Linda L Restifo

Interests

Research

Developmental neurogenetics;Developmental brain disorders;Neurotoxicity;Human genetic disease;Technology development in cellular neuroscience;Drug discovery

Teaching

Human genetic disease;History of genetic technology;Translational medical research;Cultural divide between science and medicine

Courses

Scholarly Contributions

Journals/Publications

• Restifo, L. L., & Erickson, E. P. (2023).

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

  . SAGE Open Medical Case Reports, 11. doi:10.1177/2050313X231194507
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  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.
• Tello, J. A., Jiang, L., Yitshak, Z., & Restifo, L. L. (2023). Drosophila CASK regulates brain size and neuronal morphogenesis, providing a genetic model of postnatal microcephaly suitable for drug discovery. Neurobiology of Disease.
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  To enhance the value of this report, which had originally contained only the results from two chapters of my former doctoral student's dissertation, we have added a collaborative component with colleagues in Engineering. Together, we developed a method for automated dissociation of fresh CNS tissue into single viable cells for primary neuronal culture. This addition has delayed preparation of the manuscript, but as of January 2023, it is ready to submit pending final review by our engineering colleagues.
• Restifo, L. L. (2022).

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

  . Genetics, 220(1). doi:10.1093/genetics/iyab185
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  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.
• Restifo, L. L. (2022). Unraveling the Gordian knot: genetics and the troubled road to effective therapeutics for Alzheimer's disease. GENETICS, 220(1), iyab185. doi:DOI: 10.1093/genetics/iyab185
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  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.
• Shanmugaundaram, V., Bajorath, J., Christoffersen, R., Petke, J. D., Howe, W. J., Johnson, M., Agrafiotis, D., Lee, P., Kuhn, L., Goodwin, J. T., Holloway, M. K., Doman, T. N., Walters, W. P., Schreyer, S., Medina-Franco, J., Martinez-Mayorga, K., & Restifo, L. L. (2022). Epilogue to the Gerald Maggiora Festschrift: a tribute to an exemplary mentor, colleague, collaborator, and innovator. . Journal of Computer-Aided Molecular Design, 36(9), 623-638. doi:doi: 10.1007/s10822-022-00473-2
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  Abstract: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.
• Andrew, D. R., Moe, M. E., Chen, D., Tello, J. A., Doser, R. L., Conner, W. E., Ghuman, J. K., & Restifo, L. L. (2021). Spontaneous motor-behavior abnormalities in two Drosophila models of neurodevelopmental disorders.. Journal of Neurogenetics, 35(1), 1-22. doi:10.1080/01677063.2020.1833005
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  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.
• Jiang, L., Restifo, L. L., & Zohar, Y. (2015). Dissociation of brain tissue into viable single neurons in a microfluidic device.. IEEE Nano/Molecular Medicine and Engineering, 9, 29-32.
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  A microfluidic technology-based tissue-dissociation device has for the first time been designed, fabricated and characterized for the purpose of primary neuronal cell culture.The system has been utilized for controlled dissociation, under an oscillatory flow field, of freshly explanted, enzyme-treated Drosophila larval central nervous system (CNS) into individual, viable neurons capable of robust outgrowth during in vitro culture. Device dimensions, constriction height and width, and operating conditions, flow-rate amplitude and frequency, have been determined based on video microscopy as well as quantitative analyses of the subsequent neuron-culture results.
• Smrt, R. D., Lewis, S. A., Kraft, R., & Restifo, L. L. (2015). Primary culture of Drosophila larval neurons with morphological analysis using NeuronMetrics.. Drosophila Information Service, 98, 125-140.
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  This is a methods paper with a concise scholarly review of the utility of primary cultured neurons for neuroscience research.
• Ito, K., Shinomiya, K., Ito, M., Armstrong, J. D., Boyan, G., Hartenstein, V., Harzsch, S., Heisenberg, M., Homberg, U., Jenett, A., Keshishian, H., Restifo, L. L., Rössler, W., Simpson, J. H., Strausfeld, N. J., Strauss, R., Vosshall, L. B., & , I. B. (2014). A systematic nomenclature for the insect brain. Neuron, 81(4), 755-65.
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  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.
• Hammer, M. F., Restifo, L. L., Sprissler, R., Veeramah, K. R., Johnstone, L., Karafet, T. M., Wolf, D., Salogiannis, J., Barth-Maron, A., Greenberg, M. E., Stuhlmann, T., Weinert, S., Jentsch, T. J., Pazzi, M., Talwar, D., & Erickson, R. P. (2013). Exome sequencing reveals new causal mutations in children with epileptic encephalopathies. Epilepsia, 54(7), 1270-1281. doi:10.1111/epi.12201
• Restifo, L. L., Kraft, R., Kahn, A., Medina-Franco, J. L., Orlowski, M. L., Baynes, C., López-Vallejo, F., Barnard, K., & Maggiora, G. M. (2013). A cell-based fascin bioassay identifies compounds with potential anti-metastasis or cognition-enhancing functions. Disease Models & Mechanisms, 6(1), 217-235.
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  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.
• Veeramah, K. R., Johnstone, L., Karafet, T. M., Wolf, D., Sprissler, R., Salogiannis, J., Barth-Maron, A., Greenberg, M. E., Stuhlmann, T., Weinert, S., Jentsch, T. J., Pazzi, M., Restifo, L. L., Talwar, D., Erickson, R. P., & Hammer, M. F. (2013). Exome sequencing reveals new causal mutations in children with epileptic encephalopathies. Epilepsia, 54(7), 1270-81.
