The role of glial and neuronal Eph/ephrin signaling in Drosophila mushroom body development and sleep and circadian behavior.

The Eph receptor, a prototypically large receptor protein tyrosine kinase, interacts with ephrin ligands, forming a bidirectional signaling system that impacts diverse brain functions. Eph receptors and ephrins mediate forward and reverse signaling, affecting neurogenesis, axon guidance, and synaptic signaling. While mammalian studies have emphasized their roles in neurogenesis and synaptic plasticity, the Drosophila counterparts are less studied, especially in glial cells, despite structural similarities. Using RNAi to modulate Eph/ephrin expression in Drosophila neurons and glia, we studied their roles in brain development and sleep and circadian behavior. Knockdown of neuronal ephrin disrupted mushroom body development, while glial knockdown had minimal impact. Surprisingly, disrupting ephrin in neurons or glial cells altered sleep and circadian rhythms, indicating a direct involvement in these behaviors independent from developmental effects. Further analysis revealed distinct sleep phenotypes between neuronal and glial knockdowns, underscoring the intricate interplay within the neural circuits that govern behavior. Glia-specific knockdowns showed altered sleep patterns and reduced circadian rhythmicity, suggesting an intricate role of glia in sleep regulation. Our findings challenge simplistic models of Eph/ephrin signaling limited to neuron-glia communication and emphasize the complexity of the regulatory networks modulating behavior. Future investigations targeting specific glial subtypes will enhance our understanding of Eph/ephrin signaling's role in sleep regulation across species.

Roles of Drosophila fatty acid-binding protein in development and behavior.

Fatty acid-binding proteins (FABPs) are lipid chaperones that mediate the intracellular dynamics of the hydrophobic molecules that they physically bind to. FABPs are implicated in sleep and psychiatric disorders, as well as in various cellular processes, such as cell proliferation and survival. FABP is well conserved in insects, and Drosophila has one FABP ortholog, dFabp, in its genome. Although dFabp appears to be evolutionarily conserved in some brain functions, little is known about its development and physiological function. In the present study, we investigated the function of dFabp in Drosophila development and behavior. Knockdown or overexpression of dFabp in the developing brain, wing, and eye resulted in developmental defects, such as decreased survival, altered cell proliferation, and increased apoptosis. Glia-specific knockdown of dFabp affected neuronal development, and neuronal regulation of dFabp affected glial cell proliferation. Moreover, the behavioral phenotypes (circadian rhythm and locomotor activity) of flies with regulated dFabp expression in glia and flies with regulated dFabp expression in neurons were very similar. Collectively, our results suggest that dFabp is involved in the development of various tissues and brain functions to control behavior and is a mediator of neuron-glia interactions in the Drosophila nervous system.

Neuroprotective effects of linear ubiquitin E3 ligase against aging-induced DNA damage and amyloid ß neurotoxicity in the brain of Drosophila melanogaster.

E3 ubiquitin ligase, HOIL1-interacting protein (HOIP), forms the linear ubiquitin chain assembly complex (LUBAC) with HOIL and SHANK-associated RH domain interactor and catalyzes linear ubiquitination, directly linking the N- and C-termini of ubiquitin. Recently, several studies have implicated linear ubiquitination in aging and Alzheimer disease (AD). However, little is currently known about the roles of HOIP in brain aging and AD pathology. Here, we investigated the role of linear ubiquitin E3 ligase (LUBEL), a Drosophila HOIP ortholog, in brain aging and amyloid ß (Aß) pathology in a Drosophila AD model. DNA double-strand breaks (DSBs) were increased in the aged brains of neuron-specific LUBEL-knockdown flies compared to the age-matched controls. Silencing of LUBEL in the neuron of AD model flies increased the neuronal apoptosis and neurodegeneration, whereas silencing in glial cells had no such effect. Aß aggregation levels and DSBs were also increased in the LUBEL-silenced AD model fly brains, but autophagy and proteostasis were not affected by LUBEL silencing. Collectively, our results suggest that LUBEL protects neurons from aging-induced DNA damage and Aß neurotoxicity.

Roles of ZnT86D in Neurodevelopment and Pathogenesis of Alzheimer Disease in a Drosophila melanogaster Model.

