Mitochondrial Proteome of Affected Glutamatergic Neurons in a Mouse Model of Leigh Syndrome.

Alejandro Gella,Elisenda Sanz,Montserrat Carrascal,Andrea Urpi,Melania González-Torres,Albert Quintana,Patricia Prada-Dacasa,Joaquin Abian

doi:10.3389/fcell.2020.00660

Abstract

Defects in mitochondrial function lead to severe neuromuscular orphan pathologies known as mitochondrial disease. Among them, Leigh Syndrome is the most common pediatric presentation, characterized by symmetrical brain lesions, hypotonia, motor and respiratory deficits, and premature death. Mitochondrial diseases are characterized by a marked anatomical and cellular specificity. However, the molecular determinants for this susceptibility are currently unknown, hindering the efforts to find an effective treatment. Due to the complex crosstalk between mitochondria and their supporting cell, strategies to assess the underlying alterations in affected cell types in the context of mitochondrial dysfunction are critical. Here, we developed a novel virus-based tool, the AAV-mitoTag viral vector, to isolate mitochondria from genetically defined cell types. Expression of the AAV-mitoTag in the glutamatergic vestibular neurons of a mouse model of Leigh Syndrome lacking the complex I subunit Ndufs4 allowed us to assess the proteome and acetylome of a subset of susceptible neurons in a well characterized model recapitulating the human disease. Our results show a marked reduction of complex I N-module subunit abundance and an increase in the levels of the assembly factor NDUFA2. Transiently associated non-mitochondrial proteins such as PKCδ, and the complement subcomponent C1Q were also increased in Ndufs4-deficient mitochondria. Furthermore, lack of Ndufs4 induced ATP synthase complex and pyruvate dehydrogenase (PDH) subunit hyperacetylation, leading to decreased PDH activity. We provide novel insight on the pathways involved in mitochondrial disease, which could underlie potential therapeutic approaches for these pathologies.

Highlights

Severe alterations of the mitochondrial machinery involved in energy generation lead to a group of progressive and usually fatal pathologies collectively known as primary mitochondrial disease (MD), affecting 1:4,000 births (Gorman et al, 2015)
Immunofluorescence analysis for HA in cells transfected with the mitoTag viral vector construct and transduced with viral vector particles expressing Cre recombinase showed a localization of the fusion protein consistent with incorporation into mitochondria (Figure 1C)
To test whether whole mitochondria isolation from Cre-expressing cells could be accomplished by an immunocapture method, lysates from HEK293T/17 cells transduced with the associated viral vectors (AAVs)-mitoTag viral vector (AAVDIO-Tomm20·HA) along with a Cre recombinase-expressing viral vector (AAV-Cre) were used to immunoprecipitate HA-tagged mitochondria with anti-HA coated magnetic microbeads (Figure 1D)

Summary

Introduction

Severe alterations of the mitochondrial machinery involved in energy generation lead to a group of progressive and usually fatal pathologies collectively known as primary mitochondrial disease (MD), affecting 1:4,000 births (Gorman et al, 2015). There is no cure and the treatments available are mostly ineffective (Schon et al, 2010). MD predominantly affects high energyrequiring organs such as the brain (Vafai and Mootha, 2012). Neurological damage plays a prominent role in MD pathology and lethality (DiMauro and Schon, 2008). Recent reports have evidenced the remarkable regional heterogeneity in mitochondrial protein composition in the brain, which may account for the cell type specificity of MD (Pagliarini et al, 2008)

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Results

Conclusion