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  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.
• Restifo, L. L., Baynes, C., Kraft, R., Kahn, A., Medina-Franco, J. L., Orlowski, M. L., López-Vallejo, F., Barnard, K., & Maggiora, G. M. (2012). A cell-based fascin bioassay identifies compounds with potential anti-metastasis or cognition-enhancing functions. Disease Models & Mechanisms, Epub prior to print publication. doi:10.1242/dmm.008243
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  See the final publication in 2013.
• Veeramah, K. R., O'Brien, J. E., Meisler, M. H., Cheng, X., Dib-Hajj, S. D., Waxman, S. G., Talwar, D., Girirajan, S., Eichler, E. E., Restifo, L. L., Erickson, R. P., & Hammer, M. F. (2012). De novo pathogenic SCN8A mutation identified by whole-genome sequencing of a family quartet affected by infantile epileptic encephalopathy and SUDEP. American Journal of Human Genetics, 90(3), 502-10.
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  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.
• Kim, S., Jeong, J., Restifo, L. L., & Kwon, H. (2011). Drosophila as a model system for studying lifespan and neuroprotective activities of plant-derived compounds. Journal of Asia-Pacific Entomology, 14(4), 509-517.
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  The fruit fly, Drosophila melanogaster, has been intensively used as a genetic model system for basic and applied research on human neurological diseases because of advantages over mammalian model systems such as ease of laboratory maintenance and genetic manipulations. Disease-associated gene mutations, whether endogenous or transgenically-inserted, often cause phenotypes in vivo that are similar to the clinical features of the human disorder. The Drosophila genome is simpler than that of mammals, in terms of gene and chromosome number, but nonetheless demonstrates extraordinary phylogenetic conservation of gene structure and function, especially notable among the genes whose mutations cause neurodevelopmental, neuropsychiatric, or neurodegenerative disorders. In addition, its well-established neuroanatomical, developmental, and molecular genetic research techniques allow many laboratories worldwide to study complex biological and genetic processes. Based on these merits of the Drosophila model system, it has been used for screening lifespan expansion and neuroprotective activities of plant extracts or their secondary metabolites to counteract pathological events such as mitochondrial damage by oxidative stress, which may cause sporadic neurodegenerative diseases. In this review, we have summarized that the fruit fly can be used for early-stage drug discovery and development to identify novel plant-derived compounds to protect against neurodegeneration in Alzheimer's disease and Parkinson's disease, and other neurological disorders caused by oxidative stress. Thus, the Drosophila system can directly or indirectly contribute to translational research for new therapeutic strategies to prevent or ameliorate neurodegenerative diseases.
• Restifo, L. L., & Phelan, G. R. (2011). The cultural divide: exploring communication barriers between scientists and clinicians. Disease Models & Mechanisms, 4(4), 423-6.
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  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.
• Halladay, A. K., Amaral, D., Aschner, M., Bolivar, V. J., Bowman, A., DiCicco-Bloom, E., Hyman, S. L., Keller, F., Lein, P., Pessah, I., Restifo, L., & Threadgill, D. W. (2009). Animal models of autism spectrum disorders: information for neurotoxicologists. Neurotoxicology, 30(5), 811-21.
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  Recent findings derived from large-scale datasets and biobanks link multiple genes to autism spectrum disorders. Consequently, novel rodent mutants with deletions, truncations and in some cases, overexpression of these candidate genes have been developed and studied both behaviorally and biologically. At the Annual Neurotoxicology Meeting in Rochester, NY in October of 2008, a symposium of clinicians and basic scientists gathered to present the behavioral features of autism, as well as strategies to model those behavioral features in mice and primates. The aim of the symposium was to provide researchers with up-to-date information on both the genetics of autism and how they are used in differing in vivo and in vitro animal models as well as to provide a background on the environmental exposures being tested on several animal models. In addition, researchers utilizing complementary approaches, presented on cell culture, in vitro or more basic models, which target neurobiological mechanisms, including Drosophila. Following the presentation, a panel convened to explore the opportunities and challenges of using model systems to investigate genetic and environment interactions in autism spectrum disorders. The following paper represents a summary of each presentation, as well as the discussion that followed at the end of the symposium.
• Spokony, R. F., & Restifo, L. L. (2009). Broad Complex isoforms have unique distributions during central nervous system metamorphosis in Drosophila melanogaster. Journal of Comparative Neurology, 517(1), 15-36.
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  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.
• Restifo, L., Narro, M. L., Yang, F., Kraft, R., Wenk, C., Efrat, A., & Restifo, L. L. (2007). NeuronMetrics: software for semi-automated processing of cultured neuron images. Brain research, 1138.
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  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.
• Spokony, R. F., & Restifo, L. L. (2007). Anciently duplicated Broad Complex exons have distinct temporal functions during tissue morphogenesis. Development Genes and Evolution, 217(7).
