Drosophila CASK regulates brain size and neuronal morphogenesis, providing a genetic model of postnatal microcephaly suitable for drug discovery.

BACKGROUND: CASK-related neurodevelopmental disorders are untreatable. Affected children show variable severity, with microcephaly, intellectual disability (ID), and short stature as common features. X-linked human CASK shows dosage sensitivity with haploinsufficiency in females. CASK protein has multiple domains, binding partners, and proposed functions at synapses and in the nucleus. Human and Drosophila CASK show high amino-acid-sequence similarity in all functional domains. Flies homozygous for a hypomorphic CASK mutation (∆18) have motor and cognitive deficits. A Drosophila genetic model of CASK-related disorders could have great scientific and translational value. METHODS: We assessed the effects of CASK loss of function on morphological phenotypes in Drosophila using established genetic, histological, and primary neuronal culture approaches. NeuronMetrics software was used to quantify neurite-arbor morphology. Standard nonparametric statistics methods were supplemented by linear mixed effects modeling in some cases. Microfluidic devices of varied dimensions were fabricated and numerous fluid-flow parameters were used to induce oscillatory stress fields on CNS tissue. Dissociation into viable neurons and neurite outgrowth in vitro were assessed. RESULTS: We demonstrated that ∆18 homozygous flies have small brains, small heads, and short bodies. When neurons from developing CASK-mutant CNS were cultured in vitro, they grew small neurite arbors with a distinctive, quantifiable "bushy" morphology that was significantly rescued by transgenic CASK+. As in humans, the bushy phenotype showed dosage-sensitive severity. To overcome the limitations of manual tissue trituration for neuronal culture, we optimized the design and operation of a microfluidic system for standardized, automated dissociation of CNS tissue into individual viable neurons. Neurons from CASK-mutant CNS dissociated in the microfluidic system recapitulate the bushy morphology. Moreover, for any given genotype, device-dissociated neurons grew larger arbors than did manually dissociated neurons. This automated dissociation method is also effective for rodent CNS. CONCLUSIONS: These biological and engineering advances set the stage for drug discovery using the Drosophila model of CASK-related disorders. The bushy phenotype provides a cell-based assay for compound screening. Nearly a dozen genes encoding CASK-binding proteins or transcriptional targets also have brain-development mutant phenotypes, including ID. Hence, drugs that improve CASK phenotypes might also benefit children with disorders due to mutant CASK partners.

Acute cholangitis following mRNA COVID-19 vaccine booster in a patient receiving an anti-amyloid antibody for Alzheimer's disease: A case report.

A 77-year-old woman had 2 weeks of fever and flu-like symptoms starting several hours after receiving an mRNA booster for SARS-CoV-2 and the influenza vaccine, in separate shots. Laboratory tests showed cholangitis. Medical history included APOE-ε4 carrier genotype, mild Alzheimer's disease, participation in a clinical trial of aducanumab, and resolving polymyalgia rheumatica. The patient recovered with at-home supportive care. She had aducanumab-associated amyloid-related imaging abnormalities-edema (ARIA-E) both before and after the acute cholangitis. Two months following the vaccinations polymyalgia rheumatica recurred. This case raises questions about interactions among immune-mediated disease, complications of anti-amyloid monoclonal antibodies, and adverse events following SARS-CoV-2 mRNA vaccination.

Epilogue to the Gerald Maggiora Festschrift: a tribute to an exemplary mentor, colleague, collaborator, and innovator.

In May 2022, JCAMD published a Special Issue in honor of Gerald (Gerry) Maggiora, whose scientific leadership over many decades advanced the fields of computational chemistry and chemoinformatics for drug discovery. Along the way, he has impacted many researchers in both academia and the pharmaceutical industry. In this Epilogue, we explain the origins of the Festschrift and present a series of first-hand vignettes, in approximate chronological sequence, that together paint a picture of this remarkable man. Whether they highlight Gerry's endless curiosity about molecular life sciences or his willingness to challenge conventional wisdom or his generous support of junior colleagues and peers, these colleagues and collaborators are united in their appreciation of his positive influence. These tributes also reflect key trends and themes during the evolution of modern drug discovery, seen through the lens of people who worked with a visionary leader. Junior scientists will find an inspiring roadmap for creative collegiality and collaboration.