Zinc is a fundamental trace element essential for numerous biological processes, and zinc homeostasis is regulated by the Zrt-/Irt-like protein (ZIP) and zinc transporter (ZnT) families. ZnT7 is mainly localized in the Golgi apparatus and endoplasmic reticulum (ER) and transports zinc into these organelles. Although previous studies have reported the role of zinc in animal physiology, little is known about the importance of zinc in the Golgi apparatus and ER in animal development and neurodegenerative diseases. In this study, we demonstrated that ZnT86D, a Drosophila ortholog of ZnT7, plays a pivotal role in the neurodevelopment and pathogenesis of Alzheimer disease (AD). When ZnT86D was silenced in neurons, the embryo-to-adult survival rate, locomotor activity, and lifespan were dramatically reduced. The toxic phenotypes were accompanied by abnormal neurogenesis and neuronal cell death. Furthermore, knockdown of ZnT86D in the neurons of a Drosophila AD model increased apoptosis and exacerbated neurodegeneration without significant changes in the deposition of amyloid beta plaques and susceptibility to oxidative stress. Taken together, our results suggest that an appropriate distribution of zinc in the Golgi apparatus and ER is important for neuronal development and neuroprotection and that ZnT7 is a potential protective factor against AD.

The linear ubiquitin E3 ligase-Relish pathway is involved in the regulation of proteostasis in Drosophila muscle during aging.

Linear ubiquitination is an atypic ubiquitination process that directly connects the N- and C-termini of ubiquitin and is catalyzed by HOIL-1-interacting protein (HOIP). It is involved in the immune response or apoptosis by activating the nuclear factor-κB pathway and is associated with polyglucosan body myopathy 1, an autosomal recessive disorder with progressive muscle weakness and cardiomyopathy. However, little is currently known regarding the function of linear ubiquitination in muscles. Here, we investigated the role of linear ubiquitin E3 ligase (LUBEL), a DrosophilaHOIP ortholog, in the development and aging of muscles. The muscles of the flies with down-regulation of LUBEL or its downstream factors, kenny and Relish, developed normally, and there were no obvious abnormalities in function in young flies. However, the locomotor activity of the LUBEL RNAi flies was reduced compared to age-matched control, while LUBEL RNAi did not affect the increased mitochondrial fusion or myofiber disorganization during aging. Interestingly, the accumulation of polyubiquitinated protein aggregation during aging decreased in muscles by silencing LUBEL, kenny, or Relish. Meanwhile, the levels of autophagy and global translation, which are implicated in the maintenance of proteostasis, did not change due to LUBEL down-regulation. In conclusion, we propose a new role of linear ubiquitination in proteostasis in the muscle aging.

Cullin 1 (CUL1) Promotes Primary Ciliogenesis through the Induction of Ubiquitin-Proteasome-Dependent Dvl2 Degradation.

Primary cilia are nonmotile cellular signal-sensing antenna-like structures composed of microtubule-based structures that distinguish them from motile cilia in structure and function. Primary ciliogenesis is regulated by various cellular signals, such as Wnt, hedgehog (Hh), and platelet-derived growth factor (PDGF). The abnormal regulation of ciliogenesis is closely related to developing various human diseases, including ciliopathies and cancer. This study identified a novel primary ciliogenesis factor Cullin 1 (CUL1), a core component of Skp1-Cullin-F-box (SCF) E3 ubiquitin ligase complex, which regulates the proteolysis of dishevelled 2 (Dvl2) through the ubiquitin-proteasome system. Through immunoprecipitation-tandem mass spectrometry analysis, 176 Dvl2 interacting candidates were identified, of which CUL1 is a novel Dvl2 modulator that induces Dvl2 ubiquitination-dependent degradation. Neddylation-dependent CUL1 activity at the centrosomes was essential for centrosomal Dvl2 degradation and primary ciliogenesis. Therefore, this study provides a new mechanism of Dvl2 degradation by CUL1, which ultimately leads to primary ciliogenesis, and suggest a novel target for primary cilia-related human diseases.

Physical Exercise-Induced Myokines in Neurodegenerative Diseases.

Neurodegenerative diseases (NDs), such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS), are disorders characterized by progressive degeneration of the nervous system. Currently, there is no disease-modifying treatments for most NDs. Meanwhile, numerous studies conducted on human and animal models over the past decades have showed that exercises had beneficial effects on NDs. Inter-tissue communication by myokine, a peptide produced and secreted by skeletal muscles during exercise, is thought to be an important underlying mechanism for the advantages. Here, we reviewed studies about the effects of myokines regulated by exercise on NDs and their mechanisms. Myokines could exert beneficial effects on NDs through a variety of regulatory mechanisms, including cell survival, neurogenesis, neuroinflammation, proteostasis, oxidative stress, and protein modification. Studies on exercise-induced myokines are expected to provide a novel strategy for treating NDs, for which there are no adequate treatments nowadays. To date, only a few myokines have been investigated for their effects on NDs and studies on mechanisms involved in them are in their infancy. Therefore, future studies are needed to discover more myokines and test their effects on NDs.

