Adult expression of the cell adhesion protein Fasciclin 3 is required for the maintenance of adult olfactory interneurons.

The proper functioning of the nervous system is dependent on the establishment and maintenance of intricate networks of neurons that form functional neural circuits. Once neural circuits are assembled during development, a distinct set of molecular programs is likely required to maintain their connectivity throughout the lifetime of the organism. Here, we demonstrate that Fasciclin 3 (Fas3), an axon guidance cell adhesion protein, is necessary for the maintenance of the olfactory circuit in adult Drosophila. We utilized the TARGET system to spatiotemporally knockdown Fas3 in selected populations of adult neurons. Our findings show that Fas3 knockdown results in the death of olfactory circuit neurons and reduced survival of adults. We also demonstrated that Fas3 knockdown activates caspase-3-mediated cell death in olfactory local interneurons, which can be rescued by overexpressing baculovirus p35, an anti-apoptotic protein. This work adds to the growing set of evidence indicating a crucial role for axon guidance proteins in the maintenance of neuronal circuits in adults.

Centrosome-organized plasma membrane infoldings linked to growth of a cortical actin domain.

Regulated cell shape change requires the induction of cortical cytoskeletal domains. Often, local changes to plasma membrane (PM) topography are involved. Centrosomes organize cortical domains and can affect PM topography by locally pulling the PM inward. Are these centrosome effects coupled? At the syncytial Drosophila embryo cortex, centrosome-induced actin caps grow into dome-like compartments for mitoses. We found the nascent cap to be a collection of PM folds and tubules formed over the astral centrosomal MT array. The localized infoldings require centrosome and dynein activities, and myosin-based surface tension prevents them elsewhere. Centrosome-engaged PM infoldings become specifically enriched with an Arp2/3 induction pathway. Arp2/3 actin network growth between the infoldings counterbalances centrosomal pulling forces and disperses the folds for actin cap expansion. Abnormal domain topography with either centrosome or Arp2/3 disruption correlates with decreased exocytic vesicle association. Together, our data implicate centrosome-organized PM infoldings in coordinating Arp2/3 network growth and exocytosis for cortical domain assembly.

CCT and Cullin1 Regulate the TORC1 Pathway to Promote Dendritic Arborization in Health and Disease.

The development of cell-type-specific dendritic arbors is integral to the proper functioning of neurons within their circuit networks. In this study, we examine the regulatory relationship between the cytosolic chaperonin CCT, key insulin pathway genes, and an E3 ubiquitin ligase (Cullin1) in dendritic development. CCT loss of function (LOF) results in dendritic hypotrophy in Drosophila Class IV (CIV) multi-dendritic larval sensory neurons, and CCT has recently been shown to fold components of the TOR (Target of Rapamycin) complex 1 (TORC1) in vitro. Through targeted genetic manipulations, we confirm that an LOF of CCT and the TORC1 pathway reduces dendritic complexity, while overexpression of key TORC1 pathway genes increases the dendritic complexity in CIV neurons. Furthermore, both CCT and TORC1 LOF significantly reduce microtubule (MT) stability. CCT has been previously implicated in regulating proteinopathic aggregation, thus, we examine CIV dendritic development in disease conditions as well. The expression of mutant Huntingtin leads to dendritic hypotrophy in a repeat-length-dependent manner, which can be rescued by Cullin1 LOF. Together, our data suggest that Cullin1 and CCT influence dendritic arborization through the regulation of TORC1 in both health and disease.

Dietary sucrose determines the regulatory activity of lithium on gene expression and lifespan in Drosophila melanogaster.

The amount of dietary sugars and the administration of lithium both impact the lifespan of the fruit fly Drosophila melanogaster. It is noteworthy that lithium is attributed with insulin-like activity as it stimulates protein kinase B/Akt and suppresses the activity of glycogen synthase kinase-3 (GSK-3). However, its interaction with dietary sugar has largely remained unexplored. Therefore, we investigated the effects of lithium supplementation on known lithium-sensitive parameters in fruit flies, such as lifespan, body composition, GSK-3 phosphorylation, and the transcriptome, while varying the dietary sugar concentration. For all these parameters, we observed that the efficacy of lithium was significantly influenced by the sucrose content in the diet. Overall, we found that lithium was most effective in enhancing longevity and altering body composition when added to a low-sucrose diet. Whole-body RNA sequencing revealed a remarkably similar transcriptional response when either increasing dietary sucrose from 1% to 10% or adding 1 mM LiCl to a 1% sucrose diet, characterized by a substantial overlap of nearly 500 differentially expressed genes. Hence, dietary sugar supply is suggested as a key factor in understanding lithium bioactivity, which could hold relevance for its therapeutic applications.