Unraveling the Gordian knot: genetics and the troubled road to effective therapeutics for Alzheimer's disease.

In the late 20th century, identification of the major protein components of amyloid plaques and neurofibrillary tangles provided a window into the molecular pathology of Alzheimer's disease, ushering in an era of optimism that targeted therapeutics would soon follow. The amyloid-cascade hypothesis took hold very early, supported by discoveries that dominant mutations in APP, PSEN1, and PSEN2 cause the very rare, early-onset, familial forms of the disease. However, in the past decade, a stunning series of failed Phase-3 clinical trials, testing anti-amyloid antibodies or processing-enzyme inhibitors, prompts the question, What went wrong? The FDA's recent controversial approval of aducanumab, despite widespread concerns about efficacy and safety, only amplifies the question. The assumption that common, late-onset Alzheimer's is a milder form of familial disease was not adequately questioned. The differential timing of discoveries, including blood-brain-barrier-penetrant tracers for imaging of plaques and tangles, made it easy to focus on amyloid. Furthermore, the neuropathology community initially implemented Alzheimer's diagnostic criteria based on plaques only. The discovery that MAPT mutations cause frontotemporal dementia with tauopathy made it even easier to overlook the tangles in Alzheimer's. Many important findings were simply ignored. The accepted mouse models did not predict the human clinical trials data. Given this lack of pharmacological validity, input from geneticists in collaboration with neuroscientists is needed to establish criteria for valid models of Alzheimer's disease. More generally, scientists using genetic model organisms as whole-animal bioassays can contribute to building the pathogenesis network map of Alzheimer's disease.

Spontaneous motor-behavior abnormalities in two Drosophila models of neurodevelopmental disorders.

Mutations in hundreds of genes cause neurodevelopmental disorders with abnormal motor behavior alongside cognitive deficits. Boys with fragile X syndrome (FXS), a leading monogenic cause of intellectual disability, often display repetitive behaviors, a core feature of autism. By direct observation and manual analysis, we characterized spontaneous-motor-behavior phenotypes of Drosophila dfmr1 mutants, an established model for FXS. We recorded individual 1-day-old adult flies, with mature nervous systems and prior to the onset of aging, in small arenas. We scored behavior using open-source video-annotation software to generate continuous activity timelines, which were represented graphically and quantitatively. Young dfmr1 mutants spent excessive time grooming, with increased bout number and duration; both were rescued by transgenic wild-type dfmr1+. By two grooming-pattern measures, dfmr1-mutant flies showed elevated repetitions consistent with perseveration, which is common in FXS. In addition, the mutant flies display a preference for grooming posterior body structures, and an increased rate of grooming transitions from one site to another. We raise the possibility that courtship and circadian rhythm defects, previously reported for dfmr1 mutants, are complicated by excessive grooming. We also observed significantly increased grooming in CASK mutants, despite their dramatically decreased walking phenotype. The mutant flies, a model for human CASK-related neurodevelopmental disorders, displayed consistently elevated grooming indices throughout the assay, but transient locomotory activation immediately after placement in the arena. Based on published data identifying FMRP-target transcripts and functional analyses of mutations causing human genetic neurodevelopmental disorders, we propose the following proteins as candidate mediators of excessive repetitive behaviors in FXS: CaMKIIα, NMDA receptor subunits 2A and 2B, NLGN3, and SHANK3. Together, these fly-mutant phenotypes and mechanistic insights provide starting points for drug discovery to identify compounds that reduce dysfunctional repetitive behaviors.

A systematic nomenclature for the insect brain.

Despite the importance of the insect nervous system for functional and developmental neuroscience, descriptions of insect brains have suffered from a lack of uniform nomenclature. Ambiguous definitions of brain regions and fiber bundles have contributed to the variation of names used to describe the same structure. The lack of clearly determined neuropil boundaries has made it difficult to document precise locations of neuronal projections for connectomics study. To address such issues, a consortium of neurobiologists studying arthropod brains, the Insect Brain Name Working Group, has established the present hierarchical nomenclature system, using the brain of Drosophila melanogaster as the reference framework, while taking the brains of other taxa into careful consideration for maximum consistency and expandability. The following summarizes the consortium's nomenclature system and highlights examples of existing ambiguities and remedies for them. This nomenclature is intended to serve as a standard of reference for the study of the brain of Drosophila and other insects.

