LKB1 induces apical trafficking of Silnoon, a monocarboxylate transporter, in Drosophila melanogaster

Silnoon (Sln) is a monocarboxylate transporter (MCT) that mediates active transport of metabolic monocarboxylates such as butyrate and lactate. Here, we identify Sln as a novel LKB1-interacting protein using Drosophila melanogaster genetic modifier screening. Sln expression does not affect cell cycle progression or cell size but specifically enhances LKB1-dependent apoptosis and tissue size reduction. Conversely, down-regulation of Sln suppresses LKB1-dependent apoptosis, implicating Sln as a downstream mediator of LKB1. The kinase activity of LKB1 induces apical trafficking of Sln in polarized cells, and LKB1-dependent Sln trafficking is crucial for triggering apoptosis induced by extracellular butyrate. Given that LKB1 functions to control both epithelial polarity and cell death, we propose Sln is an important downstream target of LKB1

Drosophila Porin/VDAC Affects Mitochondrial Morphology

Voltage-dependent anion channel (VDAC) has been suggested to be a mediator of mitochondrial-dependent cell death induced by Ca2+ overload, oxidative stress and Bax-Bid activation. To confirm this hypothesis in vivo, we generated and characterized Drosophila VDAC (porin) mutants and found that Porin is not required for mitochondrial apoptosis, which is consistent with the previous mouse studies. We also reported a novel physiological role of Porin. Loss of porin resulted in locomotive defects and male sterility. Intriguingly, porin mutants exhibited elongated mitochondria in indirect flight muscle, whereas Porin overexpression produced fragmented mitochondria. Through genetic analysis with the components of mitochondrial fission and fusion, we found that the elongated mitochondria phenotype in porin mutants were suppressed by increased mitochondrial fission, but enhanced by increased mitochondrial fusion. Furthermore, increased mitochondrial fission by Drp1 expression suppressed the flight defects in the porin mutants. Collectively, our study showed that loss of Drosophila Porin results in mitochondrial morphological defects and suggested that the defective mitochondrial function by Porin deficiency affects the mitochondrial remodeling process

Peroxiredoxin 3 deficiency induces cardiac hypertrophy and dysfunction by impaired mitochondrial quality control

Mitochondrial quality control (MQC) consists of multiple processes: the prevention of mitochondrial oxidative damage, the elimination of damaged mitochondria via mitophagy and mitochondrial fusion and fission. Several studies proved that MQC impairment causes a plethora of pathological conditions including cardiovascular diseases. However, the precise molecular mechanism by which MQC reverses mitochondrial dysfunction, especially in the heart, is unclear. The mitochondria-specific peroxidase Peroxiredoxin 3 (Prdx3) plays a protective role against mitochondrial dysfunction by removing mitochondrial reactive oxygen species. Therefore, we investigated whether Prdx3-deficiency directly leads to heart failure via mitochondrial dysfunction. Fifty-two-week-old Prdx3-deficient mice exhibited cardiac hypertrophy and dysfunction with giant and damaged mitochondria. Mitophagy was markedly suppressed in the hearts of Prdx3-deficient mice compared to the findings in wild-type and Pink1-deficient mice despite the increased mitochondrial damage induced by Prdx3 deficiency. Under conditions inducing mitophagy, we identified that the damaged mitochondrial accumulation of PINK1 was completely inhibited by the ablation of Prdx3. We propose that Prdx3 interacts with the N-terminus of PINK1, thereby protecting PINK1 from proteolytic cleavage in damaged mitochondria undergoing mitophagy. Our results provide evidence of a direct association between MQC dysfunction and cardiac function. The dual function of Prdx3 in mitophagy regulation and mitochondrial oxidative stress elimination further clarifies the mechanism of MQC in vivo and thereby provides new insights into developing a therapeutic strategy for mitochondria-related cardiovascular diseases such as heart failure. © 20221

Misato underlies visceral myopathy in Drosophila

Genetic mechanisms for the pathogenesis of visceral myopathy (VM) have been rarely demonstrated. Here we report the visceral role of misato (mst) in Drosophila and its implications for the pathogenesis of VM. Depletion of mst using three independent RNAi lines expressed by a pan-muscular driver elicited characteristic symptoms of VM, such as abnormal dilation of intestinal tracts, reduced gut motility, feeding defects, and decreased life span. By contrast, exaggerated expression of mst reduced intestine diameters, but increased intestinal motilities along with thickened muscle fibers, demonstrating a critical role of mst in the visceral muscle. Mst expression was detected in the adult intestine with its prominent localization to actin filaments and was required for maintenance of intestinal tubulin and actomyosin structures. Consistent with the subcellular localization of Mst, the intestinal defects induced by mst depletion were dramatically rescued by exogenous expression of an actin member. Upon ageing the intestinal defects were deteriorative with marked increase of apoptotic responses in the visceral muscle. Taken together, we propose the impairment of actomyosin structures induced by mst depletion in the visceral muscle as a pathogenic mechanism for VM