Confinement promotes nematic alignment of spindle-shaped cells during Drosophila embryogenesis.

Tissue morphogenesis is often controlled by actomyosin networks pulling on adherens junctions (AJs), but junctional myosin levels vary. At an extreme, the Drosophila embryo amnioserosa forms a horseshoe-shaped strip of aligned, spindle-shaped cells lacking junctional myosin. What are the bases of amnioserosal cell interactions and alignment? Compared with surrounding tissue, we find that amnioserosal AJ continuity has lesser dependence on α-catenin, the mediator of AJ-actomyosin association, and greater dependence on Bazooka/Par-3, a junction-associated scaffold protein. Microtubule bundles also run along amnioserosal AJs and support their long-range curvilinearity. Amnioserosal confinement is apparent from partial overlap of its spindle-shaped cells, its outward bulging from surrounding tissue and from compressive stress detected within the amnioserosa. Genetic manipulations that alter amnioserosal confinement by surrounding tissue also result in amnioserosal cells losing alignment and gaining topological defects characteristic of nematically ordered systems. With Bazooka depletion, confinement by surrounding tissue appears to be relatively normal and amnioserosal cells align despite their AJ fragmentation. Overall, the fully elongated amnioserosa appears to form through tissue-autonomous generation of spindle-shaped cells that nematically align in response to confinement by surrounding tissue.

Recurrent Duplication and Diversification of a Vital DNA Repair Gene Family Across Drosophila.

Maintaining genome integrity is vital for organismal survival and reproduction. Essential, broadly conserved DNA repair pathways actively preserve genome integrity. However, many DNA repair proteins evolve adaptively. Ecological forces like UV exposure are classically cited drivers of DNA repair evolution. Intrinsic forces like repetitive DNA, which also imperil genome integrity, have received less attention. We recently reported that a Drosophila melanogaster-specific DNA satellite array triggered species-specific, adaptive evolution of a DNA repair protein called Spartan/MH. The Spartan family of proteases cleave hazardous, covalent crosslinks that form between DNA and proteins ("DNA-protein crosslink repair"). Appreciating that DNA satellites are both ubiquitous and universally fast-evolving, we hypothesized that satellite DNA turnover spurs adaptive evolution of DNA-protein crosslink repair beyond a single gene and beyond the D. melanogaster lineage. This hypothesis predicts pervasive Spartan gene family diversification across Drosophila species. To study the evolutionary history of the Drosophila Spartan gene family, we conducted population genetic, molecular evolution, phylogenomic, and tissue-specific expression analyses. We uncovered widespread signals of positive selection across multiple Spartan family genes and across multiple evolutionary timescales. We also detected recurrent Spartan family gene duplication, divergence, and gene loss. Finally, we found that ovary-enriched parent genes consistently birthed functionally diverged, testis-enriched daughter genes. To account for Spartan family diversification, we introduce a novel mechanistic model of antagonistic coevolution that links DNA satellite evolution and adaptive regulation of Spartan protease activity. This framework promises to accelerate our understanding of how DNA repeats drive recurrent evolutionary innovation to preserve genome integrity.

Co-transcriptional splicing facilitates transcription of gigantic genes.

Although introns are typically tens to thousands of nucleotides, there are notable exceptions. In flies as well as humans, a small number of genes contain introns that are more than 1000 times larger than typical introns, exceeding hundreds of kilobases (kb) to megabases (Mb). It remains unknown why gigantic introns exist and how cells overcome the challenges associated with their transcription and RNA processing. The Drosophila Y chromosome contains some of the largest genes identified to date: multiple genes exceed 4Mb, with introns accounting for over 99% of the gene span. Here we demonstrate that co-transcriptional splicing of these gigantic Y-linked genes is important to ensure successful transcription: perturbation of splicing led to the attenuation of transcription, leading to a failure to produce mature mRNA. Cytologically, defective splicing of the Y-linked gigantic genes resulted in disorganization of transcripts within the nucleus suggestive of entanglement of transcripts, likely resulting from unspliced long RNAs. We propose that co-transcriptional splicing maintains the length of nascent transcripts of gigantic genes under a critical threshold, preventing their entanglement and ensuring proper gene expression. Our study reveals a novel biological significance of co-transcriptional splicing.