Exome sequencing reveals new causal mutations in children with epileptic encephalopathies.

PURPOSE: The management of epilepsy in children is particularly challenging when seizures are resistant to antiepileptic medications, or undergo many changes in seizure type over time, or have comorbid cognitive, behavioral, or motor deficits. Despite efforts to classify such epilepsies based on clinical and electroencephalographic criteria, many children never receive a definitive etiologic diagnosis. Whole exome sequencing (WES) is proving to be a highly effective method for identifying de novo variants that cause neurologic disorders, especially those associated with abnormal brain development. Herein we explore the utility of WES for identifying candidate causal de novo variants in a cohort of children with heterogeneous sporadic epilepsies without etiologic diagnoses. METHODS: We performed WES (mean coverage approximately 40×) on 10 trios comprised of unaffected parents and a child with sporadic epilepsy characterized by difficult-to-control seizures and some combination of developmental delay, epileptic encephalopathy, autistic features, cognitive impairment, or motor deficits. Sequence processing and variant calling were performed using standard bioinformatics tools. A custom filtering system was used to prioritize de novo variants of possible functional significance for validation by Sanger sequencing. KEY FINDINGS: In 9 of 10 probands, we identified one or more de novo variants predicted to alter protein function, for a total of 15. Four probands had de novo mutations in genes previously shown to harbor heterozygous mutations in patients with severe, early onset epilepsies (two in SCN1A, and one each in CDKL5 and EEF1A2). In three children, the de novo variants were in genes with functional roles that are plausibly relevant to epilepsy (KCNH5, CLCN4, and ARHGEF15). The variant in KCNH5 alters one of the highly conserved arginine residues of the voltage sensor of the encoded voltage-gated potassium channel. In vitro analyses using cell-based assays revealed that the CLCN4 mutation greatly impaired ion transport by the ClC-4 2Cl(-) /H(+) -exchanger and that the mutation in ARHGEF15 reduced GEF exchange activity of the gene product, Ephexin5, by about 50%. Of interest, these seven probands all presented with seizures within the first 6 months of life, and six of these have intractable seizures. SIGNIFICANCE: The finding that 7 of 10 children carried de novo mutations in genes of known or plausible clinical significance to neuronal excitability suggests that WES will be of use for the molecular genetic diagnosis of sporadic epilepsies in children, especially when seizures are of early onset and difficult to control.

A cell-based fascin bioassay identifies compounds with potential anti-metastasis or cognition-enhancing functions.

The actin-bundling protein fascin is a key mediator of tumor invasion and metastasis and its activity drives filopodia formation, cell-shape changes and cell migration. Small-molecule inhibitors of fascin block tumor metastasis in animal models. Conversely, fascin deficiency might underlie the pathogenesis of some developmental brain disorders. To identify fascin-pathway modulators we devised a cell-based assay for fascin function and used it in a bidirectional drug screen. The screen utilized cultured fascin-deficient mutant Drosophila neurons, whose neurite arbors manifest the 'filagree' phenotype. Taking a repurposing approach, we screened a library of 1040 known compounds, many of them FDA-approved drugs, for filagree modifiers. Based on scaffold distribution, molecular-fingerprint similarities, and chemical-space distribution, this library has high structural diversity, supporting its utility as a screening tool. We identified 34 fascin-pathway blockers (with potential anti-metastasis activity) and 48 fascin-pathway enhancers (with potential cognitive-enhancer activity). The structural diversity of the active compounds suggests multiple molecular targets. Comparisons of active and inactive compounds provided preliminary structure-activity relationship information. The screen also revealed diverse neurotoxic effects of other drugs, notably the 'beads-on-a-string' defect, which is induced solely by statins. Statin-induced neurotoxicity is enhanced by fascin deficiency. In summary, we provide evidence that primary neuron culture using a genetic model organism can be valuable for early-stage drug discovery and developmental neurotoxicity testing. Furthermore, we propose that, given an appropriate assay for target-pathway function, bidirectional screening for brain-development disorders and invasive cancers represents an efficient, multipurpose strategy for drug discovery.

De novo pathogenic SCN8A mutation identified by whole-genome sequencing of a family quartet affected by infantile epileptic encephalopathy and SUDEP.

