Daniela C Zarnescu
- Professor, Molecular and Cellular Biology
- Professor, Neuroscience
- Professor, Neurology
- Professor, Cellular and Molecular Medicine
- Professor, Applied BioSciences - GIDP
- Professor, Genetics - GIDP
- Professor, Neuroscience - GIDP
- Professor, BIO5 Institute
PhD, Biochemistry and Molecular Biology, The Pennsylvania State University, State College PA (1995-2000)
“Epithelial polarity and morphogenesis in Drosophila”
BS, Physics, Bucharest University, Romania (1989)
Postdoctoral fellow, Emory University School of Medicine, Atlanta GA (2000-2005)
“Modeling Fragile X syndrome in Drosophila”
Pella Fay Braucher Scholarship (Penn State) (1995)
Fred Wedler Award for outstanding doctoral dissertation, Penn State (2000)
Fragile-X Syndrome Research Foundation (FRAXA) Postdoctoral fellowship (2002)
Ruth Kirschstein NRSA Individual Fellowship (NIH) (2003-2005)
Scientist Development Grant, American Heart Association (2009-2012)
Outstanding Mentor Award (UBRP, U of A) (2017)
- Ph.D. Biochemistry and Molecular Biology
- Pennsylvania State University, State College, Pennsylvania, United States
- Invited Plenary Speaker at Annual Drosophila Research Conference
- GSA, Spring 2018
- Outstanding Mentor Award
- UBRP, Fall 2017
My research focuses on RNA processing during normal development and aging, and dysregulation of RNA metabolism in disease. In addition to these basic studies we seek to identify therapeutic strategies for diseases linked to RNA and metabolic dysregulation in the nervous system. My research group utilizes a combination of genetic, molecular, bioinformatics and pharmacological approaches in Drosophila (fruit flies), cultured cells and patient tissues. This “fly-to-man” approach takes advantage of the powerful, genetically tractable fruit fly model to uncover molecular mechanisms that we can subsequently validate in patient tissues.
I teach in multiple settings including in the laboratory, traditional classroom and the community. I use active learning approaches and focus on individual student growth. In my lectures and outreach activities I try to convey my own enthusiasm for science, highlight the need for interdisciplinary collaboration and the importance of science literacy among the public.
Directed ResearchBIOC 392 (Spring 2018)
Directed RsrchMCB 392 (Spring 2018)
DissertationMCB 920 (Spring 2018)
Honors Independent StudyBIOC 399H (Spring 2018)
Honors Independent StudyBIOC 499H (Spring 2018)
Honors Independent StudyMCB 399H (Spring 2018)
Honors Independent StudyNSCS 399H (Spring 2018)
Honors ThesisMCB 498H (Spring 2018)
Honors ThesisPSIO 498H (Spring 2018)
Internship in Applied BiosciABS 593A (Spring 2018)
Lab Presentations & DiscussionMCB 696A (Spring 2018)
Modeling Human DiseaseMCB 482 (Spring 2018)
Modeling Human DiseaseMCB 582 (Spring 2018)
ResearchMCB 900 (Spring 2018)
Science,Society + EthicsCMM 695E (Spring 2018)
Science,Society + EthicsMCB 695E (Spring 2018)
Directed RsrchMCB 392 (Fall 2017)
DissertationMCB 920 (Fall 2017)
Honors Independent StudyBIOC 399H (Fall 2017)
Honors Independent StudyMCB 399H (Fall 2017)
Honors Independent StudyMCB 499H (Fall 2017)
Honors Independent StudyNSCS 399H (Fall 2017)
Honors Independent StudyPSIO 499H (Fall 2017)
Honors ThesisMCB 498H (Fall 2017)
Honors ThesisPSIO 498H (Fall 2017)
Introduction to ResearchMCB 795A (Fall 2017)
Lab Presentations & DiscussionMCB 696A (Fall 2017)
ResearchMCB 900 (Fall 2017)
Directed ResearchBIOC 392 (Spring 2017)
Directed ResearchNSCS 392 (Spring 2017)
DissertationMCB 920 (Spring 2017)
Honors Independent StudyBIOC 299H (Spring 2017)
Honors Independent StudyMCB 299H (Spring 2017)
Honors Independent StudyMCB 399H (Spring 2017)
Honors Independent StudyMCB 499H (Spring 2017)
Honors ThesisBIOC 498H (Spring 2017)
Honors ThesisMCB 498H (Spring 2017)
Honors ThesisNSCS 498H (Spring 2017)
Honors ThesisPSIO 498H (Spring 2017)
Introduction to ResearchMCB 795A (Spring 2017)
Lab Presentations & DiscussionMCB 696A (Spring 2017)
Modeling Human DiseaseMCB 482 (Spring 2017)
Modeling Human DiseaseMCB 582 (Spring 2017)
Directed ResearchBIOC 392 (Fall 2016)
Directed ResearchNSCS 392 (Fall 2016)
DissertationMCB 920 (Fall 2016)
DissertationNRSC 920 (Fall 2016)
Honors Independent StudyBIOC 299H (Fall 2016)
Honors Independent StudyMCB 299H (Fall 2016)
Honors Independent StudyMCB 499H (Fall 2016)
Honors Independent StudyNSCS 499H (Fall 2016)
Honors ThesisBIOC 498H (Fall 2016)
Honors ThesisMCB 498H (Fall 2016)
Honors ThesisNSCS 498H (Fall 2016)
Honors ThesisPSIO 498H (Fall 2016)
Introduction to ResearchMCB 795A (Fall 2016)
Lab Presentations & DiscussionMCB 696A (Fall 2016)
Scientific CommunicationMCB 575 (Fall 2016)
Topic Molec BiologyMCB 595A (Fall 2016)
- Chou, C. C., Zhang, Y., Umoh, M. E., Vaughan, S. W., Lorenzini, I., Liu, F., Sayegh, M., Donlin-Asp, P. G., Chen, Y. H., Duong, D. M., Seyfried, N. T., Powers, M. A., Kukar, T., Hales, C. M., Gearing, M., Cairns, N. J., Boylan, K. B., Dickson, D. W., Rademakers, R., , Zhang, Y. J., et al. (2018). TDP-43 pathology disrupts nuclear pore complexes and nucleocytoplasmic transport in ALS/FTD. Nature neuroscience, 21(2), 228-239.More infoThe cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a common histopathological hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia disease spectrum (ALS/FTD). However, the composition of aggregates and their contribution to the disease process remain unknown. Here we used proximity-dependent biotin identification (BioID) to interrogate the interactome of detergent-insoluble TDP-43 aggregates and found them enriched for components of the nuclear pore complex and nucleocytoplasmic transport machinery. Aggregated and disease-linked mutant TDP-43 triggered the sequestration and/or mislocalization of nucleoporins and transport factors, and interfered with nuclear protein import and RNA export in mouse primary cortical neurons, human fibroblasts and induced pluripotent stem cell-derived neurons. Nuclear pore pathology is present in brain tissue in cases of sporadic ALS and those involving genetic mutations in TARDBP and C9orf72. Our data strongly implicate TDP-43-mediated nucleocytoplasmic transport defects as a common disease mechanism in ALS/FTD.
- Chu, M., Novak, S. M., Cover, C., Wang, A. A., Chinyere, I. R., Juneman, E. B., Zarnescu, D. C., Wong, P. K., & Gregorio, C. C. (2018). Increased Cardiac Arrhythmogenesis Associated With Gap Junction Remodeling With Upregulation of RNA-Binding Protein FXR1. Circulation, 137(6), 605-618.More infoGap junction remodeling is well established as a consistent feature of human heart disease involving spontaneous ventricular arrhythmia. The mechanisms responsible for gap junction remodeling that include alterations in the distribution of, and protein expression within, gap junctions are still debated. Studies reveal that multiple transcriptional and posttranscriptional regulatory pathways are triggered in response to cardiac disease, such as those involving RNA-binding proteins. The expression levels of FXR1 (fragile X mental retardation autosomal homolog 1), an RNA-binding protein, are critical to maintain proper cardiac muscle function; however, the connection between FXR1 and disease is not clear.