Phospholipid scramblase 1: an essential component of the nephrocyte slit diaphragm.

Blood ultrafiltration in nephrons critically depends on specialized intercellular junctions between podocytes, named slit diaphragms (SDs). Here, by studying a homologous structure found in Drosophila nephrocytes, we identify the phospholipid scramblase Scramb1 as an essential component of the SD, uncovering a novel link between membrane dynamics and SD formation. In scramb1 mutants, SDs fail to form. Instead, the SD components Sticks and stones/nephrin, Polychaetoid/ZO-1, and the Src-kinase Src64B/Fyn associate in cortical foci lacking the key SD protein Dumbfounded/NEPH1. Scramb1 interaction with Polychaetoid/ZO-1 and Flotillin2, the presence of essential putative palmitoylation sites and its capacity to oligomerize, suggest a function in promoting SD assembly within lipid raft microdomains. Furthermore, Scramb1 interactors as well as its functional sensitivity to temperature, suggest an active involvement in membrane remodeling processes during SD assembly. Remarkably, putative Ca2+-binding sites in Scramb1 are essential for its activity raising the possibility that Ca2+ signaling may control the assembly of SDs by impacting on Scramb1 activity.

Social setting interacts with hyper dopamine to boost the stimulant effect of ethanol.

Alcohol consumption occurring in a social or solitary setting often yields different behavioural responses in human subjects. For example, social drinking is associated with positive effects while solitary drinking is linked to negative effects. However, the neurobiological mechanism by which the social environment during alcohol intake impacts on behavioural responses remains poorly understood. We investigated whether distinct social environments affect behavioural responses to ethanol and the role of the dopamine system in this phenomenon in the fruit fly Drosophila melanogaster. The wild-type Canton-S (CS) flies showed higher locomotor response when exposed to ethanol in a group setting than a solitary setting, and there was no difference in females and males. Dopamine signalling is crucial for the locomotor stimulating effect of ethanol. When subjected to ethanol exposure alone, the dopamine transport mutant flies fumin (fmn) with hyper dopamine displayed the locomotor response similar to CS. When subjected to ethanol in a group setting, however, the fmn's response to the locomotor stimulating effect was substantially augmented compared with CS, indicating synergistic interaction of dopamine signalling and social setting. To identify the dopamine signalling pathway important for the social effect, we examined the flies defective in individual dopamine receptors and found that the D1 receptor dDA1/Dop1R1 is the major receptor mediating the social effect. Taken together, this study underscores the influence of social context on the neural and behavioural responses to ethanol.

The Alk receptor tyrosine kinase regulates Sparkly, a novel activity regulating neuropeptide precursor in the Drosophila central nervous system.

Numerous roles for the Alk receptor tyrosine kinase have been described in Drosophila, including functions in the central nervous system (CNS), however the molecular details are poorly understood. To gain mechanistic insight, we employed Targeted DamID (TaDa) transcriptional profiling to identify targets of Alk signaling in the larval CNS. TaDa was employed in larval CNS tissues, while genetically manipulating Alk signaling output. The resulting TaDa data were analyzed together with larval CNS scRNA-seq datasets performed under similar conditions, identifying a role for Alk in the transcriptional regulation of neuroendocrine gene expression. Further integration with bulk and scRNA-seq datasets from larval brains in which Alk signaling was manipulated identified a previously uncharacterized Drosophila neuropeptide precursor encoded by CG4577 as an Alk signaling transcriptional target. CG4577, which we named Sparkly (Spar), is expressed in a subset of Alk-positive neuroendocrine cells in the developing larval CNS, including circadian clock neurons. In agreement with our TaDa analysis, overexpression of the Drosophila Alk ligand Jeb resulted in increased levels of Spar protein in the larval CNS. We show that Spar protein is expressed in circadian (clock) neurons, and flies lacking Spar exhibit defects in sleep and circadian activity control. In summary, we report a novel activity regulating neuropeptide precursor gene that is regulated by Alk signaling in the Drosophila CNS.

Multiple isoforms of the Activin-like receptor baboon differentially regulate proliferation and conversion behaviors of neuroblasts and neuroepithelial cells in the Drosophila larval brain.

