Analysis of the LETM1 Protein Family in the Development of Zebrafish

LETM1 has been recently identified among the ~ 2000 essential genes for cell fitness and survival. It encodes a conserved protein with crucial regulatory functions in maintaining the mitochondrial volume and ion homeostasis. The key function of mitochondria relates to their vital role in energy conservation, metabolism, iron-sulfur cluster and cell death decision. Mitochondrial dysfunctions are associated with numerous diseases. LETM1 is involved in the Wolf-Hirschhorn Syndrome (WHS), a severe and complex childhood disorder with growth and mental retardation, heart and kidney failures and seizures. Seizures in WHS patients strongly correlate with loss of LETM1. The implication of LETM1 and thus, mitochondrial dysfunctions, in the pathogenesis of seizures led us to propose studying LETM1 in the developmental model system Danio rerio. In this project we will investigate for the first time the role of LETM1-related functions in the development using the zebrafish Danio rerio, a strong model for vertebrate development. Human LETM1 has two homologs in Danio rerio: letm1 and letm2. We aim to clarify when during early development and in which tissues letm1 and letm2 are expressed. We will determine the letm1 and letm2 knockout (KO) phenotypes in the development of the zebrafish with focus on the brain and neural functions. Using edge cutting technologies, we therefore generated letm1 and letm2 KO fish using different fish lines which either express green fluorescent mitochondria or a fluorescent calcium reporter in neural cells. In these fish lines we can monitor the mitochondrial morphology or the neural activities, respectively, in function of letm1 and letm2. We will study the letm1 and letm2- dependent mitochondrial respiration and metabolism and neuronal functions in the embryo, larval and adult fish and study their swimming behaviour. Our data will reveal if and how LETM1 and mitochondrial related (dys)functions correlate with pathogenic neural or locomotory symptoms. We will test if drugs that alleviate mitochondrial dysfunctions can rescue the observed developmental or behavioural phenotypes. Furthermore, our zebrafish model has the potential to serve for future drug screenings aiming to identify novel anti-epileptic agents. Altogether, the study is expected to reveal if and how letm1 - and potentially letm2 - caused mitochondrial osmotic deregulation impede the early development and the nervous system activity and establish a WHS seizure model in the zebrafish.

Mitochondria buffer intracellular cations and supply our cells with energy and metabolites day and night, keeping cells alive and enabling them to perform their highly specialized functions at the right time. Disturbance of their metabolic functions is a danger for the cell and for the entire organism. Functional mitochondria require controlled cation transport activities as these are keeping the organellar volume in shape. LETM1 is the major osmotic regulator of mitochondrial volume and its absence causes swollen and damaged mitochondria. In human, loss of LETM1 is associated with a number of diseases including seizures, diabetes and cancer. To investigate how the loss of LETM1 leads to such organismal disorders, we took advantage of the high evolutionary gene conservation of LETM1 and the diurnal nature of the vertebrate zebrafish, which like humans, is active at day and rests at night. We generated a zebrafish model expressing letm1 wildtype (+/+) or deleted for letm1 (-/-) on both chromosomal alleles by using new gene editing technologies. Our study has uncovered unexpected aspects of letm1 regulation and dysfunction. We showed that letm1 is highly expressed in embryos, as well as in juvenile and adult letm1 +/+ zebrafish, with increased concentration in the brain. We confirming that Letm1 localises to mitochondria and that its concentration is under circadian regulation, being highest at night and lowest around noon. Elimination of Letm1 resulted in high letm1-/- embryonic mortality, although a small population of offspring survived. This unique opportunity allowed to study deletion phenotypes as well as differences between non-surviving (non-compensatory) and surviving (compensatory) letm1-/- larvae and provided new functional insights at the interface between mitochondrial biology and chronobiology. Developmental malformation, disturbed mitochondrial morphology and altered metabolism of nicotinamide adenine dinucleotide (NAD) and its reduced form NADH emerged as very early signs of letm1 deficiency. NAD(H) is an important co-factor involved in the regulation of the day/night rhythms, and was decreased in letm1-/- embryos. However, we found significantly altered expression patterns of nampt1/2, a crucial factor for NAD metabolism, and of biological clock genes in the compensatory letm1-/- larvae. nampt1/2 genes were upregulated in the morning, possibly to replenish the NAD(H) pools depleted at night. Also, the expression of the clock genes clock1a, per1b, and per2 was significantly increased at their natural peaks. The coupled upregulation of NAD metabolism and circadian clocks appears to contribute to the survival of letm1-/- compensators, and their development into healthy looking adult zebrafish. Global gene expression analysis to identify potential other transcriptional survival mechanisms of letm1-/- compensators confirmed the role of NAD metabolism and indicated strong immune system changes in the letm1-/- non-compensators. These new findings are of great clinical importance for human LETM1-related diseases and are the subject of future studies.