- Daniel, S. G., Russ, A. D., Guthridge, K. M., Raina, A. I., Estes, P. S., Parsons, L. M., Richardson, H. E., Schroeder, J. A., & Zarnescu, D. C. (2017). MiR-9 mediates the role of Lethal giant larvae as an epithelial growth inhibitor in. Biology open, 7(1).More info() encodes a conserved tumor suppressor with established roles in cell polarity, asymmetric division, and proliferation control. Lgl's human orthologs, HUGL1 and HUGL2, are altered in human cancers, however, its mechanistic role as a tumor suppressor remains poorly understood. Based on a previously established connection between Lgl and Fragile X protein (FMRP), a miRNA-associated translational regulator, we hypothesized that Lgl may exert its role as a tumor suppressor by interacting with the miRNA pathway. Consistent with this model, we found thatis a dominant modifier of Argonaute1 overexpression in the eye neuroepithelium. Using microarray profiling we identified a core set of ten miRNAs that are altered throughout tumorigenesis inmutants. Among these are several miRNAs previously linked to human cancers including, which we found to be downregulated inneuroepithelial tissues. To determine whethercan act as an effector of Lgl, we overexpressed it in the context ofknock-down by RNAi and found it able to reduce the overgrowth phenotype caused by Lgl loss in epithelia. Furthermore, cross-comparisons between miRNA and mRNA profiling inmutant tissues and human breast cancer cells identified() as a common factor altered in both fly and human breast cancer tumorigenesis models. Our work provides the first evidence of a functional connection between Lgl and the miRNA pathway, demonstrates thatmediates Lgl's role in restricting epithelial proliferation, and provides novel insights into pathways controlled by Lgl during tumor progression.
- Ferro, D., Yao, S., & Zarnescu, D. C. (2018). Dynamic duo - FMRP and TDP-43: Regulating common targets, causing different diseases. Brain research, 1693(Pt A), 37-42.More infoRNA binding proteins play essential roles during development and aging, and are also involved in disease pathomechanisms. RNA sequencing and omics analyses have provided a window into systems level alterations in neurological disease, and have identified RNA processing defects among notable disease mechanisms. This review focuses on two seemingly distinct neurological disorders, the RNA binding proteins they are linked to, and their newly discovered functional relationship. When deficient, Fragile X Mental Retardation Protein (FMRP) causes developmental deficits and autistic behaviors while TAR-DNA Binding Protein (TDP-43) dysregulation causes age dependent neuronal degeneration. Recent findings that FMRP and TDP-43 associate in ribonuclear protein particles and share mRNA targets in neurons highlight the critical importance of translation regulation in synaptic plasticity and provide new perspectives on neuronal vulnerability during lifespan.
- Lehmkuhl, E. M., & Zarnescu, D. C. (2018). Lost in Translation: Evidence for Protein Synthesis Deficits in ALS/FTD and Related Neurodegenerative Diseases. Advances in neurobiology, 20, 283-301.More infoCells utilize a complex network of proteins to regulate translation, involving post-transcriptional processing of RNA and assembly of the ribosomal unit. Although the complexity provides robust regulation of proteostasis, it also offers several opportunities for translational dysregulation, as has been observed in many neurodegenerative disorders. Defective mRNA localization, mRNA sequatration, inhibited ribogenesis, mutant tRNA synthetases, and translation of hexanucleotide expansions have all been associated with neurodegenerative disease. Here, we review dysregulation of translation in the context of age-related neurodegeneration and discuss novel methods to interrogate translation. This review primarily focuses on amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), a spectrum disorder heavily associated with RNA metabolism, while also analyzing translational inhibition in the context of related neurodegenerative disorders such as Alzheimer's disease and Huntington's disease and the translation-related pathomechanisms common in neurodegenerative disease.
- Manzo, E., O'Conner, A. G., Barrows, J. M., Shreiner, D. D., Birchak, G. J., & Zarnescu, D. C. (2018). Medium-Chain Fatty Acids, Beta-Hydroxybutyric Acid and Genetic Modulation of the Carnitine Shuttle Are Protective in a Model of ALS Based on TDP-43. Frontiers in molecular neuroscience, 11, 182.More infoALS patients exhibit dyslipidemia, hypermetabolism and weight loss; in addition, cellular energetics deficits have been detected prior to denervation. Although evidence that metabolism is altered in ALS is compelling, the mechanisms underlying metabolic dysregulation and the contribution of altered metabolic pathways to disease remain poorly understood. Here we use a model of ALS based on TDP-43 that recapitulates hallmark features of the disease including locomotor dysfunction and reduced lifespan. We performed a global, unbiased metabolomic profiling of larvae expressing TDP-43 (wild-type, TDP or disease-associated mutant, TDP) and identified several lipid metabolism associated alterations. Among these, we found a significant increase in carnitine conjugated long-chain fatty acids and a significant decrease in carnitine, acetyl-carnitine and beta-hydroxybutyrate, a ketone precursor. Taken together these data suggest a deficit in the function of the carnitine shuttle and reduced lipid beta oxidation. To test this possibility we used a combined genetic and dietary approach in . Our findings indicate that components of the carnitine shuttle are misexpressed in the context of TDP-43 proteinopathy and that genetic modulation of CPT1 or CPT2 expression, two core components of the carnitine shuttle, mitigates TDP-43 dependent locomotor dysfunction, in a variant dependent manner. In addition, feeding medium-chain fatty acids or beta-hydroxybutyrate improves locomotor function, consistent with the notion that bypassing the carnitine shuttle deficit is neuroprotective. Taken together, our findings highlight the potential contribution of the carnitine shuttle and lipid beta oxidation in ALS and suggest strategies for therapeutic intervention based on restoring lipid metabolism in motor neurons.
- Coyne, A. N., Lorenzini, I., Chou, C. C., Torvund, M., Rogers, R. S., Starr, A., Zaepfel, B. L., Levy, J., Johannesmeyer, J., Schwartz, J. C., Nishimune, H., Zinsmaier, K., Rossoll, W., Sattler, R., & Zarnescu, D. C. (2017). Post-transcriptional Inhibition of Hsc70-4/HSPA8 Expression Leads to Synaptic Vesicle Cycling Defects in Multiple Models of ALS. Cell reports, 21(1), 110-125.More infoAmyotrophic lateral sclerosis (ALS) is a synaptopathy accompanied by the presence of cytoplasmic aggregates containing TDP-43, an RNA-binding protein linked to ∼97% of ALS cases. Using a Drosophila model of ALS, we show that TDP-43 overexpression (OE) in motor neurons results in decreased expression of the Hsc70-4 chaperone at the neuromuscular junction (NMJ). Mechanistically, mutant TDP-43 sequesters hsc70-4 mRNA and impairs its translation. Expression of the Hsc70-4 ortholog, HSPA8, is also reduced in primary motor neurons and NMJs of mice expressing mutant TDP-43. Electrophysiology, imaging, and genetic interaction experiments reveal TDP-43-dependent defects in synaptic vesicle endocytosis. These deficits can be partially restored by OE of Hsc70-4, cysteine-string protein (Csp), or dynamin. This suggests that TDP-43 toxicity results in part from impaired activity of the synaptic CSP/Hsc70 chaperone complex impacting dynamin function. Finally, Hsc70-4/HSPA8 expression is also post-transcriptionally reduced in fly and human induced pluripotent stem cell (iPSC) C9orf72 models, suggesting a common disease pathomechanism.
- Coyne, A. N., Zaepfel, B. L., & Zarnescu, D. C. (2017). Failure to Deliver and Translate-New Insights into RNA Dysregulation in ALS. Frontiers in cellular neuroscience, 11, 243.More infoAmyotrophic Lateral Sclerosis (ALS) is a progressive and fatal neurodegenerative disease affecting both upper and lower motor neurons. The molecular mechanisms underlying disease pathogenesis remain largely unknown. Multiple genetic loci including genes involved in proteostasis and ribostasis have been linked to ALS providing key insights into the molecular mechanisms underlying disease. In particular, the identification of the RNA binding proteins TDP-43 and fused in sarcoma (FUS) as causative factors of ALS resulted in a paradigm shift centered on the study of RNA dysregulation as a major mechanism of disease. With wild-type TDP-43 pathology being found in ~97% of ALS cases and the identification of disease causing mutations within its sequence, TDP-43 has emerged as a prominent player in ALS. More recently, studies of the newly discoveredrepeat expansion are lending further support to the notion of defects in RNA metabolism as a key factor underlying ALS. RNA binding proteins are involved in all aspects of RNA metabolism ranging from splicing, transcription, transport, storage into RNA/protein granules, and translation. How these processes are affected by disease-associated mutations is just beginning to be understood. Considerable work has gone into the identification of splicing and transcription defects resulting from mutations in RNA binding proteins associated with disease. More recently, defects in RNA transport and translation have been shown to be involved in the pathomechanism of ALS. A central hypothesis in the field is that disease causing mutations lead to the persistence of RNA/protein complexes known as stress granules. Under times of prolonged cellular stress these granules sequester specific mRNAs preventing them from translation, and are thought to evolve into pathological aggregates. Here we will review recent efforts directed at understanding how altered RNA metabolism contributes to ALS pathogenesis.
