Gene Expression Dysregulation in Familial Amyotrophic Lateral Sclerosis
Researchers have identified a new molecular player involved in the pathology of the neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS), and it’s all thanks to the humble fruit fly.
A recent study by Mallik et al in the Journal of Cell Biology took advantage of the tractable genetics of the model organism Drosophila melanogaster to uncover novel molecular components contributing to the familial form of ALS. Prior research has shown that mutations in a few RNA-binding proteins, and one in particular called FUS, cause familial ALS [1, 2]. Mutations in the fly gene cabeza (caz), the fly version of FUS, result in flies exhibiting features similar to ALS: motor defects in larval stages and the inability to emerge from their pupa, resulting in premature death .
Identifying other genes that interact with caz will allow for a better understanding of the underlying molecular mechanisms that contribute to ALS in humans. To do this, the researchers performed a genetic suppressor screen where caz mutant flies were crossed with flies with different mutations in genes throughout the fly genome. A fly with a mutation in a second gene (suppressor) that interacts with caz would rescue the ability of flies to survive through pupation, so when the scientists looked for flies that survived, they found that those with mutations in a gene called Xrp1 were the only viable ones, surviving passed the pupal stage.
Flies that were mutant for caz and had lost just one copy of Xrp1, called caz mutant Xrp1 heterozygous flies, showed improved motor success and were able to emerge from their pupa. Although these flies survived to adulthood, they still had a reduced lifespan as compared to normal flies. Looking specifically in neurons, which are the primary cells affected in ALS, the researchers found that caz mutant flies expressed elevated levels of Xrp1 protein. Thus, by reducing the expression of Xrp1 by 50% in neurons, caz mutant flies restored motor success to a level close to that of healthy flies.
To better understand the function of Xrp1, the researchers used immunofluorescence to localize Xrp1 to the nucleus and showed that it binds to chromatin, specifically euchromatic bands, puffs, and centromeric β-heterochromatin on the Polytene chromosomes, suggesting a role of Xrp1 in regulating gene expression. Gene ontology analysis of proteins that interact with Xrp1 and are identified by mass spectrometry showed that 55.4% of Xrp1-interacting proteins are also involved in gene expression regulation. In fact, inactivation of Xrp1 function by mutating its AT-hook DNA-binding domain led to pupal lethality, which further supports the role of Xrp1 in regulation of gene expression.
To independently assess the role of Xrp1 in regulating gene expression, RNAseq analysis of transcripts from the central nervous system of caz mutants was compared to those of healthy flies. This analysis showed a significant amount of gene expression dysregulation in caz mutant flies with over 1,600 genes being both up and down regulated in the mutant when compared to healthy flies. However, when caz and Xrp1 were mutated together, very few genes (about 300) were differentially expressed, demonstrating that Xrp1 is responsible for the observed dysregulation in gene expression.
Finally, the authors address the relevance of these findings for human ALS. Using flies that express a mutant human FUS protein in motor neurons, they show that losing one copy of Xrp1 rescues the motor defects and lifespan shortening seen in the FUS mutant. While the human version of Xrp1 has yet to be found, the authors identified 27 candidate genes that may function in a similar way to Xrp1. More work is needed to identify the functional ortholog of Xrp1 in humans, but the groundwork is there, all riding on the back of the intrepid fruit fly.
Original article: Mallik M, Catinozzi M, Hug CB, Zhang L, Wagner M, Bussmann J, Bittern J, Mersmann S, Klämbt C, Drexler HCA, Huynen MA, Vaquerizas JM, Storkebaum E (2018). Xrp1 genetically interacts with the ALS-associated FUS orthologue caz and mediates its toxicity. J Cell Biol, jcb.201802151. https://doi.org/10.1083/jcb.201802151.
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