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  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.
• Kraft, R., Escobar, M. M., Narro, M. L., Kurtis, J. L., Efrat, A., Barnard, K., & Restifo, L. L. (2006). Phenotypes of Drosophila brain neurons in primary culture reveal a role for fascin in neurite shape and trajectory. Journal of Neuroscience, 26(34), 8734-8747.
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  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.
• Warren, J. T., Yerushalmi, Y., Shimell, M. J., O'connor, M. B., Restifo, L. L., & Gilbert, L. I. (2006). Discrete pulses of molting hormone, 20-hydroxyecdysone, during late larval development of Drosophila melanogaster: correlations with changes in gene activity.. Developmental Dynamics, 235(2), 315-26. doi:10.1002/dvdy.20626
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  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.
• Wilson, T. G., Yerushalmi, Y., Donnell, D. M., & Restifo, L. L. (2006). Interaction between hormonal signaling pathways in Drosophila melanogaster as revealed by genetic interaction between methoprene-tolerant and broad-complex.. Genetics, 172(1), 253-64. doi:10.1534/genetics.105.046631
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  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.
• Restifo, L. L. (2005). Mental retardation genes in Drosophila: New approaches to understanding and treating developmental brain disorders. Mental Retardation and Developmental Disabilities Research Reviews, 11(4), 286-294.
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  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.
• Restifo, L., Consoulas, C., Levine, R. B., & Restifo, L. L. (2005). The steroid hormone-regulated gene Broad Complex is required for dendritic growth of motoneurons during metamorphosis of Drosophila. The Journal of comparative neurology, 485(4).
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  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.
• Restifo, L. L., & Inlow, J. K. (2004). Molecular and Comparative Genetics of Mental Retardation. Genetics, 166(2), 835-881. doi:10.1093/genetics/166.2.835
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  Abstract Affecting 1-3% of the population, mental retardation (MR) poses significant challenges for clinicians and scientists. Understanding the biology of MR is complicated by the extraordinary heterogeneity of genetic MR disorders. Detailed analyses of >1000 Online Mendelian Inheritance in Man (OMIM) database entries and literature searches through September 2003 revealed 282 molecularly identified MR genes. We estimate that hundreds more MR genes remain to be identified. A novel test, in which we distributed unmapped MR disorders proportionately across the autosomes, failed to eliminate the well-known X-chromosome overrepresentation of MR genes and candidate genes. This evidence argues against ascertainment bias as the main cause of the skewed distribution. On the basis of a synthesis of clinical and laboratory data, we developed a biological functions classification scheme for MR genes. Metabolic pathways, signaling pathways, and transcription are the most common functions, but numerous other aspects of neuronal and glial biology are controlled by MR genes as well. Using protein sequence and domain-organization comparisons, we found a striking conservation of MR genes and genetic pathways across the approximately 700 million years that separate Homo sapiens and Drosophila melanogaster. Eighty-seven percent have one or more fruit fly homologs and 76% have at least one candidate functional ortholog. We propose that D. melanogaster can be used in a systematic manner to study MR and possibly to develop bioassays for therapeutic drug discovery. We selected 42 Drosophila orthologs as most likely to reveal molecular and cellular mechanisms of nervous system development or plasticity relevant to MR.  
• Restifo, L. L., Michel, C. I., & Kraft, R. (2004). Defective neuronal development in the mushroom bodies of Drosophila fragile X mental retardation 1 mutants. Journal of Neuroscience, 24(25), 5798-5809.
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  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.
• Restifo, L., Helvig, C., Tijet, N., Feyereisen, R., Walker, F. A., & Restifo, L. L. (2004). Drosophila melanogaster CYP6A8, an insect P450 that catalyzes lauric acid (omega-1)-hydroxylation. Biochemical and biophysical research communications, 325(4).
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  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.
• Restifo, L., Inlow, J. K., & Restifo, L. L. (2004). Molecular and comparative genetics of mental retardation. Genetics, 166(2).
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  Affecting 1-3% of the population, mental retardation (MR) poses significant challenges for clinicians and scientists. Understanding the biology of MR is complicated by the extraordinary heterogeneity of genetic MR disorders. Detailed analyses of >1000 Online Mendelian Inheritance in Man (OMIM) database entries and literature searches through September 2003 revealed 282 molecularly identified MR genes. We estimate that hundreds more MR genes remain to be identified. A novel test, in which we distributed unmapped MR disorders proportionately across the autosomes, failed to eliminate the well-known X-chromosome overrepresentation of MR genes and candidate genes. This evidence argues against ascertainment bias as the main cause of the skewed distribution. On the basis of a synthesis of clinical and laboratory data, we developed a biological functions classification scheme for MR genes. Metabolic pathways, signaling pathways, and transcription are the most common functions, but numerous other aspects of neuronal and glial biology are controlled by MR genes as well. Using protein sequence and domain-organization comparisons, we found a striking conservation of MR genes and genetic pathways across the approximately 700 million years that separate Homo sapiens and Drosophila melanogaster. Eighty-seven percent have one or more fruit fly homologs and 76% have at least one candidate functional ortholog. We propose that D. melanogaster can be used in a systematic manner to study MR and possibly to develop bioassays for therapeutic drug discovery. We selected 42 Drosophila orthologs as most likely to reveal molecular and cellular mechanisms of nervous system development or plasticity relevant to MR.