- Joardar, A., Manzo, E., & Zarnescu, D. C. (2017). Metabolic Dysregulation in Amyotrophic Lateral Sclerosis: Challenges and Opportunities. Current genetic medicine reports, 5(2), 108-114.More infoAmyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease for which there is no cure and treatments are at best palliative. Several genes have been linked to ALS, which highlight defects in multiple cellular processes including RNA processing, proteostasis and metabolism. Clinical observations have identified glucose intolerance and dyslipidemia as key features of ALS however the causes of these metabolic alterations remain elusive.
- Liu, G., Coyne, A. N., Pei, F., Vaughan, S., Chaung, M., Zarnescu, D. C., & Buchan, J. R. (2017). Endocytosis regulates TDP-43 toxicity and turnover. Nature communications, 8(1), 2092.More infoAmyotrophic lateral sclerosis (ALS) is a fatal motor neuron degenerative disease. ALS-affected motor neurons exhibit aberrant localization of a nuclear RNA binding protein, TDP-43, into cytoplasmic aggregates, which contributes to pathology via unclear mechanisms. Here, we demonstrate that TDP-43 turnover and toxicity depend in part upon the endocytosis pathway. TDP-43 inhibits endocytosis, and co-localizes strongly with endocytic proteins, including in ALS patient tissue. Impairing endocytosis increases TDP-43 toxicity, aggregation, and protein levels, whereas enhancing endocytosis reverses these phenotypes. Locomotor dysfunction in a TDP-43 ALS fly model is also exacerbated and suppressed by impairment and enhancement of endocytic function, respectively. Thus, endocytosis dysfunction may be an underlying cause of ALS pathology.
- Chou, C., Alexeeva, O. M., Yamada, S., Pribadi, A., Zhang, Y., Mo, B., Williams, K. R., Zarnescu, D. C., & Rossoll, W. (2015). PABPN1 suppresses TDP-43 toxicity in ALS disease models. Human molecular genetics, 24(18), 5154-73.More infoTAR DNA-binding protein 43 (TDP-43) is a major disease protein in amyotrophic lateral sclerosis (ALS) and related neurodegenerative diseases. Both the cytoplasmic accumulation of toxic ubiquitinated and hyperphosphorylated TDP-43 fragments and the loss of normal TDP-43 from the nucleus may contribute to the disease progression by impairing normal RNA and protein homeostasis. Therefore, both the removal of pathological protein and the rescue of TDP-43 mislocalization may be critical for halting or reversing TDP-43 proteinopathies. Here, we report poly(A)-binding protein nuclear 1 (PABPN1) as a novel TDP-43 interaction partner that acts as a potent suppressor of TDP-43 toxicity. Overexpression of full-length PABPN1 but not a truncated version lacking the nuclear localization signal protects from pathogenic TDP-43-mediated toxicity, promotes the degradation of pathological TDP-43 and restores normal solubility and nuclear localization of endogenous TDP-43. Reduced levels of PABPN1 enhances the phenotypes in several cell culture and Drosophila models of ALS and results in the cytoplasmic mislocalization of TDP-43. Moreover, PABPN1 rescues the dysregulated stress granule (SG) dynamics and facilitates the removal of persistent SGs in TDP-43-mediated disease conditions. These findings demonstrate a role for PABPN1 in rescuing several cytopathological features of TDP-43 proteinopathy by increasing the turnover of pathologic proteins.
- Coyne, A. N., Yamada, S. B., Siddegowda, B. B., Estes, P. S., Zaepfel, B. L., Johannesmeyer, J. S., Lockwood, D. B., Pham, L. T., Hart, M. P., Cassel, J. A., Freibaum, B., Boehringer, A. V., Taylor, J. P., Reitz, A. B., Gitler, A. D., & Zarnescu, D. C. (2015). Fragile X protein mitigates TDP-43 toxicity by remodeling RNA granules and restoring translation. Human molecular genetics, 24(24), 6886-98.More infoRNA dysregulation is a newly recognized disease mechanism in amyotrophic lateral sclerosis (ALS). Here we identify Drosophila fragile X mental retardation protein (dFMRP) as a robust genetic modifier of TDP-43-dependent toxicity in a Drosophila model of ALS. We find that dFMRP overexpression (dFMRP OE) mitigates TDP-43 dependent locomotor defects and reduced lifespan in Drosophila. TDP-43 and FMRP form a complex in flies and human cells. In motor neurons, TDP-43 expression increases the association of dFMRP with stress granules and colocalizes with polyA binding protein in a variant-dependent manner. Furthermore, dFMRP dosage modulates TDP-43 solubility and molecular mobility with overexpression of dFMRP resulting in a significant reduction of TDP-43 in the aggregate fraction. Polysome fractionation experiments indicate that dFMRP OE also relieves the translation inhibition of futsch mRNA, a TDP-43 target mRNA, which regulates neuromuscular synapse architecture. Restoration of futsch translation by dFMRP OE mitigates Futsch-dependent morphological phenotypes at the neuromuscular junction including synaptic size and presence of satellite boutons. Our data suggest a model whereby dFMRP is neuroprotective by remodeling TDP-43 containing RNA granules, reducing aggregation and restoring the translation of specific mRNAs in motor neurons.
- Joardar, A., Menzl, J., Podolsky, T. C., Manzo, E., Estes, P. S., Ashford, S., & Zarnescu, D. C. (2015). PPAR gamma activation is neuroprotective in a Drosophila model of ALS based on TDP-43. Human molecular genetics.More infoAmyotrophic Lateral Sclerosis (ALS) is a progressive neuromuscular disease for which there is no cure. We have previously developed a Drosophila model of ALS based on TDP-43 that recapitulates several aspects of disease pathophysiology. Using this model, we designed a drug screening strategy based on the pupal lethality phenotype induced by TDP-43 when expressed in motor neurons. In screening 1200 FDA-approved compounds, we identified the PPARγ agonist pioglitazone as neuroprotective in Drosophila. Here, we show that pioglitazone can rescue TDP-43-dependent locomotor dysfunction in motor neurons and glia but not in muscles. Testing additional models of ALS, we find that pioglitazone is also neuroprotective when FUS, but not SOD1, is expressed in motor neurons. Interestingly, survival analyses of TDP or FUS models show no increase in lifespan, which is consistent with recent clinical trials. Using a pharmacogenetic approach, we show that the predicted Drosophila PPARγ homologs, E75 and E78, are in vivo targets of pioglitazone. Finally, using a global metabolomic approach, we identify a set of metabolites that pioglitazone can restore in the context of TDP-43 expression in motor neurons. Taken together, our data provide evidence that modulating PPARγ activity, although not effective at improving lifespan, provides a molecular target for mitigating locomotor dysfunction in TDP-43 and FUS but not SOD1 models of ALS in Drosophila. Furthermore, our data also identify several 'biomarkers' of the disease that may be useful in developing therapeutics and in future clinical trials.
- Novak, S. M., Joardar, A., Gregorio, C. C., & Zarnescu, D. C. (2015). Regulation of Heart Rate in Drosophila via Fragile X Mental Retardation Protein. PloS one, 10(11), e0142836.More infoRNA binding proteins play a pivotal role in post-transcriptional gene expression regulation, however little is understood about their role in cardiac function. The Fragile X (FraX) family of RNA binding proteins is most commonly studied in the context of neurological disorders, as mutations in Fragile X Mental Retardation 1 (FMR1) are the leading cause of inherited mental retardation. More recently, alterations in the levels of Fragile X Related 1 protein, FXR1, the predominant FraX member expressed in vertebrate striated muscle, have been linked to structural and functional defects in mice and zebrafish models. FraX proteins are established regulators of translation and are known to regulate specific targets in different tissues. To decipher the direct role of FraX proteins in the heart in vivo, we turned to Drosophila, which harbors a sole, functionally conserved and ubiquitously expressed FraX protein, dFmr1. Using classical loss of function alleles as well as muscle specific RNAi knockdown, we show that Drosophila FMRP, dFmr1, is required for proper heart rate during development. Functional analyses in the context of cardiac-specific dFmr1 knockdown by RNAi demonstrate that dFmr1 is required cell autonomously in cardiac cells for regulating heart rate. Interestingly, these functional defects are not accompanied by any obvious structural abnormalities, suggesting that dFmr1 may regulate a different repertoire of targets in Drosophila than in vertebrates. Taken together, our findings support the hypothesis that dFmr1 protein is essential for proper cardiac function and establish the fly as a new model for studying the role(s) of FraX proteins in the heart.