Overexpression of H3K36 methyltransferase NSD in glial cells affects brain development in Drosophila.

NSD1 is a histone methyltransferase that methylates the lysine 36 at histone H3. NSD duplication is associated with short stature, microcephaly, intellectual disability, and behavioral defects in humans. Ectopic overexpression of NSD, an NSD1 homolog in Drosophila, was shown to induce developmental abnormalities via apoptosis. In this study, to investigate the effects of NSD overexpression on Drosophila brain development, we first examined the typical NSD expression pattern in larval brains and found that endogenous NSD promoter activity was detected only in subsets of glial cells. Pan-glial, but not pan-neuronal, NSD overexpression induced apoptosis in larval brain cells. However, pan-glial NSD overexpression also induced caspase-3 cleavage in neuronal cells. Among the various glial cell types, NSD overexpression in only astrocytic glia induced apoptosis and abnormal learning defects in the larval stage. Furthermore, NSD overexpression downregulated the expression of various astrocyte-specific genes, including draper (drpr), possibly owing to an indirect effect of NSD overexpression-induced astrocytic apoptosis. Since drpr plays a role in axon pruning during mushroom body (MB) formation in Drosophila astrocytes, we examined the effect of astrocytic NSD overexpression on this process and found that it disrupted the clearance of γ-neurons in the MB, subsequently inducing arrhythmic locomotor activity of the fly. Thus, these results suggest that aberrant NSD overexpression may cause neurodevelopmental disorders by interfering with crucial functions of astrocytes in the brain, underlining the importance of the tightly controlled astrocytic NSD expression for proper neurodevelopment.

Neuroprotective Effects of Limonene (+) against Aß42-Induced Neurotoxicity in a Drosophila Model of Alzheimer's Disease.

Forest bathing is suggested to have beneficial effects on various aspects of human health. Terpenes, isoprene based-phytochemicals emitted from trees, are largely responsible for these beneficial effects of forest bathing. Although the therapeutic effects of terpenes on various diseases have been revealed, their effects on neuronal health have not yet been studied in detail. Here, we screened 16 terpenes that are the main components of Korean forests using Drosophila Alzheimer's disease (AD) models to identify which terpenes have neuroprotective effects. Six out of the 16 terpenes, ρ-cymene, limonene (+), limonene (-), linalool, α-pinene (+), and ß-pinene (-), partially suppressed the beta amyloid 42 (Aß42)-induced rough eye phenotype when fed to Aß42-expressing flies. Among them, limonene (+) restored the decreased survival of flies expressing Aß42 in neurons during development. Limonene (+) treatment did not affect Aß42 accumulation and aggregation, but did cause to decrease cell death, reactive oxygen species levels, extracellular signal-regulated kinase phosphorylation, and inflammation in the brains or the eye imaginal discs of Aß42-expressing flies. This neuroprotective effect of limonene (+) was not associated with autophagic activity. Our results suggest that limonene (+) has a neuroprotective function against the neurotoxicity of Aß42 and, thus, is a possible therapeutic reagent for AD.

Therapeutic Potential of Volatile Terpenes and Terpenoids from Forests for Inflammatory Diseases.

Forest trees are a major source of biogenic volatile organic compounds (BVOCs). Terpenes and terpenoids are known as the main BVOCs of forest aerosols. These compounds have been shown to display a broad range of biological activities in various human disease models, thus implying that forest aerosols containing these compounds may be related to beneficial effects of forest bathing. In this review, we surveyed studies analyzing BVOCs and selected the most abundant 23 terpenes and terpenoids emitted in forested areas of the Northern Hemisphere, which were reported to display anti-inflammatory activities. We categorized anti-inflammatory processes related to the functions of these compounds into six groups and summarized their molecular mechanisms of action. Finally, among the major 23 compounds, we examined the therapeutic potentials of 12 compounds known to be effective against respiratory inflammation, atopic dermatitis, arthritis, and neuroinflammation among various inflammatory diseases. In conclusion, the updated studies support the beneficial effects of forest aerosols and propose their potential use as chemopreventive and therapeutic agents for treating various inflammatory diseases.

