strategies, and to date there are no disease-modifying drugs.
This project allowed me to obtain new knowledge from the state-of-the-art techniques in neuroscience, including more understanding of the mechanisms behind the neuronal homeostasis in PD. Obtaining this Marie Curie fellowship definitively contributed to consolidate my research career. In this sense, I have recently obtained a Maria Zambrano postdoctoral fellowship to join the University of Málaga and IBIMA. This fellowship is competitive at national level and it will allow me to study the Type I interferon signalling in Neurological diseases.
This project allowed me to obtain new knowledge from the state-of-the-art techniques in neuroscience, including more understanding of the mechanisms behind the neuronal homeostasis in PD. Obtaining this Marie Curie fellowship definitively contributed to consolidate my research career. In this sense, I have recently obtained a Maria Zambrano postdoctoral fellowship to join the University of Málaga and IBIMA. This fellowship is competitive at national level and it will allow me to study the Type I interferon signalling in Neurological diseases.