- Zarnescu, D. C., Coyne, A., Yamada, S., Bagevalu Siddegowda, B., Estes, P., Zaepfel, B., Johannesmeyer, J., Lockwood, D., Pham, L., Hart, M., Cassell, J., Freibaum, B., Boehringer, A., Taylor, P., Reitz, A., & Gitler, A. (2015). Fragile X protein is neuroprotective by remodeling TDP-43 containing RNA granules and restoring the translation of specific mRNA targets. Human Molecular Genetics.More infoRNA dysregulation is a newly recognized disease mechanism in amyotrophic lateral sclerosis (ALS). Here we identify Fragile X Mental Retardation Protein (FMRP) as a robust modifier of TDP-43 dependent toxicity in a Drosophila model of ALS. TDP-43 and FMRP form a complex in flies and human cells, and colocalize with PABP in RNA stress granules in motor neurons. Here we show that FMRP overexpression mitigates TDP-43 dependent locomotor defects and reduced lifespan in Drosophila by modulating TDP-43 solubility and molecular mobility. Polysome fractionation experiments indicate that FMRP overexpression relieves the translation inhibition of futsch mRNA, a TDP-43 target that regulates neuromuscular synapse architecture. Restoration of futsch translation by FMRP overexpression not only rescues Futsch expression at the neuromuscular junction, but also mitigates Futsch dependent morphological phenotypes. Our data show that FMRP is neuroprotective by remodeling TDP-43 containing RNA granules and restoring translation of specific mRNAs in motor neurons.
- Coyne, A. N., Siddegowda, B. B., Estes, P. S., Johannesmeyer, J., Kovalik, T., Daniel, S. G., Pearson, A., Bowser, R., & Zarnescu, D. C. (2014). Futsch/MAP1B mRNA is a translational target of TDP-43 and is neuroprotective in a Drosophila model of amyotrophic lateral sclerosis. The Journal of neuroscience : the official journal of the Society for Neuroscience, 34(48), 15962-74.More infoTDP-43 is an RNA-binding protein linked to amyotrophic lateral sclerosis (ALS) that is known to regulate the splicing, transport, and storage of specific mRNAs into stress granules. Although TDP-43 has been shown to interact with translation factors, its role in protein synthesis remains unclear, and no in vivo translation targets have been reported to date. Here we provide evidence that TDP-43 associates with futsch mRNA in a complex and regulates its expression at the neuromuscular junction (NMJ) in Drosophila. In the context of TDP-43-induced proteinopathy, there is a significant reduction of futsch mRNA at the NMJ compared with motor neuron cell bodies where we find higher levels of transcript compared with controls. TDP-43 also leads to a significant reduction in Futsch protein expression at the NMJ. Polysome fractionations coupled with quantitative PCR experiments indicate that TDP-43 leads to a futsch mRNA shift from actively translating polysomes to nontranslating ribonuclear protein particles, suggesting that in addition to its effect on localization, TDP-43 also regulates the translation of futsch mRNA. We also show that futsch overexpression is neuroprotective by extending life span, reducing TDP-43 aggregation, and suppressing ALS-like locomotor dysfunction as well as NMJ abnormalities linked to microtubule and synaptic stabilization. Furthermore, the localization of MAP1B, the mammalian homolog of Futsch, is altered in ALS spinal cords in a manner similar to our observations in Drosophila motor neurons. Together, our results suggest a microtubule-dependent mechanism in motor neuron disease caused by TDP-43-dependent alterations in futsch mRNA localization and translation in vivo.
- Estes, P. S., Daniel, S. G., McCallum, A. P., Boehringer, A. V., Sukhina, A. S., Zwick, R. A., & Zarnescu, D. C. (2013). Motor neurons and glia exhibit specific individualized responses to TDP-43 expression in a Drosophila model of amyotrophic lateral sclerosis. Disease models & mechanisms, 6(3), 721-33.More infoAmyotrophic lateral sclerosis (ALS) is a fatal disease characterized by complex neuronal and glial phenotypes. Recently, RNA-based mechanisms have been linked to ALS via RNA-binding proteins such as TDP-43, which has been studied in vivo using models ranging from yeast to rodents. We have developed a Drosophila model of ALS based on TDP-43 that recapitulates several aspects of pathology, including motor neuron loss, locomotor dysfunction and reduced survival. Here we report the phenotypic consequences of expressing wild-type and four different ALS-linked TDP-43 mutations in neurons and glia. We show that TDP-43-driven neurodegeneration phenotypes are dose- and age-dependent. In motor neurons, TDP-43 appears restricted to nuclei, which are significantly misshapen due to mutant but not wild-type protein expression. In glia and in the developing neuroepithelium, TDP-43 associates with cytoplasmic puncta. TDP-43-containing RNA granules are motile in cultured motor neurons, although wild-type and mutant variants exhibit different kinetic properties. At the neuromuscular junction, the expression of TDP-43 in motor neurons versus glia leads to seemingly opposite synaptic phenotypes that, surprisingly, translate into comparable locomotor defects. Finally, we explore sleep as a behavioral readout of TDP-43 expression and find evidence of sleep fragmentation consistent with hyperexcitability, a suggested mechanism in ALS. These findings support the notion that although motor neurons and glia are both involved in ALS pathology, at the cellular level they can exhibit different responses to TDP-43. In addition, our data suggest that individual TDP-43 alleles utilize distinct molecular mechanisms, which will be important for developing therapeutic strategies.
- Morera, A. A., Coyne, A., & Zarnescu, D. C. (2013). Flies in motion: What drosophila can tell us about amyotrophic lateral sclerosis. Drosophila Melanogaster Models of Motor Neuron Disease, 57-83.More infoAbstract: Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder with a prevalence of 1 in 30,000 individuals. Generally diagnosed between 40 and 70 years of age, ALS is accompanied by progressive loss of motor neuron function and a life expectancy of 2-5 years. Although age is considered to be the highest risk factor for ALS, it is clear that the disease has a genetic basis and may also be influenced by environmental factors. Familial ALS (fALS) affects 10% of patients and has been linked to several loci, the most common of which is C9ORF72, a gene of unknown function. The remaining 90% of ALS cases are sporadic (sALS) and remain poorly understood, although some loci have been linked to both fALS and sALS. In recent years a dramatic shift in our thinking about ALS has been catalysed by findings that the RNA binding proteins TAR DNA binding protein 43 (TDP-43) and fused in sarcoma/translocated in liposarcoma (FUS/TLS) constitute markers of pathology and when mutated, neural degeneration occurs in human patients. Studies in a wide range of model systems including worms, flies, zebrafish and rodents support the notion that alterations in these RNA binding proteins and RNA metabolism cause motor neuron disease. Together with the recent discovery of GGGGCC repeat expansions in C9ORF72, these studies led to the hypothesis that ALS is a disease of RNA dysregulation. Here we will review the contributions of the fruit fly Drosophila melanogaster to our understanding of ALS with a focus on TDP-43, FUS/TLS and RNA dysregulation as a disease mechanism.© 2013 by Nova Science Publishers, Inc. All rights reserved.
- Zarnescu, D. C., & Gregorio, C. C. (2013). Fragile hearts: new insights into translational control in cardiac muscle. Trends in cardiovascular medicine, 23(8), 275-81.More infoCurrent investigations focused on RNA-binding proteins in striated muscle, which provide a scenario whereby muscle function and development are governed by the interplay of post-transcriptional RNA regulation, including transcript localization, splicing, stability, and translational control. New data have recently emerged, linking the RNA-binding protein FXR1 to the translation of key cytoskeletal components such as talin and desmoplakin in heart muscle. These findings, together with a plethora of recent reports implicating RNA-binding proteins and their RNA targets in both basic aspects of muscle development and differentiation as well as heart disease and muscular dystrophies, point to a critical role of RNA-based regulatory mechanisms in muscle biology. Here we focus on FXR1, the striated muscle-specific member of the Fragile X family of RNA-binding proteins and discuss its newly reported cytoskeletal targets as well as potential implications for heart disease.