Individuals with severe, sporadic disorders of infantile onset represent an important class of disease for which discovery of the underlying genetic architecture is not amenable to traditional genetic analysis. Full-genome sequencing of affected individuals and their parents provides a powerful alternative strategy for gene discovery. We performed whole-genome sequencing (WGS) on a family quartet containing an affected proband and her unaffected parents and sibling. The 15-year-old female proband had a severe epileptic encephalopathy consisting of early-onset seizures, features of autism, intellectual disability, ataxia, and sudden unexplained death in epilepsy. We discovered a de novo heterozygous missense mutation (c.5302A>G [p.Asn1768Asp]) in the voltage-gated sodium-channel gene SCN8A in the proband. This mutation alters an evolutionarily conserved residue in Nav1.6, one of the most abundant sodium channels in the brain. Analysis of the biophysical properties of the mutant channel demonstrated a dramatic increase in persistent sodium current, incomplete channel inactivation, and a depolarizing shift in the voltage dependence of steady-state fast inactivation. Current-clamp analysis in hippocampal neurons transfected with p.Asn1768Asp channels revealed increased spontaneous firing, paroxysmal-depolarizing-shift-like complexes, and an increased firing frequency, consistent with a dominant gain-of-function phenotype in the heterozygous proband. This work identifies SCN8A as the fifth sodium-channel gene to be mutated in epilepsy and demonstrates the value of WGS for the identification of pathogenic mutations causing severe, sporadic neurological disorders.

The cultural divide: exploring communication barriers between scientists and clinicians.

Despite remarkable advances in basic biomedical science that have led to improved patient care, there is a wide and persistent gap in the abilities of researchers and clinicians to understand and appreciate each other. In this Editorial, the authors, a scientist and a clinician, discuss the rift between practitioners of laboratory research and clinical medicine. Using their first-hand experience and numerous interviews throughout the United States, they explore the causes of this 'cultural divide'. Members of both professions use advanced problem-solving skills and typically embark on their career paths with a deeply felt sense of purpose. Nonetheless, differences in classroom education, professional training environments, reward mechanisms and sources of drive contribute to obstacles that inhibit communication, mutual respect and productive collaboration. More than a sociological curiosity, the cultural divide is a significant barrier to the bench-to-bedside goals of translational medicine. Understanding its roots is the first step towards bridging the gap.

Broad Complex isoforms have unique distributions during central nervous system metamorphosis in Drosophila melanogaster.

Broad Complex (BRC) is a highly conserved, ecdysone-pathway gene essential for metamorphosis in Drosophila melanogaster, and possibly all holometabolous insects. Alternative splicing among duplicated exons produces several BRC isoforms, each with one zinc-finger DNA-binding domain (Z1, Z2, Z3, or Z4), highly expressed at the onset of metamorphosis. BRC-Z1, BRC-Z2, and BRC-Z3 represent distinct genetic functions (BRC complementation groups rbp, br, and 2Bc, respectively) and are required at discrete stages spanning final-instar larva through very young pupa. We showed previously that morphogenetic movements necessary for adult CNS maturation require BRC-Z1, -Z2, and -Z3, but not at the same time: BRC-Z1 is required in the mid-prepupa, BRC-Z2 and -Z3 are required earlier, at the larval-prepupal transition. To explore how BRC isoforms controlling the same morphogenesis events do so at different times, we examined their central nervous system (CNS) expression patterns during the approximately 16 hours bracketing the hormone-regulated start of metamorphosis. Each isoform had a unique pattern, with BRC-Z3 being the most distinctive. There was some colocalization of isoform pairs, but no three-way overlap of BRC-Z1, -Z2, and -Z3. Instead, their most prominent expression was in glia (BRC-Z1), neuroblasts (BRC-Z2), or neurons (BRC-Z3). Despite sequence similarity to BRC-Z1, BRC-Z4 was expressed in a unique subset of neurons. These data suggest a switch in BRC isoform choice, from BRC-Z2 in proliferating cells to BRC-Z1, BRC-Z3, or BRC-Z4 in differentiating cells. Together with isoform-selective temporal requirements and phenotype considerations, this cell-type-selective expression suggests a model of BRC-dependent CNS morphogenesis resulting from intercellular interactions, culminating in BRC-Z1-controlled, glia-mediated CNS movements in late prepupa.

Anciently duplicated Broad Complex exons have distinct temporal functions during tissue morphogenesis.

