Disease-Associated PNPLA6 Mutations Maintain Partial Functions When Analyzed in Drosophila

Abstract

Mutations in patatin-like phospholipase domain-containing protein 6 (PNPLA6) have been linked with a number of inherited diseases with clinical symptoms that include spastic paraplegia, ataxia, and chorioretinal dystrophy. PNPLA6 is an evolutionary conserved protein whose ortholog in Drosophila is Swiss-Cheese (SWS). Both proteins are phospholipases hydrolyzing lysophosphatidylcholine (LPC) and phosphatidylcholine (PC). Consequently, loss of SWS/PNPLA6 in flies and mice increases both lipids and leads to locomotion deficits and neurodegeneration. PNPLA6 knock-out mice are embryonic lethal, and a mutation creating an early stop codon in human PNPLA6 has only been identified in compound heterozygote patients. In contrast, disease-causing point mutations are found in homozygous patients, with some localized in the phospholipase domain while others are in a region that contains several cNMP binding sites. To investigate how different mutations affect the function of PNPLA6 in an in vivo model, we expressed them in the Drosophila sws1 null mutant. Expressing wild-type PNPLA6 suppressed the locomotion and degenerative phenotypes in sws1 and restored lipid levels, confirming that the human protein can replace fly SWS. In contrast, none of the mutant proteins restored lipid levels, although they suppressed the behavioral and degenerative phenotypes, at least in early stages. These results show that these mutant forms of PNPLA6 retain some biological function, indicating that disruption of lipid homeostasis is only part of the pathogenic mechanism. Furthermore, our finding that mutations in the cNMP binding sites prevented the restoration of normal lipid levels supports previous evidence that cNMP regulates the phospholipase activity of PNPLA6.

Loss of swiss cheese in Neurons Contributes to Neurodegeneration with Mitochondria Abnormalities, Reactive Oxygen Species Acceleration and Accumulation of Lipid Droplets in Drosophila Brain

“…In this study, we elucidate mitochondria alterations, ROS acceleration, induction of antioxidant-associated genes expression and LD gene/protein overrepresentation upon sws dysfunction. In addition, SWS is known to be a (lyso)phospholipase itself, acting in lipid metabolism [ 21 , 57 ]. Therefore, we decided to test whether LDs could accumulate in the fly brain in the case of the neuronal sws knockdown.…”

Characterization of the Interaction of Neuropathy Target Esterase with the Endoplasmic Reticulum and Lipid Droplets

“…Our finding that the catalytic domain of NTE associates with LD and alters LD structure and cellular TAG may point to a previously unrecognized role of LDs in NTE-associated disorders. Most disease-associated NTE mutations were found in the catalytic region and some of these mutations have been linked to decreased NTE activity and altered LPC levels [18,20,[41][42][43]. This suggests impaired enzyme activity as the major pathomechanism of NTE-associated disorders.…”

Neuropathy target esterase (NTE/PNPLA6) and organophosphorus compound-induced delayed neurotoxicity (OPIDN)

“…Recent elegant experiments in Drosophila have shown that certain mutations in SWS, the fruit-fly orthologue of NTE, produce neurodegenerative disease and disruption of phospholipid homeostasis that can be rescued by expressing wild-type NTE in the flies (Sunderhaus et al, 2019b). However, expression of NTE that contained disease-causing mutations in either the catalytic domain or the CNB domains suppressed neurological deficits but did not restore lipid homeostasis.…”

“…The human NTE mutants included some with mutations in the catalytic domain and others with mutations in the region containing CNB domains. The results were interpreted to indicate that CNB regions could modulate NTE catalytic activity and that the mutant forms of human NTE retained some degree of their biological function, suggesting that disruption of the catalytic function of SWS was not the only factor in disease pathogenesis (Sunderhaus et al, 2019b).…”

“…In humans, NTE mutations cause a variety of neurodegenerative conditions resulting in a range of deficits including spastic paraplegia and blindness (Hufnagel et al, 2015;Kmoch et al, 2015;Synofzik et al, 2014). Mutations in the Drosophila NTE orthologue SwissCheese (SWS) produce neurodegeneration characterized by vacuolization that can be partially rescued by expression of wild-type human NTE, suggesting a potential therapeutic approach for certain human neurological disorders (Sujkowski et al, 2015;Sunderhaus et al, 2019b).…”