- Russ, A., Louderbough, J. M., Zarnescu, D., & Schroeder, J. A. (2012). Hugl1 and Hugl2 in mammary epithelial cells: polarity, proliferation, and differentiation. PloS one, 7(10), e47734.More infoLoss of epithelial polarity is described as a hallmark of epithelial cancer. To determine the role of Hugl1 and Hugl2 expression in the breast, we investigated their localization in human mammary duct tissue and the effects of expression modulation in normal and cancer cell lines on polarity, proliferation and differentiation. Expression of Hugl1 and Hugl2 was silenced in both MCF10A cells and Human Mammary Epithelial Cells and cell lines were grown in 2-D on plastic and in 3-D in Matrigel to form acini. Cells in monolayer were compared for proliferative and phenotypic changes while acini were examined for differences in size, ability to form a hollow lumen, nuclear size and shape, and localization of key domain-specific proteins as a measure of polarity. We detected overlapping but distinct localization of Hugl1 and Hugl2 in the human mammary gland, with Hugl1 expressed in both luminal and myoepithelium and Hugl2 largely restricted to myoepithelium. On a plastic surface, loss of Hugl1 or Hugl2 in normal epithelium induced a mesenchymal phenotype, and these cells formed large cellular masses when grown in Matrigel. In addition, loss of Hugl1 or Hugl2 expression in MCF10A cells resulted in increased proliferation on Matrigel, while gain of Hugl1 expression in tumor cells suppressed proliferation. Loss of polarity was also observed with knockdown of either Hugl1 or Hugl2, with cells growing in Matrigel appearing as a multilayered epithelium, with randomly oriented Golgi and multiple enlarged nuclei. Furthermore, Hugl1 knock down resulted in a loss of membrane identity and the development of cellular asymmetries in Human Mammary Epithelial Cells. Overall, these data demonstrate an essential role for both Hugl1 and Hugl2 in the maintenance of breast epithelial polarity and differentiated cell morphology, as well as growth control.
- Zarnescu, D., Callan, M. A., Clements, N., Ahrendt, N., & Zarnescu, D. C. (2012). Fragile X Protein is required for inhibition of insulin signaling and regulates glial-dependent neuroblast reactivation in the developing brain. Brain research, 1462.More infoFragile X syndrome (FXS) is the most common form of inherited mental disability and known cause of autism. It is caused by loss of function for the RNA binding protein FMRP, which has been demonstrated to regulate several aspects of RNA metabolism including transport, stability and translation at synapses. Recently, FMRP has been implicated in neural stem cell proliferation and differentiation both in cultured neurospheres as well as in vivo mouse and fly models of FXS. We have previously shown that FMRP deficient Drosophila neuroblasts upregulate Cyclin E, prematurely exit quiescence, and overproliferate to generate on average 16% more neurons. Here we further investigate FMRP's role during early development using the Drosophila larval brain as a model. Using tissue specific RNAi we find that FMRP is required sequentially, first in neuroblasts and then in glia, to regulate exit from quiescence as measured by Cyclin E expression in the brain. Furthermore, we tested the hypothesis that FMRP controls brain development by regulating the insulin signaling pathway, which has been recently shown to regulate neuroblast exit from quiescence. Our data indicate that phosphoAkt, a readout of insulin signaling, is upregulated in dFmr1 brains at the time when FMRP is required in glia for neuroblast reactivation. In addition, dFmr1 interacts genetically with dFoxO, a transcriptional regulator of insulin signaling. Our results provide the first evidence that FMRP is required in vivo, in glia for neuroblast reactivation and suggest that it may do so by regulating the output of the insulin signaling pathway. This article is part of a Special Issue entitled: RNA-Binding Proteins.
- Whitman, S. A., Cover, C., Yu, L., Nelson, D. L., Zarnescu, D. C., & Gregorio, C. C. (2011). Desmoplakin and talin2 are novel mRNA targets of fragile X-related protein-1 in cardiac muscle. Circulation research, 109(3), 262-71.More infoThe proper function of cardiac muscle requires the precise assembly and interactions of numerous cytoskeletal and regulatory proteins into specialized structures that orchestrate contraction and force transmission. Evidence suggests that posttranscriptional regulation is critical for muscle function, but the mechanisms involved remain understudied.
- Zarnescu, D., Callan, M. A., & Zarnescu, D. C. (2011). Heads-up: new roles for the fragile X mental retardation protein in neural stem and progenitor cells. Genesis (New York, N.Y. : 2000), 49(6).More infoFragile X syndrome (FXS) is the most common form of inherited mental retardation and is caused by the loss of function for Fragile X Mental Retardation Protein (FMRP), a selective RNA-binding protein with a demonstrated role in the localized translation of target mRNAs at synapses. Several recent studies provide compelling evidence for a new role of FMRP in the development of the nervous system, during neurogenesis. Using a multi-faceted approach and a variety of model systems ranging from cultured neurospheres and progenitor cells to in vivo Drosophila and mouse models these reports indicate that FMRP is required for neural stem and progenitor cell proliferation, differentiation, survival, as well as regulation of gene expression. Here we compare and contrast these recent reports and discuss the implications of FMRP's new role in embryonic and adult neurogenesis, including the development of novel therapeutic approaches to FXS and related neurological disorders such as autism.
- Zarnescu, D., Estes, P. S., Boehringer, A., Zwick, R., Tang, J. E., Grigsby, B., & Zarnescu, D. C. (2011). Wild-type and A315T mutant TDP-43 exert differential neurotoxicity in a Drosophila model of ALS. Human molecular genetics, 20(12).More infoThe RNA-binding protein TDP-43 has been linked to amyotrophic lateral sclerosis (ALS) both as a causative locus and as a marker of pathology. With several missense mutations being identified within TDP-43, efforts have been directed towards generating animal models of ALS in mouse, zebrafish, Drosophila and worms. Previous loss of function and overexpression studies have shown that alterations in TDP-43 dosage recapitulate hallmark features of ALS pathology, including neuronal loss and locomotor dysfunction. Here we report a direct in vivo comparison between wild-type and A315T mutant TDP-43 overexpression in Drosophila neurons. We found that when expressed at comparable levels, wild-type TDP-43 exerts more severe effects on neuromuscular junction architecture, viability and motor neuron loss compared with the A315T allele. A subset of these differences can be compensated by higher levels of A315T expression, indicating a direct correlation between dosage and neurotoxic phenotypes. Interestingly, larval locomotion is the sole parameter that is more affected by the A315T allele than wild-type TDP-43. RNA interference and genetic interaction experiments indicate that TDP-43 overexpression mimics a loss-of-function phenotype and suggest a dominant-negative effect. Furthermore, we show that neuronal apoptosis does not require the cytoplasmic localization of TDP-43 and that its neurotoxicity is modulated by the proteasome, the HSP70 chaperone and the apoptosis pathway. Taken together, our findings provide novel insights into the phenotypic consequences of the A315T TDP-43 missense mutation and suggest that studies of individual mutations are critical for elucidating the molecular mechanisms of ALS and related neurodegenerative disorders.
- Lee, H. G., Zarnescu, D. C., MacIver, B., & Thomas, G. H. (2010). The cell adhesion molecule Roughest depends on beta(Heavy)-spectrin during eye morphogenesis in Drosophila. Journal of cell science, 123(Pt 2), 277-85.More infoCell junctions have both structural and morphogenetic roles, and contain complex mixtures of proteins whose interdependencies are still largely unknown. Junctions are also major signaling centers that signify correct integration into a tissue, and modulate cell survival. During Drosophila eye development, the activity of the immunoglobulin cell adhesion molecule Roughest (also known as Irregular chiasm C-roughest protein) mediates interommatidial cell (IOC) reorganization, leading to an apoptotic event that refines the retinal lattice. Roughest and the cadherin-based zonula adherens (ZA) are interdependent and both are modulated by the apical polarity determinant, Crumbs. Here we describe a novel relationship between the Crumbs partner beta(Heavy)-spectrin (beta(H)), the ZA and Roughest. Ectopic expression of the C-terminal segment 33 of beta(H) (betaH33) induces defects in retinal morphogenesis, resulting the preferential loss of IOC. This effect is associated with ZA disruption and Roughest displacement. In addition, loss-of-function karst and roughest mutations interact to cause a synergistic and catastrophic effect on retinal development. Finally, we show that beta(H) coimmunoprecipitates with Roughest and that the distribution of Roughest protein is disrupted in karst mutant tissue. These results suggest that the apical spectrin membrane skeleton helps to coordinate the Cadherin-based ZA with Roughest-based morphogenesis.
- Mounsef, J., Karam, L., Estes, P., & Zarnescu, D. (2010). Shape analysis and classification of lgl-type and wild-type neurons. 2010 ACM International Conference on Bioinformatics and Computational Biology, ACM-BCB 2010, 362-365.More infoAbstract: Among the tumor suppressors identified in Drosophila, lgl is one of a few whose proliferative phenotype is shown to be secondary to a loss of cell polarity. Studies of lgl mutant phenotypes are likely to contribute to our understanding of both tumorigenesis as well as neural development mechanisms. However, alterations of neuronal development as a result of key protein mutations are not easy to describe without quantifiable parameters. This work presents a fully automated imaging technique that involves skeletonization and histogram-based features such as kurtosis to find and quantify discriminative morphological phenotypes that can be used to automatically distinguish normal wild-type neurons from lgl mutant neurons. Copyright © 2010 ACM.