Genetic Dissection of Alzheimer's Disease Using Drosophila Models.

Alzheimer's disease (AD), a main cause of dementia, is the most common neurodegenerative disease that is related to abnormal accumulation of the amyloid ß (Aß) protein. Despite decades of intensive research, the mechanisms underlying AD remain elusive, and the only available treatment remains symptomatic. Molecular understanding of the pathogenesis and progression of AD is necessary to develop disease-modifying treatment. Drosophila, as the most advanced genetic model, has been used to explore the molecular mechanisms of AD in the last few decades. Here, we introduce Drosophila AD models based on human Aß and summarize the results of their genetic dissection. We also discuss the utility of functional genomics using the Drosophila system in the search for AD-associated molecular mechanisms in the post-genomic era.

Overexpression of histone methyltransferase NSD in Drosophila induces apoptotic cell death via the Jun-N-terminal kinase pathway.

The nuclear receptor-binding SET domain protein gene (NSD) family encodes a group of highly conserved SET domain-containing histone lysine methyltransferases that are important in multiple aspects of development in various organisms. The association of NSD1 duplications has been reported with growth retardation diseases in humans. In this study, to gain insight into the molecular mechanisms by which the overexpression of NSD1 influences the disease progression, we analyzed the gain-of-function mutant phenotypes of the Drosophila NSD using the GAL4/UAS system. Ubiquitous overexpression of NSD in the fly caused developmental delay and reduced body size at the larval stage, resulting in pupal lethality. Moreover, targeted overexpression in various developing tissues led to significant phenotype alterations, and the gain-of-function phenotypes were rescued by NSD RNAi knockdown. We also demonstrated that NSD overexpression not only enhanced the transcription of pro-apoptotic genes but also activated caspase. The atrophied phenotype of NSD-overexpressing wing was strongly suppressed by a loss-of-function mutation in hemipterous, which encodes a Drosophila Jun N-terminal kinase. Taken together, our findings suggest that NSD induces apoptosis via the activation of JNK, and thus contributes to the understanding of the molecular mechanisms involved in NSD1-related diseases in humans.

Nardostachys jatamansi Ethanol Extract Ameliorates Aß42 Cytotoxicity.

The Nardostachys jatamansi DC (NJ) root has been used as a sedative or analgesic to treat neurological symptoms and pain in traditional Korean medicine. Here, we investigate the potential effects of NJ on Alzheimer's disease (AD) and reveal the molecular mechanism through which NJ exerts its effects. The neuroprotective effect of the NJ root ethanol extract against ß amyloid (Aß) toxicity was examined in vitro using a cell culture system and in vivo using a Drosophila AD model. The NJ extract and chlorogenic acid, a major component of NJ, inhibited Aß-induced cell death in SH-SY5Y cells. Moreover, the NJ extract rescued the neurological phenotypes of the Aß42-expressing flies (decreased survival and pupariation rate and a locomotor defect) and suppressed Aß42-induced cell death in the brain. We also found that NJ extract intake reduced glial cell number, reactive oxygen species level, extracellular-signal-regulated kinase (ERK) phosphorylation, and nitric oxide level in Aß42-expressing flies, without affecting Aß accumulation. These data suggest that the neuroprotective activity of NJ might be associated with its antioxidant and anti-inflammatory properties, as well as its inhibitory action against ERK signaling; thus, NJ is a promising medicinal plant for the development of AD treatment.

Drosophila tensin plays an essential role in cell migration and planar polarity formation during oogenesis by mediating integrin-dependent extracellular signals to actin organization.

Oogenesis in Drosophila involves very dynamic cellular changes such as cell migration and polarity formation inside an ovary during short period. Previous studies identified a number of membrane-bound receptors directly receiving certain types of extracellular inputs as well as intracellular signalings to be involved in the regulation of these dynamic cellular changes. However, yet our understanding on exactly how these receptor-mediated extracellular inputs lead to dynamic cellular changes remains largely unclear. Here, we identified Drosophila tensin encoded by blistery (by) as a novel regulator of cell migration and planar polarity formation and characterized the genetic interaction between tensin and integrin during oogenesis. Eggs from by mutant showed decreased hatching rate and morphological abnormality, a round-shape, compared to the wild-type eggs. Further analyses revealed that obvious cellular defects such as defective border cell migration and planar polarity formation might be primarily associated with the decreased hatching rate and the round-shape phenotype of by mutant eggs, respectively. Moreover, by mutation also induced marked defects in F-actin organization closely associated with both cell migration and planar polarity formation during oogenesis of Drosophila. Notably, all these defective phenotypes observed in by mutant eggs became much severer by reduced level of integrin, indicative of a close functional association between integrin and tensin during oogenesis. Collectively, our findings suggest that tensin acts as a crucial regulator of dynamic cellular changes during oogenesis by bridging integrin-dependent extracellular signals to intracellular cytoskeletal organization.