• Consoulas, C., Restifo, L. L., & Levine, R. B. (2002). Dendritic remodeling and growth of motoneurons during metamorphosis of Drosophila melanogaster.. The Journal of Neuroscience, 22(12), 4906-4917. doi:10.1523/jneurosci.22-12-04906.2002
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  Insect motoneurons display dramatic dendritic plasticity during metamorphosis. Many larval motoneurons survive to adulthood but undergo dendritic regression and outgrowth as they are incorporated into developing circuits. This study explores the dendritic remodeling and development of Drosophila motoneurons MN1–MN5, which innervate indirect flight muscles of the adult. MN1–MN5 are persistent larval neurons exhibiting two distinct metamorphic histories. MN1–MN4 are born in the embryo, innervate larval muscles, and undergo dendritic regression and regrowth during metamorphosis. MN5, which was identified through a combination of intracellular dye injection and retrograde staining at all stages, is also born embryonically but remains developmentally arrested until the onset of metamorphosis. In the larva, MN5 lacks dendrites, and its axon stops in the mesothoracic nerve without innervating a target muscle. It is dye coupled to the peripherally synapsing interneuron, which will become part of the giant fiber escape circuit of the adult fly. During pupal development, MN5 undergoes de novo dendritic growth and extension of its axon to innervate the developing target muscle. Its unique developmental history and identifiability make MN5 well suited for the study of dendritic growth using genetic and neurophysiological approaches.
• Sandstrom, D. J., & Restifo, L. L. (1999). Epidermal tendon cells require Broad Complex function for correct attachment of the indirect flight muscles in Drosophila melanogaster.. Journal of Cell Science, 112(22), 4051-4065. doi:10.1242/jcs.112.22.4051
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  Drosophila Broad Complex, a primary response gene in the ecdysone cascade, encodes a family of zinc-finger transcription factors essential for metamorphosis. Broad Complex mutations of the rbp complementation group disrupt attachment of the dorsoventral indirect flight muscles during pupal development. We previously demonstrated that isoform BRC-Z1 mediates the muscle attachment function of rbp(+) and is expressed in both developing muscle fibers and their epidermal attachment sites. We now report two complementary studies to determine the cellular site and mode of action of rbp(+) during maturation of the myotendinous junctions of dorsoventral indirect flight muscles. First, genetic mosaics, produced using the paternal loss method, revealed that the muscle attachment phenotype is determined primarily by the genotype of the dorsal epidermis, with the muscle fiber and the ventral epidermis exerting little or no influence. When the dorsal epidermis was mutant, the vast majority of muscles detached or chose ectopic attachment sites, regardless of the muscle genotype. Conversely, wild-type dorsal epidermis could support attachment of mutant muscles. Second, ultrastructural analysis corroborated and extended these results, revealing defective and delayed differentiation of rbp mutant epidermal tendon cells in the dorsal attachment sites. Tendon cell processes, the stress-bearing links between the epidermis and muscle, were reduced in number and showed delayed appearance of microtubule bundles. In contrast, mutant muscle and ventral epidermis resembled the wild type. In conclusion, BRC-Z1 acts in the dorsal epidermis to ensure differentiation of the myotendinous junction. By analogy with the cell-cell interaction essential for embryonic muscle attachment, we propose that BRC-Z1 regulates one or more components of the epidermal response to a signal from the developing muscle.
• Kraft, R., Levine, R. B., Levine, R. B., Restifo, L. L., & Restifo, L. L. (1998). The steroid hormone 20-hydroxyecdysone enhances neurite growth of Drosophila mushroom body neurons isolated during metamorphosis.. Journal of Neuroscience, 18(21), 8886-8899. doi:10.1523/jneurosci.18-21-08886.1998
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  Mushroom bodies (MBs) are symmetrically paired neuropils in the insect brain that are of critical importance for associative olfactory learning and memory. In Drosophila melanogaster, the MB intrinsic neurons (Kenyon cells) undergo extensive reorganization at the onset of metamorphosis. A phase of rapid axonal degeneration without cell death is followed by axonal regeneration. This re-elaboration occurs as levels of the steroid hormone 20-hydroxyecdysone (20E) are rising during the pupal stage. Based on the known role of 20E in directing many features of CNS remodeling during insect metamorphosis, we hypothesized that the outgrowth of MB axonal processes is promoted by 20E. Using a GAL4 enhancer trap line (201Y) that drives MB-restricted reporter gene expression, we identified Kenyon cells in primary cultures dissociated from early pupal CNS. Paired cultures derived from single brains isolated before the 20E pupal peak were incubated in medium with or without 20E for 2-4 d. Morphometric analysis demonstrated that MB neurons exposed to 20E had significantly greater total neurite length and branch number compared with that of MB neurons grown without hormone. The relationship between branch number and total neurite length remained constant regardless of hormone treatment in vitro, suggesting that 20E enhances the rate of outgrowth from pupal MB neurons in a proportionate manner and does not selectively increase neuritic branching. These results implicate 20E in enhancing axonal outgrowth of Kenyon cells to support MB remodeling during metamorphosis.