- Zarnescu, D., Callan, M. A., Cabernard, C., Heck, J., Luois, S., Doe, C. Q., & Zarnescu, D. C. (2010). Fragile X protein controls neural stem cell proliferation in the Drosophila brain. Human molecular genetics, 19(15).More infoFragile X syndrome (FXS) is the most common form of inherited mental retardation and is caused by the loss of function for Fragile X protein (FMRP), an RNA-binding protein thought to regulate synaptic plasticity by controlling the localization and translation of specific mRNAs. We have recently shown that FMRP is required to control the proliferation of the germline in Drosophila. To determine whether FMRP is also required for proliferation during brain development, we examined the distribution of cell cycle markers in dFmr1 brains compared with wild-type throughout larval development. Our results indicate that the loss of dFmr1 leads to a significant increase in the number of mitotic neuroblasts (NB) and BrdU incorporation in the brain, consistent with the notion that FMRP controls proliferation during neurogenesis. Developmental studies suggest that FMRP also inhibits neuroblast exit from quiescence in early larval brains, as indicated by misexpression of Cyclin E. Live imaging experiments indicate that by the third instar larval stage, the length of the cell cycle is unaffected, although more cells are found in S and G2/M in dFmr1 brains compared with wild-type. To determine the role of FMRP in neuroblast division and differentiation, we used Mosaic Analysis with a Repressible Marker (MARCM) approaches in the developing larval brain and found that single dFmr1 NB generate significantly more neurons than controls. Our results demonstrate that FMRP is required during brain development to control the exit from quiescence and proliferative capacity of NB as well as neuron production, which may provide insights into the autistic component of FXS.
- Zarnescu, D., Epstein, A. M., Bauer, C. R., Ho, A., Bosco, G., & Zarnescu, D. C. (2009). Drosophila Fragile X protein controls cellular proliferation by regulating cbl levels in the ovary. Developmental biology, 330(1).More infoFMRP is an RNA binding protein linked to the most common form of inherited mental retardation, Fragile X syndrome (FraX). In addition to severe cognitive deficits, FraX etiology includes postpubescent macroorchidism, which is thought to result from overproliferation. Using a Drosophila FraX model, we show that FMRP controls germline proliferation during oogenesis. dFmr1 null ovaries contain egg chambers with both fewer and supranumerary germ cells. The mutant germaria contain a significantly increased number of cyclin E and PhosphoHistone H3 positive cells, suggesting that loss of FMRP leads to defects in cell cycle progression. BrdU incorporation and flow cytometry data suggest that, in addition to proliferation, germline endoreplication and ploidy are also affected by the loss of FMRP during ovary development. Here we report that FMRP controls the levels of cbl mRNA in the ovary and that reducing cbl gene dosage by half rescues the dFmr1 oogenesis phenotypes. These data support a model whereby FMRP controls germline proliferation by regulating the expression of cbl in the developing ovary.
- Bauer, C. R., Epstein, A. M., Sweeney, S. J., Zarnescu, D. C., & Bosco, G. (2008). Genetic and systems level analysis of Drosophila sticky/citron kinase and dFmr1 mutants reveals common regulation of genetic networks. BMC Systems Biology, 2.More infoPMID: 19032789;PMCID: PMC2610033;Abstract: Background: In Drosophila, the genes sticky and dFmr1 have both been shown to regulate cytoskeletal dynamics and chromatin structure. These genes also genetically interact with Argonaute family microRNA regulators. Furthermore, in mammalian systems, both genes have been implicated in neuronal development. Given these genetic and functional similarities, we tested Drosophila sticky and dFmr1 for a genetic interaction and measured whole genome expression in both mutants to assess similarities in gene regulation. Results: We found that sticky mutations can dominantly suppress a dFmr1 gain-of-function phenotype in the developing eye, while phenotypes produced by RNAi knock-down of sticky were enhanced by dFmr1 RNAi and a dFmr1 loss-of-function mutation. We also identified a large number of transcripts that were misexpressed in both mutants suggesting that sticky and dFmr1 gene products similarly regulate gene expression. By integrating gene expression data with a protein-protein interaction network, we found that mutations in sticky and dFmr1 resulted in misexpression of common gene networks, and consequently predicted additional specific phenotypes previously not known to be associated with either gene. Further phenotypic analyses validated these predictions. Conclusion: These findings establish a functional link between two previously unrelated genes. Microarray analysis indicates that sticky and dFmr1 are both required for regulation of many developmental genes in a variety of cell types. The diversity of transcripts regulated by these two genes suggests a clear cause of the pleiotropy that sticky and dFmr1 mutants display and provides many novel, testable hypotheses about the functions of these genes. As both of these genes are implicated in the development and function of the mammalian brain, these results have relevance to human health as well as to understanding more general biological processes. © 2008 Bauer et al; licensee BioMed Central Ltd.
- Chang, S., Bray, S. M., Zigang, L. i., Zarnescu, D. C., Chuan, H. e., Jin, P., & Warren, S. T. (2008). Identification of small molecules rescuing fragile X syndrome phenotypes in Drosophila. Nature Chemical Biology, 4(4), 256-263.More infoPMID: 18327252;Abstract: Fragile X syndrome is caused by the functional loss of the fragile X mental retardation 1 (FMR1) gene. Deletion of the FMR1 ortholog in Drosophila melanogaster (Fmr1) recapitulates many phenotypes associated with fragile X syndrome. We have discovered that Fmr1 mutant Drosophila die during development when reared on food containing increased levels of glutamate, which is consistent with the theory that FMR1 loss results in excess glutamate signaling. Using this lethal phenotype, we screened a chemical library of 2,000 compounds and identified nine molecules that rescued the lethality, including three that implicate the GABAergic inhibitory pathway. Indeed, GABA treatment rescued several known Fmr1 mutant phenotypes in flies, including mushroom bodies defects, excess Futsch translation and abnormal male courtship behavior. These data are consistent with GABAergic inhibition of the enhanced excitatory pathway in fragile X syndrome. In addition, our screen reveals that the muscarinic cholinergic receptors may have a role in fragile X syndrome in parallel to the GABAergic pathway. These results point to potential therapeutic approaches for treating fragile X syndrome. © 2008 Nature Publishing Group.
- Zarnescu, D., Estes, P. S., O'Shea, M., Clasen, S., & Zarnescu, D. C. (2008). Fragile X protein controls the efficacy of mRNA transport in Drosophila neurons. Molecular and cellular neurosciences, 39(2).More infoFragile X syndrome, the most common form of inherited mental retardation is caused by mutations in the FMR1 gene. FMR1 encodes an RNA-binding protein thought to control the transport and translation of target mRNAs. While the function of FMRP in translational control has been clearly demonstrated, its role in mRNA transport and localization in neurons remains elusive. Using a genetically encoded mRNA imaging system in Drosophila we provide the first demonstration that FMRP controls mRNA transport. Live imaging of FMRP associated mRNAs show that mRNA granules are less motile and exhibit decreased directional movement in dFmr1 mutant neurons. Furthermore, Fluorescence Recovery After Photobleaching experiments show that the mobile fraction of mRNA molecules within neurites is dependent on FMRP dosage. These data support a model whereby FMRP regulates transport efficacy, by regulating the association between mRNA cargo and microtubules and suggest a new mechanism for the disease.
- Zarnescu, D. C., Jin, P., Betschinger, J., Nakamoto, M., Wang, Y., Dockendorff, T. C., Feng, Y., Jongens, T. A., Sisson, J. C., Knoblich, J. A., Warren, S. T., & Moses, K. (2005). Fragile X protein functions with Lgl and the PAR complex in flies and mice. Developmental Cell, 8(1), 43-52.More infoPMID: 15621528;Abstract: Fragile X syndrome, the most common form of inherited mental retardation, is caused by loss of function for the Fragile X Mental Retardation 1 gene (FMR1). FMR1 protein (FMRP) has specific mRNA targets and is thought to be involved in their transport to subsynaptic sites as well as translation regulation. We report a saturating genetic screen of the Drosophila autosomal genome to identify functional partners of dFmr1. We recovered 19 mutations in the tumor suppressor lethal (2) giant larvae (dlgl) gene and 90 mutations at other loci. dlgl encodes a cytoskeletal protein involved in cellular polarity and cytoplasmic transport and is regulated by the PAR complex through phosphorylation. We provide direct evidence for a Fmrp/Lgl/mRNA complex, which functions in neural development in flies and is developmentally regulated in mice. Our data suggest that Lgl may regulate Fmrp/mRNA sorting, transport, and anchoring via the PAR complex. Copyright © 2005 by Elsevier Inc.