Histone Lysine Methylation and Neurodevelopmental Disorders.

Methylation of several lysine residues of histones is a crucial mechanism for relatively long-term regulation of genomic activity. Recent molecular biological studies have demonstrated that the function of histone methylation is more diverse and complex than previously thought. Moreover, studies using newly available genomics techniques, such as exome sequencing, have identified an increasing number of histone lysine methylation-related genes as intellectual disability-associated genes, which highlights the importance of accurate control of histone methylation during neurogenesis. However, given the functional diversity and complexity of histone methylation within the cell, the study of the molecular basis of histone methylation-related neurodevelopmental disorders is currently still in its infancy. Here, we review the latest studies that revealed the pathological implications of alterations in histone methylation status in the context of various neurodevelopmental disorders and propose possible therapeutic application of epigenetic compounds regulating histone methylation status for the treatment of these diseases.

Drosophila DJ-1 decreases neural sensitivity to stress by negatively regulating Daxx-like protein through dFOXO.

DJ-1, a Parkinson's disease (PD)-associated gene, has been shown to protect against oxidative stress in Drosophila. However, the molecular mechanism underlying oxidative stress-induced phenotypes, including apoptosis, locomotive defects, and lethality, in DJ-1-deficient flies is not fully understood. Here we showed that Daxx-like protein (DLP), a Drosophila homologue of the mammalian Death domain-associated protein (Daxx), was upregulated under oxidative stress conditions in the loss-of-function mutants of Drosophila DJ-1ß, a Drosophila homologue of DJ-1. DLP overexpression induced apoptosis via the c-Jun N-terminal kinase (JNK)/Drosophila forkhead box subgroup O (dFOXO) pathway, whereas loss of DLP increased resistance to oxidative stress and UV irradiation. Moreover, the oxidative stress-induced phenotypes of DJ-1ß mutants were dramatically rescued by DLP deficiency, suggesting that enhanced expression of DLP contributes to the DJ-1ß mutant phenotypes. Interestingly, we found that dFOXO was required for the increase in DLP expression in DJ-1ß mutants and that dFOXO activity was increased in the heads of DJ-1ß mutants. In addition, subcellular localization of DLP appeared to be influenced by DJ-1 expression so that cytosolic DLP was increased in DJ-1ß mutants. Similarly, in mammalian cells, Daxx translocation from the nucleus to the cytosol was suppressed by overexpressed DJ-1ß under oxidative stress conditions; and, furthermore, targeted expression of DJ-1ß to mitochondria efficiently inhibited the Daxx translocation. Taken together, our findings demonstrate that DJ-1ß protects flies against oxidative stress- and UV-induced apoptosis by regulating the subcellular localization and gene expression of DLP, thus implying that Daxx-induced apoptosis is involved in the pathogenesis of DJ-1-associated PD.

In Vivo Screening of Traditional Medicinal Plants for Neuroprotective Activity against Aß42 Cytotoxicity by Using Drosophila Models of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder, characterized by progressive neuronal loss with amyloid ß-peptide (Aß) plaques. Despite several drugs currently used to treat AD, their beneficial effects on AD progress remains under debate. Here, we established a rapid in vivo screening system using Drosophila AD models to assess the neuroprotective activities of medicinal plants that have been used in traditional Chinese medicine. Among 23 medicinal plants tested, the extracts from five plants, Coriandrum sativum, Nardostachys jatamansi, Polygonum multiflorum (P. multiflorum), Rehmannia glutinosa, and Sorbus commixta (S. commixta), showed protective effects against the Aß42 neurotoxicity. We further characterized the neuroprotective activity of ethanol extracts from P. multiflorum and S. commixta. Aß42-expressing flies that we used showed AD neurological phenotypes, such as decreased survival and motility and increased cell death and reactive oxygen species level. However, feeding these flies extracts from P. multiflorum or S. commixta showed strong suppression of such phenotypes. Similar results were observed in human cells, so that the treatment of P. multiflorum and S. commixta extracts increased the viability of Aß-treated SH-SY5Y cells. Moreover, 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside, one of the main constituents of P. multiflorum, also showed similar protective activity against Aß42 cytotoxicity in both Drosophila and human cells. Taken together, our results suggest that both P. multiflorum and S. commixta have therapeutic potential for the treatment of neurodegenerative diseases, such as AD.