In Drosophila coordinated proliferation of two neural stem cells, neuroblasts (NB) and neuroepithelial (NE) cells, is pivotal for proper larval brain growth that ultimately determines the final size and performance of an adult brain. The larval brain growth displays two phases based on behaviors of NB and NEs: the first one in early larval stages, influenced by nutritional status and the second one in the last larval stage, promoted by ecdysone signaling after critical weight checkpoint. Mutations of the baboon (babo) gene that produces three isoforms (BaboA-C), all acting as type-I receptors of Activin-type transforming growth factor ß (TGF-ß) signaling, cause a small brain phenotype due to severely reduced proliferation of the neural stem cells. In this study we show that loss of babo function severely affects proliferation of NBs and NEs as well as conversion of NEs from both phases. By analyzing babo-null and newly generated isoform-specific mutants by CRISPR mutagenesis as well as isoform-specific RNAi knockdowns in a cell- and stage-specific manner, our data support differential contributions of the isoforms for these cellular events with BaboA playing the major role. Stage-specific expression of EcR-B1 in the brain is also regulated primarily by BaboA along with function of the other isoforms. Blocking EcR function in both neural stem cells results in a small brain phenotype that is more severe than baboA-knockdown alone. In summary, our study proposes that the Babo-mediated signaling promotes proper behaviors of the neural stem cells in both phases and achieves this by acting upstream of EcR-B1 expression in the second phase.

Altered circadian rhythm, sleep, and rhodopsin 7-dependent shade preference during diapause in Drosophila melanogaster.

To survive adverse environments, many animals enter a dormant state such as hibernation, dauer, or diapause. Various Drosophila species undergo adult reproductive diapause in response to cool temperatures and/or short day-length. While flies are less active during diapause, it is unclear how adverse environmental conditions affect circadian rhythms and sleep. Here we show that in diapause-inducing cool temperatures, Drosophila melanogaster exhibit altered circadian activity profiles, including severely reduced morning activity and an advanced evening activity peak. Consequently, the flies have a single activity peak at a time similar to when nondiapausing flies take a siesta. Temperatures ≤15 °C, rather than photoperiod, primarily drive this behavior. At cool temperatures, flies rapidly enter a deep-sleep state that lacks the sleep cycles of flies at higher temperatures and require high levels of stimulation for arousal. Furthermore, we show that at 25 °C, flies prefer to siesta in the shade, a preference that is virtually eliminated at 10 °C. Resting in the shade is driven by an aversion to blue light that is sensed by Rhodopsin 7 outside of the eyes. Flies at 10 °C show neuronal markers of elevated sleep pressure, including increased expression of Bruchpilot and elevated Ca2+ in the R5 ellipsoid body neurons. Therefore, sleep pressure might overcome blue light aversion. Thus, at the same temperatures that cause reproductive arrest, preserve germline stem cells, and extend lifespan, D. melanogaster are prone to deep sleep and exhibit dramatically altered, yet rhythmic, daily activity patterns.

Re-assessment of the subcellular localization of Bazooka/Par-3 in Drosophila: no evidence for localization to the nucleus and the neuromuscular junction.

Bazooka/Par-3 (Baz) is an evolutionarily conserved scaffold protein that functions as a master regulator for the establishment and maintenance of cell polarity in many different cell types. In the vast majority of published research papers Baz has been reported to localize at the cell cortex and at intercellular junctions. However, there have also been several reports showing localization and function of Baz at additional subcellular sites, in particular the nuclear envelope and the neuromuscular junction. In this study we have re-assessed the localization of Baz to these subcellular sites in a systematic manner. We used antibodies raised in different host animals against different epitopes of Baz for confocal imaging of Drosophila tissues. We tested the specificity of these antisera by mosaic analysis with null mutant baz alleles and tissue-specific RNAi against baz. In addition, we used a GFP-tagged gene trap line for Baz and a bacterial artificial chromosome (BAC) expressing GFP-tagged Baz under control of its endogenous promoter in a baz mutant background to compare the subcellular localization of the GFP-Baz fusion proteins to the staining with anti-Baz antisera. Together, these experiments did not provide evidence for specific localization of Baz to the nucleus or the neuromuscular junction.

Dan forms condensates in neuroblasts and regulates nuclear architecture and progenitor competence in vivo.

