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

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.

Ndufs4 knockout mouse models of Leigh syndrome: pathophysiology and intervention

“…Remarkably, absence of the NDUFS4 protein induced near complete absence of the NDUFA12 subunit and increased the protein level of NDUFA2 (an assembly factor of CI) in brain, liver, heart, kidney, diaphragm and skeletal muscle of Ndufs4 −/− -WB animals. 64 This finding was confirmed in Ndufs4 −/− -WB-derived mouse embryonic fibroblasts (MEFs; see below), Ndufs4 −/− brainstem glutamatergic neurons 94 and NDUFS4 -mutated Leigh syndrome patient cells. Compatible with previous data, 91 Ndufs4 −/− -WB-derived MEFs displayed in situ CI activity, but BN–PAGE analysis revealed that NDUFAF2 attached to an inactive CI subcomplex (CI-830) and inactive assemblies of higher molecular weight.…”

Inflammatory capacity of exosomes released in the early stages of acute pancreatitis predicts the severity of the disease

“…Peptides were analyzed using an Orbitrap Fusion Lumos Tribrid mass spectrometer coupled to a Thermo Scientific Dionex Ultimate 3000 ultrahigh‐pressure chromatographic system (Thermo Fisher Scientific) as described previously [17].…”

Isolation and Characterization of NpCI, a New Metallocarboxypeptidase Inhibitor from the Marine Snail Nerita peloronta with Anti-Plasmodium falciparum Activity

“…Peptide alignment and protein sequence analyses from the fragmentation spectra were performed using the Bruker Daltonics software and Mascot search engine, with incorporation of the EDMAN degradation data. LC.ESI-MS/MS analysis were performed in an Orbitrap Fusion Lumos Tribid spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), using a μ-precolumn Acclaim C18 PepMap100 and a C18 Acclaim PepMap RSLC (nanoViper, Waltham, MA, USA) analytical column, following the details reported in [ 44 ]. Validation of the final derived sequence was achieved by high resolution/high accuracy MALDI.MS analysis on a Bruker timsTOF fleX mass spectrometer.…”