- Zarnescu, D. C., Shan, G., Warren, S. T., & Jin, P. (2005). Come FLY with us: Toward understanding fragile X syndrome. Genes, Brain and Behavior, 4(6), 385-392.More infoPMID: 16098136;Abstract: The past few years have seen an increased number of articles using Drosophila as a model system to study fragile X syndrome. Phenotypic analyses have demonstrated an array of neuronal and behavioral defects similar to the phenotypes reported in mouse models as well as human patients. The availability of both cellular and molecular tools along with the power of genetics makes the tiny fruit fly a premiere model in elucidating the molecular basis of fragile X syndrome. Here, we summarize the advances made in recent years in the characterization of fragile X Drosophila models and the identification of new molecular partners in neural development. Copyright © Blackwell Munksgaard 2005.
- Jin, P., Zarnescu, D. C., Ceman, S., Nakamoto, M., Mowrey, J., Jongens, T. A., Nelson, D. L., Moses, K., & Warren, S. T. (2004). Biochemical and genetic interaction between the fragile X mental retardation protein 3nd the microRNA pathway. Nature Neuroscience, 7(2), 113-117.More infoPMID: 14703574;Abstract: Fragile X syndrome is caused by a loss of expression of the fragile X mental retardation protein (FMRP). FMRP is a selective RNA-binding protein which forms a messenger ribonucleoprotein (mRNP) complex that associates with polyribosomes. Recently, mRNA ligands associated with FMRP have been identified. However, the mechanism by which FMRP regulates the translation of its mRNA ligands remains unclear. MicroRNAs are small noncoding RNAs involved in translational control. Here we show that in vivo mammalian FMRP interacts with microRNAs and the components of the microRNA pathways including Dicer and the mammalian ortholog of Argonaute 1 (AGO1). Using two different Drosophila melanogaster models, we show that AGO1 is critical for FMRP function in neural development and synaptogenesis. Our results suggest that FMRP may regulate neuronal translation via microRNAs and links microRNAs with human disease.
- Zarnescu, D. C., & Moses, K. (2004). Born again at the synapse: A new function for the anaphase promoting complex/cyclosome. Developmental Cell, 7(6), 777-778.More infoPMID: 15572119;Abstract: Currently, perhaps the most significant biological problem is to understand the mechanisms of learning and memory, and many of the answers will come from molecular explanations of synaptic plasticity. Two new papers have established a surprising connection: the Anaphase Promoting Complex/Cyclosome (APC/C) has a second function in controlling local protein stability at synapses, and hence in the control of behavior. Copyright © 2004 Cell Press.
- Jin, P., Zarnescu, D. C., Zhang, F., Pearson, C. E., Lucchesi, J. C., Moses, K., & Warren, S. T. (2003). RNA-mediated neurodegeneration caused by the fragile X premutation rCGG repeats in Drosophila. Neuron, 39(5), 739-747.More infoPMID: 12948442;Abstract: Fragile X syndrome carriers have FMR1 alleles, called premutations, with an intermediate number of 5′ untranslated CGG repeats between patients (>200 repeats) and normal individuals (
- Médina, E., Williams, J., Klipfell, E., Zarnescu, D., Thomas, G., & Bivic, A. L. (2002). Crumbs interacts with moesin and βHeavy-spectrin in the apical membrane skeleton of Drosophila. Journal of Cell Biology, 158(5), 941-951.More infoPMID: 12213838;PMCID: PMC2173152;Abstract: The apical transmembrane protein Crumbs is necessary for both cell polarization and the assembly of the zonula adherens (ZA) in Drosophila epithelia. The apical spectrin-based membrane skeleton (SBMS) is a protein network that is essential for epithelial morphogenesis and ZA integrity, and exhibits close colocalization with Crumbs and the ZA in fly epithelia. These observations suggest that Crumbs may stabilize the ZA by recruiting the SBMS to the junctional region. Consistent with this hypothesis, we report that Crumbs is necessary for the organization of the apical SBMS in embryos and Schneider 2 cells, whereas the localization of Crumbs is not affected in karst mutants that eliminate the apical SBMS. Our data indicate that it is specifically the 4.1 protein/ezrin/radixin/moesin (FERM) domain binding consensus, and in particular, an arginine at position 7 in the cytoplasmic tail of Crumbs that is essential to efficiently recruit both the apical SBMS and the FERM domain protein, DMoesin. Crumbs, Discs lost, βHeavy-spectrin, and DMoesin are all coimmunoprecipitated from embryos, confirming the existence of a multimolecular complex. We propose that Crumbs stabilizes the apical SBMS via DMoesin and actin, leading to reinforcement of the ZA and effectively coupling epithelial morphogenesis and cell polarity.
- Zarnescu, D. C., & Thomas, G. H. (1999). Apical spectrin is essential for epithelial morphogenesis but not apicobasal polarity in Drosophila. Journal of Cell Biology, 146(5), 1075-1086.More infoPMID: 10477760;PMCID: PMC2169487;Abstract: Changes in cell shape and position drive morphogenesis in epithelia and depend on the polarized nature of its constituent cells. The spectrin-based membrane skeleton is thought to be a key player in the establishment and/or maintenance of cell shape and polarity. We report that apical β(Heavy)- Spectrin (β(H)), a terminal web protein that is also associated with the zonula adherens, is essential for normal epithelial morphogenesis of the Drosophila follicle cell epithelium during oogenesis. Elimination of β(H) by the karst mutation prevents apical constriction of the follicle cells during mid-oogenesis, and is accompanied by a gross breakup of the zonula adherens. We also report that the integrity of the migratory border cell cluster, a group of anterior follicle cells that delaminates from the follicle epithelium, is disrupted. Elimination of β(H) prevents the stable recruitment of α-spectrin to the apical domain, but does not result in a loss of apicobasal polarity, as would be predicted from current models describing the role of spectrin in the establishment of cell polarity. These results demonstrate a direct role for apical (αβ(H)2-spectrin in epithelial morphogenesis driven by apical contraction, and suggest that apical and basolateral spectrin do not play identical roles in the generation of apicobasal polarity.
- Thomas, G. H., Zarnescu, D. C., Juedes, A. E., Bales, M. A., Londergan, A., Korte, C. C., & Kiehart, D. P. (1998). Drosophila β(Heavy)-spectrin is essential for development and contributes to specific cell fates in the eye. Development, 125(11), 2125-2134.More infoPMID: 9570776;Abstract: The spectrin membrane skeleton is a ubiquitous cytoskeletal structure with several cellular roles, including the maintenance of cell integrity, determination of cell shape and as a contributor to cell polarity. We have isolated mutations in the gene encoding β(Heavy)-spectrin in Drosophila, and have named this essential locus karst. karst mutant individuals have a pleiotropic phenotype characterized by extensive larval lethality and, in adult escapers, rough eyes, bent wings, tracheal defects and infertility. Within karst mutant eyes, a significant number of ommatidia specifically lack photoreceptor R7 alongside more complex morphological defects. Immunolocalization of β(Heavy)-spectrin in wild-type eye-antennal and wing imaginal discs reveals that β(Heavy)-spectrin is present in a restricted subdomain of the membrane skeleton that colocalizes with DE-cadherin. We propose a model where normal levels of Sevenless signaling are dependent on tight cell-cell adhesion facilitated by the β(Heavy)-spectrin membrane skeleton. Immunolocalization of β(Heavy)-spectrin in the adult and larval midgut indicates that it is a terminal web protein, but we see no gross morphological defects in the adult apical brush border in karst mutant flies. Rhodamine phalloidin staining of karst mutant ovaries similarly reveals no conspicuous defect in the actin cytoskeleton or cellular morphology in egg chambers. This is in contrast to mutations in α-spectrin, the molecular partner of β(Heavy)-spectrin, which affect cellular structure in both the larval gut and adult ovaries. Our results emphasize the fundamental contribution of the spectrin membrane skeleton to normal development and reveals a critical interplay between the integrity of a cell's membrane skeleton, the structure of cell-cell contacts and cell signaling.
- Zarnescu, D. C. (2018, April). Lost in Translation - Synaptic and metabolic deficits in ALS. Annual Drosophila Research Conference - Plenary talk. Philadelphia: Genetics Society of America.
- Zarnescu, D. C. (2018, March). Flies in motion – what Drosophila can tell us about neurodegenerative disease. ASU Science Seminar. Phoenix: ASU.