Genome organization is thought to underlie cell type specific gene expression, yet how it is regulated in progenitors to produce cellular diversity is unknown. In Drosophila, a developmentally-timed genome reorganization in neural progenitors terminates competence to produce early-born neurons. These events require downregulation of Distal antenna (Dan), part of the conserved pipsqueak DNA-binding superfamily. Here we find that Dan forms liquid-like condensates with high protein mobility, and whose size and subnuclear distribution are balanced with its DNA-binding. Further, we identify a LARKS domain, a structural motif associated with condensate-forming proteins. Deleting just 13 amino acids from LARKS abrogates Dan's ability to retain the early-born neural fate gene, hunchback, in the neuroblast nuclear interior and maintain competence in vivo. Conversely, domain-swapping with LARKS from known phase-separating proteins rescues Dan's effects on competence. Together, we provide in vivo evidence for condensate formation and the regulation of progenitor nuclear architecture underlying neuronal diversification.

A nuclear architecture screen in Drosophila identifies Stonewall as a link between chromatin position at the nuclear periphery and germline stem cell fate.

The association of genomic loci to the nuclear periphery is proposed to facilitate cell type-specific gene repression and influence cell fate decisions. However, the interplay between gene position and expression remains incompletely understood, in part because the proteins that position genomic loci at the nuclear periphery remain unidentified. Here, we used an Oligopaint-based HiDRO screen targeting ∼1000 genes to discover novel regulators of nuclear architecture in Drosophila cells. We identified the heterochromatin-associated protein Stonewall (Stwl) as a factor promoting perinuclear chromatin positioning. In female germline stem cells (GSCs), Stwl binds and positions chromatin loci, including GSC differentiation genes, at the nuclear periphery. Strikingly, Stwl-dependent perinuclear positioning is associated with transcriptional repression, highlighting a likely mechanism for Stwl's known role in GSC maintenance and ovary homeostasis. Thus, our study identifies perinuclear anchors in Drosophila and demonstrates the importance of gene repression at the nuclear periphery for cell fate.

Genome organization regulates nuclear pore complex formation and promotes differentiation during Drosophila oogenesis.

Genome organization can regulate gene expression and promote cell fate transitions. The differentiation of germline stem cells (GSCs) to oocytes in Drosophila involves changes in genome organization mediated by heterochromatin and the nuclear pore complex (NPC). Heterochromatin represses germ cell genes during differentiation, and NPCs anchor these silenced genes to the nuclear periphery, maintaining silencing to allow for oocyte development. Surprisingly, we found that genome organization also contributes to NPC formation, mediated by the transcription factor Stonewall (Stwl). As GSCs differentiate, Stwl accumulates at boundaries between silenced and active gene compartments. Stwl at these boundaries plays a pivotal role in transitioning germ cell genes into a silenced state and activating a group of oocyte genes and nucleoporins (Nups). The upregulation of these Nups during differentiation is crucial for NPC formation and further genome organization. Thus, cross-talk between genome architecture and NPCs is essential for successful cell fate transitions.

Drosophila melanogaster Set8 and L(3)mbt function in gene expression independently of histone H4 lysine 20 methylation.

Monomethylation of lysine 20 of histone H4 (H4K20me1) is catalyzed by Set8 and thought to play important roles in many aspects of genome function that are mediated by H4K20me binding proteins. We interrogated this model in a developing animal by comparing in parallel the transcriptomes of Set8 null , H4 K20R/A , and l(3)mbt mutant Drosophila melanogaster We found that the gene expression profiles of H4 K20A and H4 K20R larvae are markedly different than Set8 null larvae despite similar reductions in H4K20me1. Set8 null mutant cells have a severely disrupted transcriptome and fail to proliferate in vivo, but these phenotypes are not recapitulated by mutation of H4 K20 , indicating that the developmental defects of Set8 null animals are largely due to H4K20me1-independent effects on gene expression. Furthermore, the H4K20me1 binding protein L(3)mbt is recruited to the transcription start sites of most genes independently of H4K20me even though genes bound by L(3)mbt have high levels of H4K20me1. Moreover, both Set8 and L(3)mbt bind to purified H4K20R nucleosomes in vitro. We conclude that gene expression changes in Set8 null and H4 K20 mutants cannot be explained by loss of H4K20me1 or L(3)mbt binding to chromatin and therefore that H4K20me1 does not play a large role in gene expression.

Beyond histones: the elusive substrates of chromatin regulators.

