Drosophila models of peroxisomal biogenesis disorder: peroxins are required for spermatogenesis and very-long-chain fatty acid metabolism

Haiyang Chen 0 1 2 Zhonghua Liu 1 2 Xun Huang 1 2 0 Graduate University of Chinese Academy of Sciences , Beijing 1 Academy of Sciences , Beijing 100101, China 2 Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology , Chinese Peroxisomes are vital eukaryotic organelles that participate in lipid metabolism, in particular the metabolism of very-long-chain fatty acids (VLCFA). The biogenesis of peroxisomes is regulated by a set of peroxin proteins (PEX). In humans, mutations affecting peroxin protein production or function result in devastating diseases classified as peroxisome biogenesis disorders (PBD). The way in which peroxisomal dysfunction leads to the pathophysiological consequences of PBD is not well understood. Here we report that Drosophila pex mutants faithfully recapitulate several key features of human PBD, including impaired peroxisomal protein import, elevated VLCFA levels and growth retardation. Moreover, disruption of pex function results in spermatogenesis defects, including spermatocyte cytokinesis failure in Drosophila. Importantly, increased VLCFA levels enhance these spermatogenesis defects whereas reduced VLCFA levels alleviate them. Thus, regulation of proper VLCFA levels by pex genes is crucial for spermatogenesis. Together our study reveals an indispensable function of pex genes during spermatogenesis and provides a causative link between the phenotypic severity of pex mutants and VLCFA levels. - INTRODUCTION Peroxisomes are single membrane bound organelles present in almost all eukaryotic cells. They are critical for the metabolism of fatty acids and many other metabolites. The amorphous peroxisomal matrix hosts more than 50 enzymes that participate in various metabolic reactions (1,2). The importance of peroxisome function is underscored by the existence of a class of devastating human diseases named peroxisomal biogenesis disorders (PBD) in which import of the peroxisomal matrix or membrane proteins is impaired (3,4). Occurring at a frequency of 1/25 000 to 1/50 000 births, PBD is characterized by metabolic homeostatic defects as well as developmental abnormalities including facial malformations, and growth and mental retardation. PBD often leads to death in infancy or childhood and currently there is no effective treatment or cure. In yeast and plants, degradation of fatty acids by b-oxidation is carried out exclusively in peroxisomes. In animals, b-oxidation of fatty acids with carbon chains shorter than 20 mainly occurs in mitochondria, although b-oxidation of very-long-chain fatty acid (VLCFA, C20 and up) occurs exclusively in peroxisomes (1). Therefore, elevated VLCFA levels, in particular of C24 and C26 VLCFAs, is a hallmark of PBD (4,5). Since peroxisomes also participate in other metabolic pathways such as H2O2 production and decomposition, ether lipid biosynthesis and glyoxylate metabolism, the contribution of elevated VLCFAs to PBD pathogenesis remains uncertain (4,6,7). Nevertheless, therapeutic treatments aiming to lower the VLCFA level have been developed for PBD and other VLCFA accumulation diseases, such as adrenoleukodystrophy (ALD), which is caused by mutations in the putative fatty acid transporter ABCD1. For example, in combination with a low VLCFA diet, Lorenzos oil, a specific mixture of oils that may inhibit fatty acid biosynthesis, has been used to treat PBD, although with limited success (7). All peroxisomal proteins are synthesized in the cytoplasm and imported into peroxisomes using peroxisome targeting signals (PTS). Protein components required for peroxisome biogenesis are collectively called peroxins. At least 12 peroxin genes (PEX genes) have been linked to PBD. Among these, PEX1, 2, 5, 6, 7, 10, 12, 13 and 14 are required for PTS1- or PTS2-containing peroxisomal matrix protein import, although PEX3, 16 and 19 are essential for peroxisomal membrane protein targeting (8 10). Three ring finger domain-containing peroxisome membrane proteins, PEX2, PEX10 and PEX12, form a complex, although the exact molecular function of this complex is unknown (11,12). The disease-related peroxins are highly conserved throughout evolution, with orthologs present in yeast, worm, flies, mice and humans. In addition to the conserved peroxins, species-specific peroxins have also been identified in yeast and humans (9,13). To gain more insight into PBD pathogenic mechanisms and to establish experimental systems for treatment development, a number of PBD models have been generated. Like PBD patients, pex2, pex5 and pex13 knockout mice exhibit growth retardation, increased VLCFA levels and neonatal lethality (14 16). How peroxin disruption leads to these phenotypes is not clear. In Caenorhabditis elegans, RNAi of the pex5 homolog, prx-5, led to postembryonic L1 stage arrest (17). In Arabidopsis, mutants of PEX2, PEX10 and PEX12 are all embryonic lethal, indicating that peroxisomes are essential for plant development (18). The Drosophila genome contains 15 peroxin genes. Their functions are largely unexplored. Here we show that pex genes are important for spermatocyte development. Increasing the amount of VLCFA but not long-chain fatty acid (LCFA) in the diet significantly enhances the spermatocyte developmental defects of pex mutants. In addition, pex genetically interacts with bond and Sc2, two VLCFA biosynthesis genes, further supporting the conclusion that VLCFA homeostasis regulated by pex genes is crucial for spermatogenesis in Drosophila. The genetic properties of pex mutants afford an opportunity for discovering pathways and processes that interact with VLCFA metabolism, which may subsequently facilitate the development of intervention strategies for PBD. Drosophila PEX2 and PEX10 are essential for male fertility We identified 15 Drosophila peroxin genes by performing Blast searches using the protein sequences of all known peroxins from yeast and humans (Supplementary Material, Table S1). Except PEX1 and PEX6, all the encoded peroxins share 30% identity and 50% similarity to their human counterparts throughout the whole protein coding region (Supplementary Material, Table S1). Although many mutants are available for these pex genes, little information regarding their mutant phenotypes and potential function during Drosophila development has been reported. Thus, as a first step in determining pex gene function in development, we examined the mutant phenotypes of several pex genes, in particular the ring finger protein-coding genes pex2, pex10 and pex12. The pex2f01899 P-element insertion mutation is homozygous lethal, but in trans to a deficiency (DfExel6112) is viable with sterility in males and reduced fertility in females (Fig. 1A and data not shown). We could separate the lethality from the male sterility through recombination, indicating that the lethality is due to a mutation in another gene. A second P-element insertion allele, pex2HP35039, is homozygous viable and male st (...truncated)