Broad Complex (BRC) is an essential ecdysone-pathway gene required for entry into and progression through metamorphosis in Drosophila melanogaster. Mutations of three BRC complementation groups cause numerous phenotypes, including a common suite of morphogenesis defects involving central nervous system (CNS), adult salivary glands (aSG), and male genitalia. These defects are phenocopied by the juvenile hormone mimic methoprene. Four BRC isoforms are produced by alternative splicing of a protein-binding BTB/POZ-encoding exon (BTBBRC) to one of four tandemly duplicated, DNA-binding zinc-finger-encoding exons (Z1BRC, Z2BRC, Z3BRC, Z4BRC). Highly conserved orthologs of BTBBRC and all four ZBRC were found among published cDNA sequences or genome databases from Diptera, Lepidoptera, Hymenoptera, and Coleoptera, indicating that BRC arose and underwent internal exon duplication before the split of holometabolous orders. Tramtrack subfamily members, abrupt, tramtrack, fruitless, longitudinals lacking (lola), and CG31666 were characterized throughout Holometabola and used to root phylogenetic analyses of ZBRC exons, which revealed that the ZBRC clade includes Zabrupt. All four ZBRC domains, including Z4BRC, which has no known essential function, are evolving in a manner consistent with selective constraint. We used transgenic rescue to explore how different BRC isoforms contribute to shared tissue-morphogenesis functions. As predicted from earlier studies, the common CNS and aSG phenotypes were rescued by BRC-Z1 in rbp mutants, BRC-Z2 in br mutants, and BRC-Z3 in 2Bc mutants. However, the isoforms are required at two different developmental stages, with BRC-Z2 and -Z3 required earlier than BRC-Z1. The sequential action of BRC isoforms indicates subfunctionalization of duplicated ZBRC exons even when they contribute to common developmental processes.

NeuronMetrics: software for semi-automated processing of cultured neuron images.

Using primary cell culture to screen for changes in neuronal morphology requires specialized analysis software. We developed NeuronMetrics for semi-automated, quantitative analysis of two-dimensional (2D) images of fluorescently labeled cultured neurons. It skeletonizes the neuron image using two complementary image-processing techniques, capturing fine terminal neurites with high fidelity. An algorithm was devised to span wide gaps in the skeleton. NeuronMetrics uses a novel strategy based on geometric features called faces to extract a branch number estimate from complex arbors with numerous neurite-to-neurite contacts, without creating a precise, contact-free representation of the neurite arbor. It estimates total neurite length, branch number, primary neurite number, territory (the area of the convex polygon bounding the skeleton and cell body), and Polarity Index (a measure of neuronal polarity). These parameters provide fundamental information about the size and shape of neurite arbors, which are critical factors for neuronal function. NeuronMetrics streamlines optional manual tasks such as removing noise, isolating the largest primary neurite, and correcting length for self-fasciculating neurites. Numeric data are output in a single text file, readily imported into other applications for further analysis. Written as modules for ImageJ, NeuronMetrics provides practical analysis tools that are easy to use and support batch processing. Depending on the need for manual intervention, processing time for a batch of approximately 60 2D images is 1.0-2.5 h, from a folder of images to a table of numeric data. NeuronMetrics' output accelerates the quantitative detection of mutations and chemical compounds that alter neurite morphology in vitro, and will contribute to the use of cultured neurons for drug discovery.

Phenotypes of Drosophila brain neurons in primary culture reveal a role for fascin in neurite shape and trajectory.