Gene transcription is intimately linked to chromatin state and histone modifications. However, the enzymes mediating these post-translational modifications have many additional, nonhistone substrates, making it difficult to ascribe the most relevant modification. In this issue of Genes & Development, Crain and colleagues (doi:10.1101/gad.351698.124) have combined a powerful histone replacement system with mutational analysis of a chromatin regulator and a chromatin reader in Drosophila melanogaster Importantly, they discovered that genes controlled by the histone 4 lysine 20 (H4K20) methyltransferase Set8 and the protein recognizing H4K20 monomethylation, L(3)mbt, differ substantially from those affected by mutation of H4K20 itself. This demonstrates that H4K20 is not the key substrate for Set8 but that methylation of other, unidentified proteins mediates its effects on transcription.

Vitamin B6 deficiency cooperates with oncogenic Ras to induce malignant tumors in Drosophila.

Vitamin B6 is a water-soluble vitamin which possesses antioxidant properties. Its catalytically active form, pyridoxal 5'-phosphate (PLP), is a crucial cofactor for DNA and amino acid metabolism. The inverse correlation between vitamin B6 and cancer risk has been observed in several studies, although dietary vitamin B6 intake sometimes failed to confirm this association. However, the molecular link between vitamin B6 and cancer remains elusive. Previous work has shown that vitamin B6 deficiency causes chromosome aberrations (CABs) in Drosophila and human cells, suggesting that genome instability may correlate the lack of this vitamin to cancer. Here we provide evidence in support of this hypothesis. Firstly, we show that PLP deficiency, induced by the PLP antagonists 4-deoxypyridoxine (4DP) or ginkgotoxin (GT), promoted tumorigenesis in eye larval discs transforming benign RasV12 tumors into aggressive forms. In contrast, PLP supplementation reduced the development of tumors. We also show that low PLP levels, induced by 4DP or by silencing the sgllPNPO gene involved in PLP biosynthesis, worsened the tumor phenotype in another Drosophila cancer model generated by concomitantly activating RasV12 and downregulating Discs-large (Dlg) gene. Moreover, we found that RasV12 eye discs from larvae reared on 4DP displayed CABs, reactive oxygen species (ROS) and low catalytic activity of serine hydroxymethyltransferase (SHMT), a PLP-dependent enzyme involved in thymidylate (dTMP) biosynthesis, in turn required for DNA replication and repair. Feeding RasV12 4DP-fed larvae with PLP or ascorbic acid (AA) plus dTMP, rescued both CABs and tumors. The same effect was produced by overexpressing catalase in RasV12 DlgRNAi 4DP-fed larvae, thus allowing to establish a relationship between PLP deficiency, CABs, and cancer. Overall, our data provide the first in vivo demonstration that PLP deficiency can impact on cancer by increasing genome instability, which is in turn mediated by ROS and reduced dTMP levels.

The protective effect and bioactive compounds of Astragalus membranaceus against neurodegenerative disorders via alleviating oxidative stress in Drosophila.

Oxidative stress is proposed as a regulatory element in various neurological disorders, which is involved in the progress of several neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Antioxidant drugs are widely used to alleviate neurodegenerative disorders. Astragalus membranaceus (Huangqi, AM) is a commonly used medicinal herb with a wide range of pharmacological effects. Here, the protective effect and mechanism of AM extract (AME) and its bioactive compounds against neurodegenerative disorders via alleviating oxidative stress were detected using adult Drosophila melanogaster. The drug safety was measured by development analysis; oxidative stress resistance ability was detected by survival rate under H2O2 environment; ROS level was detected by DHE staining and gstD1-GFP fluoresence assay; antioxidative abilitiy was represent by measuring antioxidant enzyme activity, antioxidative-related gene expression, and ATP and MFN2 levels. The neuroprotective effect was evaluated by lifespan and locomotion analysis in Aß42 transgenic and Pink1B9 mutants. AME dramatically increased the survival rates, improved the CAT activity, restored the decreased mRNA expressions of Sod1, Cat, and CncC under H2O2 stimulation, and ameliorated the neurobehavioral defects of the AD and PD. Thirteen small molecules in AM had antioxidant function, in which vanillic acid and daidzein had the most potent antioxidant effect. Vanillic acid and daidzein could increase the activities of SOD and CAT, GSH level, and the expressions of antioxidant genes. Vanillic acid could improve the levels of ATP and MFN2, and mRNA expressions of ND42 and SDHC to rescue mitochondrial dysfunction. Furthermore, vanillic acid ameliorated neurobehavioral defects of PD. Daidzein ameliorated neurobehavioral defect of Aß-induced AD mode. Taken together, AM plays a protective role in oxidative damage, thereby as a potential natural drug to treat neurodegenerative disorders.