• Liu, E., & Restifo, L. L. (1998). Identification of a Broad Complex-regulated enhancer in the developing visual system of Drosophila.. Journal of Neurobiology, 34(3), 253-70. doi:10.1002/(sici)1097-4695(19980215)34:3<253::aid-neu5>3.0.co;2-1
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  During metamorphosis, the central nervous system (CNS) is reconstructed through the concerted action of cell birth, death, and remodeling, so that it can serve the novel and complex behavioral needs of the adult insect. In Drosophila, Broad Complex (BRC) zinc-finger transcription factors are essential for many aspects of metamorphosis, including reorganization of the CNS. In particular, we showed previously that some mutant alleles disrupt the assembly of visual system synaptic neuropils. Using an enhancer-detector screen, we have now identified a candidate BRC target gene, H217, that is normally expressed in visual system neural precursor cells of the inner proliferative center. Moreover, the P-element insertion in the H217 line has caused a hypomorphic mutation in an essential gene, with an optic lobe disorganization phenotype very similar to that seen in BRC mutants. In BRC mutants of the br complementation group (but not in rbp or 2Bc mutants), the H217 enhancer is ectopically expressed in lamina precursor cells (LPCs) whose proliferation is regulated by signals from photoreceptor axons. As predicted by the current model of BRC structure-function relationships, we demonstrated that BRC-Z2 isoforms, when induced during the third larval instar, can repress H217 enhancer activity in the LPCs, whereas BRC-Z3 cannot. Taken together, the data suggest that the H217 P element has tagged an essential gene repressed by BRC-Z2 in LPCs and required for the normal architecture of the retinotopically connected visual system neuropils.
• Restifo, L. L., & Hauglum, W. (1998). Parallel molecular genetic pathways operate during CNS metamorphosis in Drosophila.. Molecular and Cellular Neurosciences, 11(3), 134-48. doi:10.1006/mcne.1998.0683
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  Insect metamorphosis provides a valuable model for studying mechanisms of steroid hormone action on the nervous system during a dynamic phase of functional remodeling. The Drosophila Broad Complex (BRC) holds a pivotal position in the gene expression cascade triggered by the molting hormone 20-hydroxyecdysone (20E) at the onset of metamorphosis. We previously demonstrated that the BRC, which encodes a family of zinc-finger transcription factors, is essential for transducing 20E signals into the morphogenetic movements and cellular assembly that alter the CNS from juvenile to adult form and function. We set out to examine the relationship of BRC to two other genes, IMP-E1 and Deformed (Dfd), involved in the metamorphic transition of the CNS. Representatives of the whole family of BRC transcript isoforms accumulate in the CNS during the larval-to-pupal transition and respond directly to 20E in vitro. IMP-E1 is also directly regulated by 20E, but its induction is independent of BRC, revealing that 20E works through at least two pathways in the CNS. DFD expression is also independent of BRC function. Surprisingly, BRC and DFD proteins are expressed in distinct, nonoverlapping subsets of neuronal nuclei of the subesophageal ganglion even though both are required for its migration into the head capsule. This suggests that the segment identity and ecdysone cascades operate in parallel to control region-specific reorganization during metamorphosis.
• Restifo, L. L., & Wilson, T. G. (1998). A juvenile hormone agonist reveals distinct developmental pathways mediated by ecdysone-inducible broad complex transcription factors.. Developmental genetics, 22(2), 141-59. doi:10.1002/(sici)1520-6408(1998)22:2<141::aid-dvg4>3.0.co;2-6
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  Juvenile hormone (JH) is an important regulator of insect development that, by unknown mechanisms, modifies molecular, cellular, and organismal responses to the molting hormone, 20-hydroxyecdysone (20E). In dipteran insects such as Drosophila, JH or JH agonists, administered at times near the onset of metamorphosis, cause lethality. We tested the hypothesis that the JH agonist methoprene acts by interfering with function of the Broad Complex (BRC), a 20E-regulated locus encoding BTB/POZ-zinc finger transcription factors essential for metamorphosis of many tissues. We found that methoprene, administered by feeding or by topical application, disrupts the metamorphic reorganization of the central nervous system, salivary glands, and musculature in a dose-dependent manner. As we predicted, methoprene phenocopies a subset of previously described BRC defects; it also phenocopies Deformed and produces abnormalities not associated with known mutations. Interestingly, methoprene specifically disrupts those metamorphic events dependent on the combined action of all BRC isoforms, while sparing those that require specific isoform subsets. Thus, our data provide independent pharmacological evidence for the model, originally based on genetic studies, that BRC proteins function in two developmental pathways. Mutations of Methoprene-tolerant (Met), a gene involved in the action of JH, protect against all features of the "methoprene syndrome." These findings have allowed us to propose novel alternative models linking BRC, juvenile hormone, and MET.