Subtle cellular phenotypes in the CNS may evade detection by routine histopathology. Here, we demonstrate the value of primary culture for revealing genetically determined neuronal phenotypes at high resolution. Gamma neurons of Drosophila melanogaster mushroom bodies (MBs) are remodeled during metamorphosis under the control of the steroid hormone 20-hydroxyecdysone (20E). In vitro, wild-type gamma neurons retain characteristic morphogenetic features, notably a single axon-like dominant primary process and an arbor of short dendrite-like processes, as determined with microtubule-polarity markers. We found three distinct genetically determined phenotypes of cultured neurons from grossly normal brains, suggesting that subtle in vivo attributes are unmasked and amplified in vitro. First, the neurite outgrowth response to 20E is sexually dimorphic, being much greater in female than in male gamma neurons. Second, the gamma neuron-specific "naked runt" phenotype results from transgenic insertion of an MB-specific promoter. Third, the recessive, pan-neuronal "filagree" phenotype maps to singed, which encodes the actin-bundling protein fascin. Fascin deficiency does not impair the 20E response, but neurites fail to maintain their normal, nearly straight trajectory, instead forming curls and hooks. This is accompanied by abnormally distributed filamentous actin. This is the first demonstration of fascin function in neuronal morphogenesis. Our findings, along with the regulation of human Fascin1 (OMIM 602689) by CREB (cAMP response element-binding protein) binding protein, suggest FSCN1 as a candidate gene for developmental brain disorders. We developed an automated method of computing neurite curvature and classifying neurons based on curvature phenotype. This will facilitate detection of genetic and pharmacological modifiers of neuronal defects resulting from fascin deficiency.

Interaction between hormonal signaling pathways in Drosophila melanogaster as revealed by genetic interaction between methoprene-tolerant and broad-complex.

Juvenile hormone (JH) regulates insect development by a poorly understood mechanism. Application of JH agonist insecticides to Drosophila melanogaster during the ecdysone-driven onset of metamorphosis results in lethality and specific morphogenetic defects, some of which resemble those in mutants of the ecdysone-regulated Broad-Complex (BR-C). The Methoprene-tolerant (Met) bHLH-PAS gene mediates JH action, and Met mutations protect against the lethality and defects. To explore relationships among these two genes and JH, double mutants were constructed between Met alleles and alleles of each of the BR-C complementation groups: broad (br), reduced bristles on palpus (rbp), and 2Bc. Defects in viability and oogenesis were consistently more severe in rbp Met or br Met double mutants than would be expected if these genes act independently. Additionally, complementation between BR-C mutant alleles often failed when MET was absent. Patterns of BRC protein accumulation during metamorphosis revealed essentially no difference between wild-type and Met-null individuals. JH agonist treatment did not block accumulation of BRC proteins. We propose that MET and BRC interact to control transcription of one or more downstream effector genes, which can be disrupted either by mutations in Met or BR-C or by application of JH/JH agonist, which alters MET interaction with BRC.

Discrete pulses of molting hormone, 20-hydroxyecdysone, during late larval development of Drosophila melanogaster: correlations with changes in gene activity.

Periodic pulses of the insect steroid molting hormone 20-hydroxyecdysone (20E), acting via its nuclear receptor complex (EcR/USP), control gene expression at many stages throughout Drosophila development. However, during the last larval instar of some lepidopteran insects, subtle changes in titers of ecdysteroids have been documented, including the so-called "commitment peak." This small elevation of 20E reprograms the larva for metamorphosis to the pupa. Similar periods of ecdysteroid immunoreactivity have been observed during the last larval instar of Drosophila. However, due to low amplitude and short duration, along with small body size and staging difficulties, their timing and ecdysteroid composition have remained uncertain. Employing a rigorous regimen of Drosophila culture and a salivary gland reporter gene, Sgs3-GFP, we used RP-HPLC and differential ecdysteroid RIA analysis to determine whole body titers of 20E during the last larval instar. Three small peaks of 20E were observed at 8, 20, and 28 hr following ecdysis, prior to the well-characterized large peak around the time of pupariation. The possible regulation of 20E levels by biosynthetic P450 enzymes and the roles of these early peaks in coordinating gene expression and late larval development are discussed.

Mental retardation genes in drosophila: New approaches to understanding and treating developmental brain disorders.

Drosophila melanogaster is emerging as a valuable genetic model system for the study of mental retardation (MR). MR genes are remarkably similar between humans and fruit flies. Cognitive behavioral assays can detect reductions in learning and memory in flies with mutations in MR genes. Neuroanatomical methods, including some at single-neuron resolution, are helping to reveal the cellular bases of faulty brain development caused by MR gene mutations. Drosophila fragile X mental retardation 1 (dfmr1) is the fly counterpart of the human gene whose malfunction causes fragile X syndrome. Research on the fly gene is leading the field in molecular mechanisms of the gene product's biological function and in pharmacological rescue of brain and behavioral phenotypes. Future work holds the promise of using genetic pathway analysis and primary neuronal culture methods in Drosophila as tools for drug discovery for a wide range of MR and related disorders.