Penetrance of biallelic SMARCAL1 mutations is associated with environmental and genetic disturbances of gene expression.

Biallelic mutations of the DNA annealing helicase SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1) cause Schimke immuno-osseous dysplasia (SIOD, MIM 242900), an incompletely penetrant autosomal recessive disorder. Using human, Drosophila and mouse models, we show that the proteins encoded by SMARCAL1 orthologs localize to transcriptionally active chromatin and modulate gene expression. We also show that, as found in SIOD patients, deficiency of the SMARCAL1 orthologs alone is insufficient to cause disease in fruit flies and mice, although such deficiency causes modest diffuse alterations in gene expression. Rather, disease manifests when SMARCAL1 deficiency interacts with genetic and environmental factors that further alter gene expression. We conclude that the SMARCAL1 annealing helicase buffers fluctuations in gene expression and that alterations in gene expression contribute to the penetrance of SIOD.

Suppressive effects of SuHeXiang Wan on amyloid-ß42-induced extracellular signal-regulated kinase hyperactivation and glial cell proliferation in a transgenic Drosophila model of Alzheimer's disease.

SuHeXiang Wan (SHXW), a Chinese traditional medicine, has been used to treat infantile convulsions, seizures and strokes. Previously, we reported that modified SHXW, called KSOP1009, suppressed the hyper-activation of c-Jun N-terminal kinase (JNK) and Alzheimer's disease (AD)-like phenotypes in amyloid-ß42 (Aß42)-expressing Drosophila AD models. In the present study, we, further, investigated the detailed mechanism by which KSOP1009 suppresses the AD-like phenotypes of the model flies. As seen in the brains of AD patients, pan-neuronal expression of Aß42 in Drosophila increased activation of extracellular signal-regulated kinase (ERK), which was monitored by its phosphorylation level, and the number of glial cells in the brain. Suppression of caspase activity did not affect these phenomena, suggesting that Aß42 induces ERK activation and glial cell proliferation independently of apoptotic processes. KSOP1009 intake significantly reduced the level of ERK activation and the number of glial cells. Moreover, KSOP1009 intake also effectively decreased the defects in the wing vein formation induced by Epidermal growth factor receptor (Egfr) overexpression in fly wings, suggesting that it may contain an inhibitory substance that inhibits the EGFR/ERK signaling pathway. In addition, the Aß42-induced locomotive defect was partially rescued by inhibition of the elevated ERK activity through its antagonistic drug treatment. Taken together, these results suggest that KSOP1009 exerts its therapeutic effect by inhibiting the EGFR/ERK pathway and glial cell proliferation and by suppressing the JNK pathway and apoptosis.

Functional Characterization of Drosophila melanogaster CYP6A8 Fatty Acid Hydroxylase.

Genomic analysis indicated that the genome of Drosophila melanogaster contains more than 80 cytochrome P450 genes. To date, the enzymatic activity of these P450s has not been extensively studied. Here, the biochemical properties of CYP6A8 were characterized. CYP6A8 was cloned into the pCW vector, and its recombinant enzyme was expressed in Escherichia coli and purified using Ni2+-nitrilotriacetate affinity chromatography. Its expression level was approximately 130 nmol per liter of culture. Purified CYP6A8 exhibited a low-spin state in the absolute spectra of the ferric forms. Binding titration analysis indicated that lauric acid and capric acid produced type І spectral changes, with Kd values 28 ± 4 and 144 ± 20 µM, respectively. Ultra-performance liquid chromatography-mass spectrometry analysis showed that the oxidation reaction of lauric acid produced (ω-1)-hydroxylated lauric acid as a major product and ω-hydroxy-lauric acid as a minor product. Steady-state kinetic analysis of lauric acid hydroxylation yielded a kcat value of 0.038 ± 0.002 min-1 and a Km value of 10 ± 2 µM. In addition, capric acid hydroxylation of CYP6A8 yielded kinetic parameters with a kcat value of 0.135 ± 0.007 min-1 and a Km value of 21 ± 4 µM. Because of the importance of various lipids as carbon sources, the metabolic analysis of fatty acids using CYP6A8 in this study can provide an understanding of the biochemical roles of P450 enzymes in many insects, including Drosophila melanogaster.