• Sandstrom, D. J., Bayer, C. A., Fristrom, J. W., & Restifo, L. L. (1997). Broad-complex transcription factors regulate thoracic muscle attachment in Drosophila.. Developmental biology, 181(2), 168-85. doi:10.1006/dbio.1996.8469
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  The Broad-Complex, a 20-hydroxyecdysone-regulated gene, is essential for the development of many tissues during metamorphosis. In Broad-Complex mutants of the rbp complementation group, dorsoventral indirect flight muscles (DVM) are largely absent, and the dorsal longitudinal indirect flight muscles, tergotrochanteral muscles, and remaining DVM often select incorrect attachment sites. The Broad-Complex encodes a family of zinc-finger-containing transcription factors, and it is hypothesized that Broad Complex proteins containing the Z1 zinc-finger pair (BRC-Z1) mediate rbp+ function. We provide additional strong support for this hypothesis by showing that heat-shock-induced BRC-Z1 expression rescues the thoracic muscle defects of rbp mutants completely. BRC-Z4 induction can also rescue the thoracic musculature, but BRC-Z2 and -Z3 can not. Thus, the effect is specific to BRC-Z1 and its closest relative, BRC-Z4. Formation of muscle primordia from imaginal myoblasts appears normal in rbp mutants. However, the myotendinous junctions linking the DVM to the dorsal epidermis are weak, and the muscles detach during pupal life and subsequently degenerate. The data indicate that rbp mutations disrupt the cell-cell interactions between developing muscles and epidermal tendon cells as they recognize and attach to one another. Using a BRC-Z1-specific monoclonal antibody, we show that both the developing muscles and epidermal tendon cells express BRC-Z1 at the time of pupation, before mutant muscles begin to detach. We conclude that 20-hydroxyecdysone acts through the Broad-Complex to control the development of thoracic myotendinous junctions.
• Levine, R. B., Levine, R. B., Morton, D. B., Restifo, L. L., & Restifo, L. L. (1995). Remodeling of the insect nervous system.. Current Opinion in Neurobiology, 5(1), 28-35. doi:10.1016/0959-4388(95)80083-2
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  Our nervous systems and behavior are shaped by hormonally driven developmental changes that continue beyond the embryonic period. Key insights into this process have emerged from studies of the insect nervous system. During insect metamorphosis, the nervous system is remodeled through postembryonic neurogenesis, programmed cell death and the modification of persistent neurons. These changes are regulated to a large degree by gene cascades that are triggered by steroid hormones, the ecdysteroids. Current studies are attempting to reveal the molecular mechanisms involved in regulating these dramatic examples of developmental plasticity.
• Restifo, L. L., & Merrill, V. K. (1994). Two Drosophila regulatory genes, Deformed and the Broad-Complex, share common functions in development of adult CNS, head, and salivary glands.. Developmental Biology, 162(2), 465-85. doi:10.1006/dbio.1994.1102
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  Deformed (Dfd), a homeotic selector gene required for segment identity in the head, and the Broad-Complex (BR-C), a steroid hormone-regulated locus required for metamorphosis of the epidermis and multiple internal tissues, are members of distinct genetic regulatory hierarchies. Their protein products contain DNA-binding domains (of the homeodomain and zinc-finger variety, respectively) and are believed to act by regulating the transcription of target genes. In this study we demonstrate that Dfd and BR-C mutants dying during metamorphosis share defects of CNS reorganization, ventral adult head development, and adult salivary gland morphogenesis. Specifically, the shared phenotypes are (i) failure to separate the subesophageal ganglion (SEG) from the thoracic ganglion (TG); (ii) structural and functional abnormalities of the proboscis and maxillary palps, innervated by the SEG; and (iii) failure of the adult salivary glands to extend into the thorax. Experiments performed with a conditional allele demonstrate that Dfd+ function during either larval life or metamorphosis is sufficient to rescue the SEG-TG separation phenotype. BR-C;Dfd double mutants show synergistic enhancement of the ventral head defects. This genetic interaction suggests that the segment identity and steroid hormone-sensitive regulatory hierarchies intersect during postembryonic development.
• Restifo, L. L., & White, K. (1992). Mutations in a steroid hormone-regulated gene disrupt the metamorphosis of internal tissues in Drosophila: salivary glands, muscle, and gut.. Roux's Archives of Developmental Biology, 201(4), 221-234. doi:10.1007/bf00188753
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  In holometabolous insects, the steroid molting hormone 20-OH-ecdysone (ecdysterone) orchestrates the diverse developmental events of metamorphosis, in large part by regulating gene expression. In Drosophila, the Broad Complex (BR-C) is one of the first loci to be induced by ecdysterone at the end of larval life, and is essential for translating the hormonal signal into the behavioral and anatomical events which herald the onset of metamorphosis. BR-C products are believed to act by binding to and modifying the transcriptional activities of other hormone-sensitive genes. In addition to abnormalities of the epidermis, BR-C mutants dying during metamorphosis manifest a syndrome of multiple internal tissue defects which represent a failure of the larval-to-adult transition. We have reported features of central nervous system metamorphosis requiring BR-C function, notably morphogenetic movements and optic lobe organization. In this paper we describe defective development of salivary glands, flight muscles, and gut in BR-C mutants, including: persistence of larval salivary glands; failure of the adult salivary glands to extend into the thorax; abnormalities of midgut transition and of proventriculus structure and location; and absence of dorsal-ventral indirect flight muscles. Some of these abnormalities represent defects in programmed cell death. Distinct patterns of phenotypes were seen in mutants of each of the three lethal complementation groups comprising the BR-C. The patterns of phenotypes suggest overlapping but distinct functions encoded by this complex locus.