Discovery of levodopa-induced dyskinesia-associated genes using genomic studies in patients and Drosophila behavioral analyses

Although levodopa is the most effective medication for Parkinson's disease, long-term levodopa treatment is largely compromised due to late motor complications, including levodopa-induced dyskinesia (LID). However, the genetic basis of LID pathogenesis has not been fully understood. Here, we discover genes pathogenic for LID using Drosophila genetics and behavioral analyses combined with genome-wide association studies on 578 patients clinically diagnosed with LID. Similar to the therapeutic effect of levodopa in patients, acute levodopa treatments restore the motor defect of Parkinson's disease model flies, while prolonged treatments cause LID-related symptoms, such as increased yawing, freezing and abrupt acceleration of locomotion. These symptoms require dopamine 1-like receptor 1 and are induced by neuronal overexpression of the receptor. Among genes selected from our analyses in the patient genome, neuronal knockdown of adenylyl cyclase 2 suppresses the levodopa-induced phenotypes and the receptor overexpression-induced symptoms in Drosophila. Together, our study provides genetic insights for LID pathogenesis through the D1-like receptor-adenylyl cyclase 2 signaling axis. A combined research approach using GWAS on Parkinson's disease patients and a Drosophila model of L-DOPA-induced dyskinesia (LID) reveals that LID is linked to ADCY2 signaling.N

Feeding and Fasting Signals Converge on the LKB1-SIK3 Pathway to Regulate Lipid Metabolism in <i>Drosophila</i>

<div><p>LKB1 plays important roles in governing energy homeostasis by regulating AMP-activated protein kinase (AMPK) and other AMPK-related kinases, including the salt-inducible kinases (SIKs). However, the roles and regulation of LKB1 in lipid metabolism are poorly understood. Here we show that <i>Drosophila</i> LKB1 mutants display decreased lipid storage and increased gene expression of <i>brummer</i>, the <i>Drosophila</i> homolog of adipose triglyceride lipase (ATGL). These phenotypes are consistent with those of SIK3 mutants and are rescued by expression of constitutively active SIK3 in the fat body, suggesting that SIK3 is a key downstream kinase of LKB1. Using genetic and biochemical analyses, we identify HDAC4, a class IIa histone deacetylase, as a lipolytic target of the LKB1-SIK3 pathway. Interestingly, we found that the LKB1-SIK3-HDAC4 signaling axis is modulated by dietary conditions. In short-term fasting, the adipokinetic hormone (AKH) pathway, related to the mammalian glucagon pathway, inhibits the kinase activity of LKB1 as shown by decreased SIK3 Thr196 phosphorylation, and consequently induces HDAC4 nuclear localization and <i>brummer</i> gene expression. However, under prolonged fasting conditions, AKH-independent signaling decreases the activity of the LKB1-SIK3 pathway to induce lipolytic responses. We also identify that the <i>Drosophila</i> insulin-like peptides (DILPs) pathway, related to mammalian insulin pathway, regulates SIK3 activity in feeding conditions independently of increasing LKB1 kinase activity. Overall, these data suggest that fasting stimuli specifically control the kinase activity of LKB1 and establish the LKB1-SIK3 pathway as a converging point between feeding and fasting signals to control lipid homeostasis in <i>Drosophila</i>.</p></div

Activation of insulin receptor increases phosphorylation of SIK3 by Akt.

<p>(A) Immunoblot analyses showing the effect of 4 hr fasting and constitutively active insulin receptor (InR^CA) on Thr196 phosphorylation of SIK3 protein in larvae (top three panels). Anti-phospho-Thr196 SIK3, -Myc (SIK3 protein), and -β-tubulin (TUB) antibodies were used. Densitometry of phospho-Thr196 SIK3 bands (bottom panel). <i>FB-Gal4</i> was used to drive transgene expression in the fat body. (B) Immunoblot analyses showing the effect of constitutively active insulin receptor (InR^CA) on Akt-dependent phosphorylation of SIK3 protein in larvae (top four panels). The lysates were immunoprecipitated with an anti-Myc (SIK3 protein) antibody, and then immunoblotted with an anti-phospho-Akt substrate antibody. Densitometry of phospho-SIK3 bands (bottom panel). (C) A schematic model for LKB1 and SIK3 function to regulate lipid homeostasis in <i>Drosophila</i> fat body. LKB1 regulates the nucleocytoplasmic localization of HDAC4 via SIK3-dependent phosphorylation. Under feeding condition, DILPs-induced Akt activation leads to SIK3 activation, thereby inhibiting HDAC4 activity by phosphorylation. Under short-term fasting conditions, the AKH pathway inhibits the kinase activity of LKB1 in phosphorylating SIK3 Thr196 residue and controls SIK3 activity via PKA-dependent phosphorylation. Unphosphorylated and nuclear localized HDAC4 deacetylates and activates FOXO to increase <i>bmm</i> expression [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005263#pgen.1005263.ref019" target="_blank">19</a>], thereby reducing lipid storage. AKH-independent signaling modulates the LKB1-SIK3-HDAC4 pathway to induce <i>bmm</i> expression when fasting is prolonged. Data are presented as mean ± SEM (*<i>P</i> < 0.05; **<i>P</i> < 0.01; NS, non-significant).</p