The steroid hormone-regulated gene Broad Complex is required for dendritic growth of motoneurons during metamorphosis of Drosophila.

Dendrites are subject to subtle modifications as well as extensive remodeling during the assembly and maturation of neural circuits in a wide variety of organisms. During metamorphosis, Drosophila flight motoneurons MN1-MN4 undergo dendritic regression, followed by regrowth, whereas MN5 differentiates de novo (Consoulas et al. [2002] J. Neurosci. 22:4906-4917). Many cellular changes during metamorphosis are triggered and orchestrated by the steroid hormone 20-hydroxyecdysone, which initiates a cascade of coordinated gene expression. Broad Complex (BRC), a primary response gene in the ecdysone cascade, encodes a family of transcription factors (BRC-Z1-Z4) that are essential for metamorphic reorganization of the central nervous system (CNS). Using neuron-filling techniques that reveal cellular morphology with very high resolution, we tested the hypothesis that BRC is required for metamorphic development of MN1-MN5. Through a combination of loss-of-function mutant analyses, genetic mapping, and transgenic rescue experiments, we found that 2Bc function, mediated by BRC-Z3, is required selectively for motoneuron dendritic regrowth (MN1-MN4) and de novo outgrowth (MN5), as well as for soma expansion of MN5. In contrast, larval development and dendritic regression of MN1-MN4 are BRC-independent. Surprisingly, BRC proteins are not expressed in the motoneurons, suggesting that BRC-Z3 exerts its effect in a non-cell-autonomous manner. The 2Bc mutants display no gross defects in overall thoracic CNS structure, or in peripheral structures such as target muscles or sensory neurons. Candidates for mediating the effect of BRC-Z3 on dendritic growth of MN1-MN5 include their synaptic inputs and non-neuronal CNS cells that interact with them through direct contact or diffusible factors.

Drosophila melanogaster CYP6A8, an insect P450 that catalyzes lauric acid (omega-1)-hydroxylation.

Only a handful of P450 genes have been functionally characterized from the approximately 90 recently identified in the genome of Drosophila melanogaster. Cyp6a8 encodes a 506-amino acid protein with 53.6% amino acid identity with CYP6A2. CYP6A2 has been shown to catalyze the metabolism of several insecticides including aldrin and heptachlor. CYP6A8 is expressed at many developmental stages as well as in adult life. CYP6A8 was produced in Saccharomyces cerevisiae and enzymatically characterized after catalytic activity was reconstituted with D. melanogaster P450 reductase and NADPH. Although several saturated or non-saturated fatty acids were not metabolized by CYP6A8, lauric acid (C12:0), a short-chain unsaturated fatty acid, was oxidized by CYP6A8 to produce 11-hydroxylauric acid with an apparent V(max) of 25 nmol/min/nmol P450. This is the first report showing that a member of the CYP6 family catalyzes the hydroxylation of lauric acid. Our data open new prospects for the CYP6 P450 enzymes, which could be involved in important physiological functions through fatty acid metabolism.

Defective neuronal development in the mushroom bodies of Drosophila fragile X mental retardation 1 mutants.

Fragile X mental retardation 1 (Fmr1) is a highly conserved gene with major roles in CNS structure and function. Its product, the RNA-binding protein FMRP, is believed to regulate translation of specific transcripts at postsynaptic sites in an activity-dependent manner. Hence, Fmr1 is central to the molecular mechanisms of synaptic plasticity required for normal neuronal maturation and cognitive ability. Mutations in its Drosophila ortholog, dfmr1, produce phenotypes of brain interneurons and axon terminals at the neuromuscular junction, as well as behavioral defects of circadian rhythms and courtship. We hypothesized that dfmr1 mutations would disrupt morphology of the mushroom bodies (MBs), highly plastic brain regions essential for many forms of learning and memory. We found developmental defects of MB lobe morphogenesis, of which the most common is a failure of beta lobes to stop at the brain midline. A similar recessive beta-lobe midline-crossing phenotype has been previously reported in the memory mutant linotte. The dfmr1 MB defects are highly sensitive to genetic background, which is reminiscent of mammalian fragile-X phenotypes. Mutations of dfmr1 also interact with one or more third-chromosome loci to promote alpha/beta-lobe maturation. These data further support the use of the Drosophila model system for study of hereditary cognitive disorders of humans.