• Restifo, L. L., & White, K. (1991). Mutations in a steroid hormone-regulated gene disrupt the metamorphosis of the central nervous system in Drosophila.. Developmental Biology, 148(1), 174-94. doi:10.1016/0012-1606(91)90328-z
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  The actions of steroid hormones on vertebrate and invertebrate nervous systems include alterations in neuronal architecture, regulation of neuronal differentiation, and programmed cell death. In particular, central nervous system (CNS) metamorphosis in insects requires a precise pattern of exposure to the steroid molting hormone 20-hydroxyecdysone (ecdysterone). To test whether the effects of steroid hormones on the insect nervous system are due to changes in patterns of gene expression, we examined Drosophila mutants of the ecdysterone-regulated locus, the Broad Complex (BR-C). This report documents aspects of CNS reorganization which are dependent on BR-C function. During wild-type metamorphosis, CNS components undergo dramatic morphogenetic movements relative to each other and to the body wall. These movements, in particular, the separation of the subesophageal ganglion from the thoracic ganglion, the positioning of the developing visual system, and the fusion of right and left brain hemispheres, are deranged in BR-C mutants. In addition, a subset of mutants shows disorganization of optic lobe neuropil, both within and among optic lobe ganglia. Optic lobe disorganization is found in mutants of the br and l(1)2Bc complementation groups, but not in those of the rbp complementation group. This suggests that the three complementation groups of this complex locus represent distinct but overlapping functions necessary for normal CNS reorganization. This study demonstrates that ecdysterone-regulated gene expression is essential for CNS metamorphosis, illustrating the utility of Drosophila as a model system for investigating the genetic basis of steroid hormone action on the nervous system.
• Restifo, L. L., & White, K. (1990). Molecular and genetic approaches to neurotransmitter and neuromodulator systems in Drosophila.. Advances in Insect Physiology, 22, 115-219. doi:10.1016/s0065-2806(08)60006-5
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  Drosophila melanogaster provides one of the most powerful experimental systems for studying genetic and molecular aspects of nervous system physiology and development. This chapter summarizes studies of compounds that are postulated to mediate or modulate neurotransmission in Drosophila with an emphasis on the genetic approaches used to study them. The premise is that the classical and molecular genetics have the potential to enhance biochemical, immunochemical, and physiological studies of neurotransmitter and neuromodulator function. This chapter covers four major classes of putative neurotransmitters or neuromodulators: (1) acetylcholine (Ach), (2) biogenic amines, (3) amino acids, and (4) neuropeptides. In no instance is there incontrovertible evidence for a role in neurotransmission, although the case for glutamate at the neuromuscular junction is quite strong. For acetylcholine, there is a great deal of circumstantial evidence that is often derived from genetic analyses. In other cases, postulated roles are based solely on analogies with work in other species.
• Restifo, L. L., & Guild, G. M. (1986). An ecdysterone-responsive puff site in Drosophila contains a cluster of seven differentially regulated genes.. Journal of molecular biology, 188(4), 517-28. doi:10.1016/s0022-2836(86)80002-x
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  We have determined the molecular organization of an ecdysterone-responsive puff site in Drosophila melanogaster. The 71E puff site contains a tightly linked cluster of at least seven genes within a neighborhood of 10 X 10(3) base-pairs. All the genes are expressed in a tissue-specific manner in either the larval or the prepupal salivary gland. However, these genes can be divided into two groups on the basis of their temporal pattern of transcription. Six of the genes are expressed only in prepupal salivary glands and are arranged as three divergently transcribed pairs. Nestled within this region is one gene expressed primarily in late third-instar salivary glands. We conclude that this developmentally complex puff site contains six members of the ecdysterone-induced "late"-gene set and one member of the ecdysterone-regulated "intermolt" -gene set. Additional complexity is found at the transcript level: a heterogeneously sized population of RNA molecules arises from each of the seven genes.
• Restifo, L. L., & Guild, G. M. (1986). Poly(A) shortening of coregulated transcripts in Drosophila.. Developmental Biology, 115(2), 507-10. doi:10.1016/0012-1606(86)90271-x
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  The 71E ecdysterone-regulated puff of Drosophila melanogaster contains a cluster of six coregulated "late" genes which are expressed in the prepupal salivary gland. The resulting transcripts exhibit a decrease in their length during the 12-hr period in which they accumulate. Using the enzyme ribonuclease H, we show that this size decrease is a result of a progressively shorter poly(A) tract and suggest that these transcripts undergo an active sequential shortening of their poly(A) tracts in prepupal salivary glands. It is interesting to note that this shortening precedes the complete loss of these transcripts from the RNA population.

