Identification of dietary alanine toxicity and trafficking dysfunction in a Drosophila model of hereditary sensory and autonomic neuropathy type 1

Human Molecular Genetics, 2015, Vol. 24, No. 24 6899–6909 doi: 10.1093/hmg/ddv390 Advance Access Publication Date: 22 September 2015 Original Article ORIGINAL ARTICLE Identification of dietary alanine toxicity and trafficking dysfunction in a Drosophila model of hereditary sensory and autonomic neuropathy type 1 1 Department of Biology and Hull-York Medical School, University of York, York YO10 5DD, UK and 2School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, UK *To whom correspondence should be addressed at: Department of Biology and Hull-York Medical School, University of York, Wentworth Way, Heslington, York YO10 5YW, UK. Tel: +44-1904-328537; Fax: +44-1904-328505; Email: Abstract Hereditary sensory and autonomic neuropathy type 1 (HSAN1) is characterized by a loss of distal peripheral sensory and motorneuronal function, neuropathic pain and tissue necrosis. The most common cause of HSAN1 is due to dominant mutations in serine palmitoyl-transferase subunit 1 (SPT1). SPT catalyses the condensation of serine with palmitoyl-CoA, the initial step in sphingolipid biogenesis. Identified mutations in SPT1 are known to both reduce sphingolipid synthesis and generate catalytic promiscuity, incorporating alanine or glycine into the precursor sphingolipid to generate a deoxysphingoid base (DSB). Why either loss of function in SPT1, or generation of DSBs should generate deficits in distal sensory function remains unclear. To address these questions, we generated a Drosophila model of HSAN1. Expression of dSpt1 bearing a disease-related mutation induced morphological deficits in synapse growth at the larval neuromuscular junction consistent with a dominantnegative action. Expression of mutant dSpt1 globally was found to be mildly toxic, but was completely toxic when the diet was supplemented with alanine, when DSBs were observed in abundance. Expression of mutant dSpt1 in sensory neurons generated developmental deficits in dendritic arborization with concomitant sensory deficits. A membrane trafficking defect was observed in soma of sensory neurons expressing mutant dSpt1, consistent with endoplasmic reticulum (ER) to Golgi block. We found that we could rescue sensory function in neurons expressing mutant dSpt1 by co-expressing an effector of ER–Golgi function, Rab1 suggesting compromised ER function in HSAN1 affected dendritic neurons. Our Drosophila model identifies a novel strategy to explore the pathological mechanisms of HSAN1. Introduction Hereditary sensory and autonomic neuropathy type 1 (HSAN1) is a rare disorder pathologically characterized by distal sensory loss and peripheral ulceration, predominantly in lower limbs (1–5). Degeneration of motorneurons is also known to occur in addition to distal limb weakness and muscle atrophy (4). The condition is caused in the majority of cases by inheritable dominant mutations in the genes encoding subunits of the enzyme serine palmitoyl-transferase (SPT). SPT function is essential for catalysis of the first step in de novo sphingolipid synthesis at the ER (6,7). SPT exists as a heterodimer of homologous subunits, SPT long chain base subunit 1 (SPTLC1/SPT1) and SPT long chain base † Present address: Department of Zoology, University of Cambridge, Downing Street, Cambridge, CB2 3EJ, UK. Received: June 23, 2015. Revised: August 30, 2015. Accepted: September 15, 2015 © The Author 2015. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 6899 Matthew C. W. Oswald1,†, Ryan J. H. West1, Emyr Lloyd-Evans2 and Sean T. Sweeney1, * 6900 | Human Molecular Genetics, 2015, Vol. 24, No. 24 Results dSpt1 is an essential gene in Drosophila HSAN1 is caused by mutations in the SPTLC1 gene, the most common mutation being C133W (6,7). The SPTLC1 gene encodes SPT1, which localizes to the ER membrane and associates with cytosolic SPT2 to form the type 1 ER protein SPT (18). SPT performs the initial step in de novo sphingolipid synthesis, catalysing the condensation of -serine and palmitoyl-coA to form ketosphinganine. Position C133 shows a high degree of evolutionary conservation (Fig. 1A) and lies within a highly conserved protein domain that has been linked to critical roles in SPT activity and substrate specificity (8). To aid our analysis, we identified alleles of Drosophila dSpt1. From the Drosophila genome project and Bloomington stock centre, we identified a P-element insertion in the 5′ regulatory region of dSpt1, dSpt1l(2)SH1626 (19). When crossed to a deficiency stock uncovering the region, Df(2)vg-B, we found that development of the P-element/deficiency transheterozygotes was delayed by 3–5 days. We used this delayed eclosion phenotype to screen previously identified but unsequenced point mutations from the genomic region in dSpt1. We identified two point mutations l(2) 49Fb1 and l(2)49Fb4 in the dSpt1 locus (20). Sequencing of the dSpt1 locus in l(2)49Fb1 allele revealed a C251Y nonsense mutation, while sequencing of the l(2)49Fb4 allele uncovered a Q90– STOP truncation. Furthermore, we used a P-element imprecise excision strategy to delete 1.6 kb (including the start codon) of the dSpt1 locus and generate the dSpt1JO8 allele (Supplementary Material, Fig. S1). When the dSpt1l(2)SH1626 mutant was crossed to the l(2)49Fb4, l(2)49Fb1 or dSpt1JO8 alleles, all mutant combinations were found to have the 3–5 day delayed development phenotype. Complementation crosses of l(2)49Fb4, l(2)49Fb1 and dSpt1JO8 to each other were lethal at first instar, confirming our dSpt1l(2)SH1626 allele as an dSpt1 hypomorph. Global expression of the UAS-dSpt1 transgene under the control of tubulin-GAL4 was also sufficient to rescue all transheterozygous dSpt1 mutant combinations, allowing survival to adulthood. Neuronal (ElavGAL4) or muscle (MHC-GAL4) driven expression of UAS-dSpt1 failed to rescue dSpt1 mutants to adulthood. We can therefore conclude that the dSpt1 locus, like the dSpt2 locus (lace) (21), is an essential gene in Drosophila and required globally. dSpt1C129W acts in a dominant-negative manner at the Drosophila larval neuromuscular synapse to alter synapse growth parameters HSAN1 patients have peripheral- and motorneuronal functional deficits (4). To try to dissect the mechanism driving SPT1C133W dysfunction in motor neurons, we examined the effects of expression of a mutated Drosophila SPT1 protein at the larval neuromuscular synapse. We obtained the cDNA of the Drosophila SPT1 (dSpt1), introduced a C to W substitution at amino acid position 129 (analogous to the human 133, Fig. 1A) by site-directed-mutagenesis and subcloned both the wild-type and mutant subunit into the pUASt vector (22). We generated multiple independent UASdSpt1 and UAS-dSpt1C129W transgenes by mic (...truncated)