Primary Cultures of Pure Embryonic Dorsal Root Ganglia Sensory Neurons as a New Cellular Model for Friedreich’s Ataxia

Peripheral neuropathies can have various origins, from genetic to acquired causes, and affect altogether a large group of people in the world. Current available therapies aim at helping the disease symptoms but not to correct or stop the development of the disease. Primary neuronal cultures represent an essential tool in the study of events related to peripheral neuropathies as they allow to isolate the affected cell types, often originating in complex tissues in which they account for only a few percentage of cells. They provide a powerful system to identifying or testing compounds with potential therapeutic effect in the treatment of those diseases. Friedreich's ataxia is an autosomal recessive neurodegenerative disorder, which is characterized by a progressive spinocerebellar and sensory ataxia. Proprioceptive neurons of the dorsal root ganglia (DRG) are the primary affected cells. The disease is triggered by a mutation in the gene FXN which leads to a reduction of the frataxin protein. In order to study the neurophysiopathology of the disease at the cellular and molecular levels, we have established a model of primary cultures of DRG sensory neurons in which we induce the loss of the frataxin protein. With such a model we can alleviate the issues related to the complexity of DRG tissues and low amount of sensory neuron material in adult mouse. Hereby, we provide a protocol of detailed and optimized methods to obtain high yield of healthy mouse DRG sensory neuron in culture

Functional reconstitution of mitochondrial Fe/S cluster synthesis on Isu1 reveals the involvement of ferredoxin

Maturation of iron-sulphur (Fe/S) proteins involves complex biosynthetic machinery. In vivo synthesis of [2Fe-2S] clusters on the mitochondrial scaffold protein Isu1 requires the cysteine desulphurase complex Nfs1-Isd11, frataxin, ferredoxin Yah1 and its reductase Arh1. The roles of Yah1-Arh1 have remained enigmatic, because they are not required for in vitro Fe/S cluster assembly. Here, we reconstitute [2Fe-2S] cluster synthesis on Isu1 in a reaction depending on Nfs1-Isd11, frataxin, Yah1, Arh1 and NADPH. Unlike in the bacterial system, frataxin is an essential part of Fe/S cluster biosynthesis and is required simultaneously and stoichiometrically to Yah1. Reduced but not oxidized Yah1 tightly interacts with apo-Isu1 indicating a dynamic interaction between Yah1-apo-Isu1. Nuclear magnetic resonance structural studies identify the Yah1-apo-Isu1 interaction surface and suggest a pathway for electron flow from reduced ferredoxin to Isu1. Together, our study defines the molecular function of the ferredoxin Yah1 and its human orthologue FDX2 in mitochondrial Fe/S cluster synthesis

Iron-Sulfur Protein Assembly in Human Cells

Iron-sulfur (Fe-S) clusters serve as a fundamental inorganic constituent of living cells ranging from bacteria to human. The importance of Fe-S clusters is underscored by their requirement as a co-factor for the functioning of different enzymes and proteins. The biogenesis of Fe-S cluster is a highly coordinated process which requires specialized cellular machinery. Presently, understanding of Fe-S cluster biogenesis in human draws meticulous attention since defects in the biogenesis process result in development of multiple diseases with unresolved solutions. Mitochondrion is the major cellular compartment of Fe-S cluster biogenesis, although cytosolic biogenesis machinery has been reported in eukaryotes, including in human. The core biogenesis pathway comprises two steps. The process initiates with the assembly of Fe-S cluster on a platform scaffold protein in the presence of iron and sulfur donor proteins. Subsequent process is the transfer and maturation of the cluster to a bonafide target protein. Human Fe-S cluster biogenesis machinery comprises the mitochondrial iron-sulfur cluster (ISC) assembly and export system along with the cytosolic Fe-S cluster assembly (CIA) machinery. Impairment in the Fe-S cluster machinery components results in cellular dysfunction leading to various mitochondrial pathophysiological consequences. The current review highlights recent developments and understanding in the domain of Fe-S cluster assembly biology in higher eukaryotes, particularly in human cells