RNA sequencing data from the guts of Drosophila wildtype and CG3740/nazo double mutant 20-day-old males

<p>Lipid dyshomeostasis has been implicated in a variety of diseases ranging from obesity to neurodegenerative disorders such as Neurodegeneration with Brain Iron Accumulation (NBIA). Here, we uncover the physiological role of Nazo, the Drosophilamelanogaster homolog of the NBIA-mutated protein – c19orf12, whose function has been elusive. Ablation of Drosophila c19orf12 homologs leads to dysregulation of multiple lipid metabolism genes. nazo mutants exhibit markedly reduced gut lipid droplet and whole-body triglyceride contents. Consequently, they are sensitive to starvation and oxidative stress. Nazo is required for maintaining normal levels of Perilipin-2, an inhibitor of the lipase – Brummer. Concurrent knockdown of Brummer or overexpression of Perilipin-2 rescues the nazo phenotype, suggesting that this defect, at least in part, may arise from diminished Perilipin-2 on lipid droplets leading to aberrant Brummer-mediated lipolysis. Our findings potentially provide novel insights into the role of c19orf12 as a possible link between lipid dyshomeostasis and neurodegeneration, particularly in the context of NBIA.</p><p>The samples( guts) were dissected in ice-cold PBS and flash-frozen in liquid nitrogen followed by RNA extraction using a standard RNA extraction kit (Qiagen) before being processed. </p&gt

Fig 4 -

nazo mutants show diminished organismal triglyceride stores along with increased sensitivity to starvation and oxidative stress (A) Confocal images of fat-body of 40-day old male adults of indicated genotypes, stained with BODIPY 493/503 dye (BODIPY 493/503 dye–green and DAPI–blue; Scale bar = 10μm) (B) Quantification of percentage area covered by lipid droplets in each fat body (N = 5–6 males; Student’s t-test, p-value ***p NP-Gal4::UAS-NazoMyc; nazo/nazo. 6 males per genotype per biological replicates were used in triplicates. (Student’s t-test **** p-value = 2O2, counted every 12 hours (N = 80),Student’s t-test, p-value ***p 2O2 quantification shows that nazo mutants along with CG3740 have increased H2O2 levels relative to WT flies. 8 males per genotypes per biological replicates were used in quadruplets. (Student’s t-test **** p-value = < 0.0001. * = 0.0188).</p

S3 Fig -

(A) Guts from 20-day-old males with relish mutation or ubiquitous knockdown of dSTING by Actin-Gal4 driven dSTING RNAi do not show gut lipid droplet depletion on BODIPY 493/503 dye staining (Scale bar 10μm). (B) Quantification of the amount of food consumed by adult males in 24-hour period laced with Blue-1 food dye color shows that nazo mutants do not have feeding defects. (N = 20, Student t-test p-value ns = not significant) (C) Quantification of gut fluorescence in flies of indicated genotypes fed with fluorescent oleic acid reveals that that nazo mutants are not defective in absorption of fluorescent labeled oleic acid (N = 20, Student t-test, p-value ns = not significant). (D) Quantification of whole-body glucose from males of indicated genotypes. 8 males per genotype per biological replicates were used in triplets. (Student t-test p-value ns = not significant) (E) Nazo microRNA leads to efficient depletion of Nazo transcript. Relative expression of nazo transcripts in guts of flies ubiquitously expressing NazomircroRNA under actin-Gal4 driver. The three transgenic lines represent different insertions of the same microRNA transgene. (Student’s t-test p-value **** = (TIF)</p

S8 Fig -

Confocal images of guts from 20-day old flies harboring a GFP trap in plin-2 (LSD-2) locus stained with GFP (green), Lipid-TOX-Red, and DAPI shows reduced levels of Perilipin-2 upon Nazo depletion (Scale bar = 10μm). (TIF)</p

<i>nazo</i> mutants show gut lipid depletion.

(A) Confocal images of guts from 20-day old male flies stained with BODIPY 493/503 dye show markedly reduced lipid droplet content in midguts. (BODIPY 493/503 dye–green and DAPI–blue; Scale bar = 10μm) (B) Quantification of percentage area occupied by lipid droplets in the midgut R4/5 region (N = 5–6 males; Student’s t-test **** p-value = nazo and nazo/CG3740 double mutants. (BODIPY 493/503 dye–green and DAPI–blue; Scale bar = 10μm).</p

Drosophila c19orf12 mutants show reduced lifespan, functional deficits, and dysregulation of lipid metabolism genes.

(A) Schematic of Drosophila Nazo and CG3740 genomic locus with deletion (open reading frame in dark blue, 5” and 3” untranslated regions in light blue, extent of deletion indicated by line) (B) Lifespan of males of indicated genotypes (N = 60; WT vs CG3740 p–ns, WT vs nazo pN  =  60 for each genotype, ***p t-test). (D) Lipid metabolism genes upregulated or downregulated (with indicated fold change) in guts of 20-day old nazo/CG3740 mutant males relative to WT flies.</p

GO molecular function analysis of upregulated and downregulated genes from the gut of WT vs <i>nazo/CG3740</i> double mutants.

GO molecular function analysis of upregulated and downregulated genes from the gut of WT vs nazo/CG3740 double mutants.</p