Presentations

• Restifo, L. L. (2022, September). Genetics and the Troubled Road to Effective Therapeutics for Alzheimer’s Disease: Can We Unravel the Gordian Knot? . 11th Annual Sanford ImaGENE Precision Medicine Summit. Sanford Center, Sioux Falls, SD: Sanford Imagenetics (Sanford Health, University of South Dakota Sanford School of Medicine).
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  I was an invited speaker. The theme of this year's symposium was “Promises and Pitfalls of Treatment of Genetic Disorders.” Sanford Imagenetics is a cutting-edge precision medicine program with a large team of clinical geneticists, pharmacogeneticists, and genetic counselors. To be blunt, it puts UAz to shame in terms of the inadequacy of clinical genetics services we provide to the community.

Poster Presentations

• Restifo, L. L. (2023, September).

  A Flexible Scheme for Teaching Neurodegenerative Disease to Graduate Students: using the spectrum of genetic causation as an organizing principle.

  . Arizona Alzheimer’s Consortium Annual Conference. ASU, Tempe, AZ: Arizona Alzheimer’s Consortium.
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  Background: The development of safe and effective treatments for neurodegenerative disorders has lagged behind that of most other types of medical conditions. There is a compelling argument for teaching the next generation of neuroscientists more about these challenging diseases, from both basic-science and clinical perspectives. The starting premise is the importance of teaching clinical diagnostic logic, including differential diagnosis and disease classification, along with the research that revealed pathophysiology, underlying mechanisms, and avenues for drug discovery. Together, these teaching goals represent a major challenge.Methods: Over a period of 30 years, a graduate-level 'disease course' at University of Arizona has been developed by repeated trials with varying course formats and content, as well as an evolving approach to prerequisites and student evaluation. I present a classroom-based strategy that avoids hyperfocus on individual molecules, signaling pathways, cellular subtypes, neuroanatomical regions, or any specific research methodology.Results: To choose which neurodegenerative diseases to cover, a two-fold approach was used. First, focus on several exemplar diseases in depth rather than cover many superficially. Second, use the spectrum of genetic causation to create a 4-part disease menu to select from. At one end of the spectrum, the Simple Monogenic Group includes Huntington's disease, spinal muscular atrophy, and Duchenne muscular dystrophy. The Heterogeneous Monogenic Group includes diseases that are caused by mutations in any of a large number of genes, e.g., peripheral neuropathies, spinocerebellar ataxias, and the degenerative subset of sensori-neural deafness. Next, the Familial-or-Sporadic Group, contains the 'big three' – Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis – as well as Lewy body disease, frontotemporal dementia and prion diseases. In this group, single genes, gene-X-environment (GXE) interactions, and polygenic inheritance can all play a role. Finally, the Complex Disease due to GXE Group includes multiple sclerosis, neuromyelitis optica, and autoimmune encephalitis.In addition to the difficulty of the material, other challenges emerged. Future neuroscientists are inclined to believe what they read in the literature. At the outset, most are unaware of the need to assess the validity of cellular and animal models used for research or to question prevailing hypotheses about disease mechanisms. Many assume that diagnostic criteria are stable and valid. Not all are familiar with basic genetics concepts. Variation in students' academic backgrounds is a double-edged sword. At times, it can promote outstanding classroom discussion. However, a substantial fraction of students is reluctant to participate; the pandemic did not help.Conclusions: Students from a wide range of life science disciplines respond well to comprehensive exploration of exemplar neurodegenerative diseases, including differential diagnosis and systems-level pathophysiology. Alongside the breadth and depth of knowledge gained, they develop essential analytical reasoning skills that can enhance their current and future research. Incorporating clinical concepts and controversies into a research-emphasis course may help bridge the cultural divide between scientists and physicians.
• Restifo, L. L. (2022, November). Bridging the cultural divide between basic science and clinical medicine: a flexible scheme for teaching neurological disorders to graduate students. Society for Neuroscience Annual Conference 2022. San Diego, CA: Society for Neuroscience.
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  Students in neuroscience doctoral programs seek coursework focused on neurological disorders for varied reasons, including direct connections to their projects, scientific and personal curiosity, and a desire to explore the medical relevance of research. Such courses, especially if they present clinical diagnostic logic as well as research discoveries, can help bridge the cultural divide between science and medicine. For graduate programs and faculty, the number and range of topics under the umbrella of neurological disease is daunting even for a full-semester course. I developed an approach based on a rotating menu of exemplar diseases from which four are chosen for in-depth analysis. With the caveat that no disease is truly "simple", the course is organized into four modules representing ever-increasing complexity. The first module covers a monogenic disease with Mendelian inheritance (e.g., Duchenne muscular dystrophy, spinal muscular atrophy). The second addresses disorders that can be either hereditary with many possible genetic causes or sporadic with uncertain etiology (e.g., peripheral neuropathies, Alzheimer's disease). During the second half of the course, the emphasis shifts to diseases with strong gene-by-environment interactions. For diseases in the third module, there is strong evidence to support a pathogenic mechanism (e.g., multiple sclerosis), whereas in the fourth module, disease pathogenesis is much less clear (e.g., autism spectrum disorder). Within each module, the starting point is the clinical picture, including epidemiology, diagnostic criteria, differential diagnosis, natural history, and data from imaging and histopathology. The relevant neuroanatomy can be addressed. This is followed by the genetic basis of or contribution to the disease, along with environmental risk factors and triggers. Laboratory evaluation of tissues or cells from patients or animal models is the next focus, with the goal of exploring disease pathogenesis and progression. The module ends by considering the current status of therapeutics and efforts to develop the first or better treatments. Readings come from the clinical, basic science, and translational science literature, providing opportunities to assess a wide range of established and emerging methodologies. Cross-cutting themes addressed in all modules include disease classification, the evolution of diagnostic criteria, the role of genetic testing, the quest for preclinical diagnosis and predictive biomarkers, validation criteria for animal models, the assumptions on which drug-discovery programs are based, and the special challenges of clinical trials.
• Restifo, L. L., & Erickson, R. P. (2022, September).

  Case Report: A severe reaction to mRNA Sars-CoV-2 vaccine booster in an APOE-e4 carrier with mild Alzheimer's disease receiving aducanumab in a clinical trial

  . Arizona Alzheimer's Consortium Annual Conference. Phoenix, AZ: Arizona Alzheimer's Consortium.
• Scherer, K., Hammer, M. F., Day, W. A., Montfort, W. R., Miller, J. H., Johnstone, L. M., Andrew, D. R., & Restifo, L. L. (2021, November). A sporadic case of severe peripheral neuropathy expands the phenotypic range of TUBB2A mutations.. Society for Neuroscience 2021.
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  Whole-exome sequencing study of patient and parents revealed a de novo mutation in TUBB2A which is predicted, based on 3D structure modeling, to disrupt the contact site between beta-tubulin and kinesin. This would explain the proband's childhood-onset severe sensorimotor peripheral neuropathy.The figures were finalized for poster presentation at the (virtual) Society Neuroscience conference in Nov. 2021. We're preparing the manuscript for Frontiers in Neurology and hope to submit in Spring 2022.
• Lewis, S. A., & Restifo, L. L. (2015, March). Pak (p21-activated kinase) mutations cause defects in brain structure and neurite-arbor morphogenesis through regulation of non-muscle myosin. 56th Annual Drosophila Research Conference. Chicago, IL: Genetics Society of America.

Others

• Zohar, Y., Restifo, L. L., & Jiang, L. (2017, December). Systems for Dissociation of Biological Tissues. US Patent & Trademark Office. https://pdfaiw.uspto.gov/.aiw?PageNum=0&docid=20170355950
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  This is a patent application. Here are the unique identifiers:PCT/US15/63978Publication No. US 2017/0355950 A1Abstract: Provided herein is technology relating to processing biological samples and particularly, but not exclusively, to systems and apparatuses for dissociating biological tissues into viable cells.