Periodic Reporting for period 1 - MitoTROJAN (Dysfunctional mitochondria as a neuronal pathogen)
Okres sprawozdawczy: 2021-12-01 do 2023-11-30
The symbiosis between the mitochondrion and the ancestor of the eukaryotic cell allowed cellular complexity. Due to its archeobacterial origin, mitochondria has been posited as a potential source of molecules that can elicit cellular responses to pathogens. Among other key functions, mitochondria generates most of the energy required by cells. Alterations in components of the mitochondrial energy-generating machinery lead to primary mitochondrial disease (MD), a group of highly invalidating human conditions with no effective treatment. High-energy-requiring cells, such as neurons, are especially affected in MD. However, not all neuronal populations are equally affected and the molecular determinants of this susceptibility are currently unknown. Recently, Quintana´s lab uncovered that GABAergic neurons in the Globus Pallidus (GPe) of a mouse model of Leigh syndrome (LS) are particularly sensitive to MD leading to fatal encephalopathy, recapitulating the human pathology. In addition, unpublished results of the host group showed that this neuronal population develop a robust cellular antiviral-like response elicited by mitochondrial double-stranded RNA (mtdsRNA). Nevertheless, the contribution of mtdsRNA release in the MD progression has never been defined. Thus, the main goal of MitoTROJAN is to identify the effect of mtdsRNA-induced neuropathology in the context of mitochondrial dysfunction with the overarching goal of providing novel targets for the treatment of MD. By combining mouse genetics with cutting-edge molecular biology and multi-omics approach, I will carry out a multidisciplinary strategy to i) parse the molecular mechanisms of mtdsRNA-induced neuropathology and ii) characterise the antiviral response-induced protein shutdown in affected neurons. Overall results will provide new insight on an uncharacterized mechanism of mitochondria-mediated neurodegeneration and will help to identify novel therapeutic targets for MD and other neurodegenerative diseases.
MitoTROJAN proposes to decipher, for the first time, the cellular mechanisms involved in mtdsRNA-induced antiviral response in susceptible neuronal populations and to establish the role of this aberrant antiviral signalling in the neuropathology of MD.
By combining mouse genetics with cutting-edge molecular biology tools, I carried out a multi-approach strategy to:
1. Parse the molecular mechanisms of mtdsRNA-induced antiviral response in GABAergic neurons.
2. Characterise the antiviral response-induced protein shutdown in affected neurons.
1. To identify whether the activation of mtdsRNA cytosolic sensor PKR in susceptible neuronal population was a key mediator of mtdsRNA-induced neurodegeneration in the Ndufs4KO mouse model of Leigh Syndrome, I blocked the activity of this kinase. To this end, I used an adeno-associated viral (AAV)-based approach using a shRNA targeting PKR. These results show that AAV-mediated silencing of PKR was sufficient to ameliorate the clinical phenotype of Ndufs4KO mice, significantly extending their lifespan. Moreover, silencing PKR decreased inflammatory markers in affected brain areas, suggesting that the antiviral response is contributing to disease likely by exacerbating the neuroimmune response.
2. The objective number 2 was to investigate if the increased abundance of mtdsRNA associated to PKR observed in affected neurons of Ndufs4KO mice halts global protein synthesis further contributing to the pathogenesis of the disease. To study protein synthesis in vivo I used NCAA labelling using azidohomoalanine (AHA) that is recognized by the endogenous methionine tRNA synthetase. Then, I performed fluorescent NCAA tagging (FUNCAT) and bio-orthogonal NCAA tagging (BONCAT) to visualized and purified de novo protein synthesis. These results points out that affected neurons of the mutant mice did not show inhibition of protein synthesis and that PKR upregulation may contribute to disease progression through the NF-κB pathway.
The scientific findings of this proposal will be published in high-quality peer reviewed international journal so that the new methodology approaches and results can be available to all the scientific community. In this regard, we aim to submit a manuscript by the end of this year. In addition, some of the results generated through the MSCA action have laid the foundation for future projects.
The results obtained from MitoTROJAN were also presented in international and national scientific meetings.
By combining mouse genetics with cutting-edge molecular biology tools, I carried out a multi-approach strategy to:
1. Parse the molecular mechanisms of mtdsRNA-induced antiviral response in GABAergic neurons.
2. Characterise the antiviral response-induced protein shutdown in affected neurons.
1. To identify whether the activation of mtdsRNA cytosolic sensor PKR in susceptible neuronal population was a key mediator of mtdsRNA-induced neurodegeneration in the Ndufs4KO mouse model of Leigh Syndrome, I blocked the activity of this kinase. To this end, I used an adeno-associated viral (AAV)-based approach using a shRNA targeting PKR. These results show that AAV-mediated silencing of PKR was sufficient to ameliorate the clinical phenotype of Ndufs4KO mice, significantly extending their lifespan. Moreover, silencing PKR decreased inflammatory markers in affected brain areas, suggesting that the antiviral response is contributing to disease likely by exacerbating the neuroimmune response.
2. The objective number 2 was to investigate if the increased abundance of mtdsRNA associated to PKR observed in affected neurons of Ndufs4KO mice halts global protein synthesis further contributing to the pathogenesis of the disease. To study protein synthesis in vivo I used NCAA labelling using azidohomoalanine (AHA) that is recognized by the endogenous methionine tRNA synthetase. Then, I performed fluorescent NCAA tagging (FUNCAT) and bio-orthogonal NCAA tagging (BONCAT) to visualized and purified de novo protein synthesis. These results points out that affected neurons of the mutant mice did not show inhibition of protein synthesis and that PKR upregulation may contribute to disease progression through the NF-κB pathway.
The scientific findings of this proposal will be published in high-quality peer reviewed international journal so that the new methodology approaches and results can be available to all the scientific community. In this regard, we aim to submit a manuscript by the end of this year. In addition, some of the results generated through the MSCA action have laid the foundation for future projects.
The results obtained from MitoTROJAN were also presented in international and national scientific meetings.
The performance of the MitoTROJAN project in Quintana’s lab, has allowed me to be involved in a multidisciplinary research program that is timely and forefront of modern molecular life sciences research and that represent an improvement in the field of mitochondrial patho-physiology and will help to develop efficient treatments on the basis of a successful transcriptomic and proteomic profiling.
Carrying out this project has enabled me to develop and implement innovative molecular biology methods and unprecedented multi-omics approach (such as cell-type specific cytosolic translatome profiling and cell-type specific de-novo protein synthesis profiling) widen my scientific knowledge on mitochondrial pathophysiology and experimental skills in translational medicine, improving my independent thinking and facilitating to reach a professional maturity as an independent mitochondrial research scientist.
Moreover, the innovative approaches and results generated here can also be applied to other pathologies associated with mitochondrial dysfunction and inflammation such as cancer or neurodegenerative diseases as Parkinson's disease, Multiple Sclerosis, Huntington's disease among others
Carrying out this project has enabled me to develop and implement innovative molecular biology methods and unprecedented multi-omics approach (such as cell-type specific cytosolic translatome profiling and cell-type specific de-novo protein synthesis profiling) widen my scientific knowledge on mitochondrial pathophysiology and experimental skills in translational medicine, improving my independent thinking and facilitating to reach a professional maturity as an independent mitochondrial research scientist.
Moreover, the innovative approaches and results generated here can also be applied to other pathologies associated with mitochondrial dysfunction and inflammation such as cancer or neurodegenerative diseases as Parkinson's disease, Multiple Sclerosis, Huntington's disease among others