LKB1 and its downstream kinase SIK3 are required for lipid homeostasis.

<p>(A) qPCR analysis of LKB1 and its cofactors required for the catalytic activity, STRAD and MO25, in <i>Drosophila</i> larvae under feeding condition. (B) TAG amounts of wild-type and <i>LKB1</i> mutant larvae (<i>n</i> = 10 per genotype). (C) qPCR analysis for lipogenic genes (<i>SREBP</i>, <i>FAS</i> and <i>ACC</i>) and lipolytic genes (<i>bmm</i> and <i>HSL</i>) in wild-type and <i>LKB1</i> mutant larvae at mid-to-late L2 (60 hr AEL) stage under feeding conditions. (D) qPCR analysis of LKB1, SIKs (SIK2 and SIK3), and AMPK complex (AMPKα, AMPKβ, and AMPKγ) in larvae. (E) TAG amounts in LKB1 mutants following fat body-specific expression of wild-type, kinase-dead (K201I) LKB1, constitutively active (T196E) SIK3 or constitutively active (T184D) AMPK. Genotypes are as follows: FB> (<i>FB-Gal4</i>/+), LKB1^X5,FB> (<i>FB-Gal4/+;LKB1</i>^<i>X5</i><i>/LKB1</i>^<i>X5</i>), LKB1^X5,FB>LKB1^WT (<i>FB-Gal4/UAS-LKB1;LKB1</i>^<i>X5</i><i>/LKB1</i>^<i>X5</i>), LKB1^X5,FB>LKB1^KI (<i>FB-Gal4/UAS-LKB1 K201I;LKB1</i>^<i>X5</i><i>/LKB1</i>^<i>X5</i>), LKB1^X5,FB>SIK3^TE (<i>FB-Gal4/UAS-SIK3 T196E;LKB1</i>^<i>X5</i><i>/LKB1</i>^<i>X5</i>), and LKB1^X5,FB>AMPK^TD (<i>FB-Gal4/UAS-AMPK T184D;LKB1</i>^<i>X5</i><i>/LKB1</i>^<i>X5</i>) (<i>n</i> = 10 per genotype). (F) qPCR analysis of <i>bmm</i> gene expression in <i>LKB1</i> mutants following fat body-specific expression of wild-type LKB1 or constitutively active (T196E) SIK3 at mid-to-late L2 stage under feeding condition. Genotypes are as follows: FB> (<i>FB-Gal4</i>/+), LKB1^X5,FB> (<i>FB-Gal4/+;LKB1</i>^<i>X5</i><i>/LKB1</i>^<i>X5</i>), LKB1^X5,FB>LKB1^WT (<i>FB-Gal4/UAS-LKB1;LKB1</i>^<i>X5</i><i>/LKB1</i>^<i>X5</i>), and LKB1^X5,FB>SIK3^TE (<i>FB-Gal4/UAS-SIK3 T196E;LKB1</i>^<i>X5</i><i>/LKB1</i>^<i>X5</i>). (G) Immunoblot analyses showing the effect of LKB1 on Thr196 phosphorylation of SIK3 protein in larvae. Wild-type and kinase-dead (K70M) SIK3 were highly phosphorylated at Thr196 by LKB1 (second panel). SIK3^T196A was used as a control. <i>FB-Gal4</i> was used to drive transgene expression in the fat body. (H) Immunoblot analyses showing relative amounts of SIK3 Thr196 phosphorylation in wild-type and <i>LKB1</i>^<i>X5</i> mutant larvae. The phosphorylation was absolutely dependent on LKB1 (first panel). <i>FB-Gal4</i> was used to drive transgene expression. (G-H) Anti-LKB1, -phospho-Thr196 SIK3, -Myc (SIK3 protein), and -β-tubulin (TUB) antibodies were used. Data are presented as mean ± SEM (*<i>P</i> < 0.05).</p