- Zarnescu, D. C. (2017, April/2017). Tales from the brain: RNA processing defects lead to synaptic dysfunction in ALS. Saint Louis University, Department of Biochemistry. Saint Louis.
- Zarnescu, D. C. (2017, December). Modeling ALS in Drosophila. AZ Wellbeing Commons - Neuroscience. Phoenix: ASU.
- Zarnescu, D. C. (2017, July/2017). Post-transcriptional Inhibition in Multiple Models of ALS. ALS Gordon Conference. Stowe VT: GRC.
- Zarnescu, D. C. (2017, November/2017). Flies on drugs: what Drosophila can tell us about human disease. UA Pharmacology seminar.
- Zarnescu, D. C. (2017, Sep/2017). What can fruit flies tell us about neurodegeneration. AZ 2nd ALS Symposium. Biosphere 2.
- Zarnescu, D. C. (2016, August). Synaptic defects caused by RNA dysregulation in ALS. Packard Center for ALS - Johns Hopkins University.
- Zarnescu, D. C. (2016, May). Synaptic and metabolic dysregulation in ALS. 1st Pittsburgh ALS Symposium.
- Zarnescu, D. C. (2016, November). Synaptic defects caused by RNA dysregulation in ALS. RNA binding proteins in neurological disorders.
- Zarnescu, D. C. (2016, October). Synaptic defects caused by RNA dysregulation in ALS. MCB seminar series.
- Zarnescu, D. C. (2016, September). Synaptic and metabolic dysregulation in ALS-what flies can tell us about ALS. 1st Arizona ALS Symposium.
- Zarnescu, D. C. (2015, March). Futsch/MAP1B is a translational target of TDP-43 and is neuroprotective in a Drosophila model of ALS. American society for Neurochemistry.
- Zarnescu, D. C. (2015, March). TDP-43 dependent translation dysregulation in ALS. American Society for Neurochemistry.
- Zarnescu, D. C. (2015, Summer). RNA dysregulation in ALS. U Mass Medical School NeurologyU Mass Medical School.
- Zarnescu, D. C. (2014, April). Drug screening in a Drosophila model of ALS. 55th Annual Drosophila Genetics Meeting.
- Zarnescu, D. C. (2014, April). Stress granules and translation dysregulation in ALS - an RNA centric view of neurodegeneration. SUNY Albany.
- Zarnescu, D. C. (2014, August). ALS research update. Himelic foundation board.
- Zarnescu, D. C. (2014, December). Futsch/MAP1B is a translational target of TDP-43 and is neuroprotective in a Drosophila model of ALS. MND International Symposium Brussels.
- Zarnescu, D. C. (2014, October). Of flies and humans: deciphering the molecular mechanisms of ALS. Neurology Grand Rounds.
- Zarnescu, D. C. (2013, April). Flying High: What Drosophila can tell us about motor neuron disease. Barrow Neurological Institute.
- Zarnescu, D. C. (2013, December). FMRP regulates TDP-43 toxicity in a Drosophila model of ALS. Motor Neuron Disease Association Annual Meeting. Milan.
- Zarnescu, D. C. (2013, February). RNA binding proteins in ALS. Northeast ALS Consortium, Science Advisory Board meeting.
- Zarnescu, D. C. (2013, June). Flies in motion: what Drosophila can tell us about ALS. Pennsylvania Drug Discovery Institute.
- Zarnescu, D. C. (2013, March). Drug discovery in a Drosophila model of ALS. 54th Annual Drosophila meeting.
- Zarnescu, D. C. (2012, October). Flies on Drugs: A Search for ALS Therapeutics. Massachusetts General Hospital, ALS group.
- Zarnescu, D. C. (2012, September). Fragile X Protein is required for inhibition of insulin signaling and regulates glial-dependent neuroblast reactivation in the developing brain. Neurofly- The European Drosophila Neurobiology Meeting. Padua.
- Zarnescu, D. C. (2012, Spring). Deciphering RNA based mechanisms in development and disease-of flies, mice and men. Boston Biomedical Research Institute.
- Zarnescu, D. C. (2012, Spring). Deciphering RNA based mechanisms in development and disease-of flies, mice and men. Department of Biology U MassUniversity if Massachusetts.
- Zarnescu, D. C. (2011, Fall). RNA based mechanisms in development and disease. Neuroscience seminar series, U Illinois, Urbana-Champaign.
- Zarnescu, D. C. (2011, Fall). RNA tales from the brain-what flies can tell us about neurological disorders. Southern CA Drosophila meeting.
- Zarnescu, D. C. (2011, Spring). Deciphering RNA based mechanisms in development and disease (of flies, mice and men). Biology Department, Drexel University.
- Zarnescu, D. C. (2008, Summer). Fragile X protein controls the efficacy of mRNA transport in neurons. Gordon Conference-Cell Biology of the neuron.
- Zarnescu, D. C. (2010, Fall). RNA tales from the brain - what can Drosophila tell us about neurological disease. Vanderbilt Cell Biology seminars.
- Zarnescu, D. C. (2010, Spring). mRNA regulation during neuronal development: a Fragile X perspective. U Penn Genetics seminar.
- Zarnescu, D. C. (2010, Summer). RNA based mechanisms in development and disease. Developmental Biology workshop, Crete.
- Zarnescu, D. C. (2009, Spring). Mechanisms controlling the localization of RNA granules in neurons. Department of Biology seminar, Drexel University.
- Zarnescu, D. C. (2009, Spring). Mechanisms for Fragile X portein function and regulation in Drosophila. Penn State BMCB Department.
- Zarnescu, D. C. (2008, Fall). Mechanisms for transporting Fragile X protein and associated mRNAs in neurons. Translation at synapse, Janelia Farm, HHMI.
- Zarnescu, D. C. (2017, June/2017). Post-transcriptional Inhibition of Hsc70-4/HSPA8 Expression Leads to Synaptic Vesicle Cycling Defects in Multiple Models of ALS. EMBO Symposium on neurodegeneration. Heidelberg, Germany: EMBO.
- Zarnescu, D. C., & Coyne, A. (2016, August). Disease associated, mutant TDP-43 causes synaptic vesicle endocytosis defects by repressing the translation of Hsc70-4 in a Drosophila model of ALS. Gordon Conference - Brain disorders.More infoAmyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease affecting upper and lower motor neurons. TDP-43, an RNA binding protein linked to the majority of ALS cases, is involved in several aspects of RNA metabolism. Using a Drosophila model of ALS we have previously identified a role for TDP-43 in the localization and translation of futsch/MAP1B mRNA, and have shown that remodeling of TDP-43 complexes by Fragile X Protein (FMRP) is neuroprotective by restoring the translation of target mRNAs. Here we use a combination of genetic, molecular, and imaging approaches to show that disease associated TDP-43G298S but not TDP-43WT regulates the translation of hsc70-4 mRNA, a molecular chaperone that functions at multiple steps in the synaptic vesicle cycle. FM1-43 dye uptake experiments reveal defects in endocytosis and a reduction in the size of the readily releasable and recycling vesicle pools in both TDP-43WT and TDP-43G298S variants. However, upon overexpression of Hsc70-4, endocytosis is restored specifically only in the disease associated mutant. Genetic interaction experiments with cysteine string protein (CSP), dynamin, clathrin, lap, and auxilin suggest that synaptic vesicle cycling defects caused by TDP-43 occur during early endocytic events at the presynaptic membrane. In addition, overexpression of Hsc70-4 but not Hsc70-1 or Hsp83 mitigates multiple aspects of TDP-43 toxicity including locomotor dysfunction and reduced lifespan. Notably, this rescue is dependent on both the chaperone and membrane bending activities of Hsc70-4 as evidenced by genetic interactions with ATPase or membrane binding mutants, respectively. Thus, both chaperone and membrane bending activities, both of which contribute to the maintenance of synaptic protein pools, are affected upon TDP-43 overexpression and contribute to ALS phenotypes and toxicity. These results highlight mechanistic differences between TDPWT and TDPG298S, and provide the first evidence for synaptic dysfunction via translation inhibition caused by ALS associated mutant TDP-43. Furthermore, the discovery of Hsc70-4, a molecular chaperone, as a translation target of mutant TDP-43 helps integrate the ribostasis and proteostasis models of neurodegeneration.
- Zarnescu, D. C., & Coyne, A. (2016, July). Disease associated, mutant TDP-43 causes synaptic vesicle endocytosis defects by repressing the translation of Hsc70-4 in a Drosophila model of ALS. Gordon Conference - Cell Biology of the Neuron.More infoAmyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease affecting upper and lower motor neurons. TDP-43, an RNA binding protein linked to the majority of ALS cases, is involved in several aspects of RNA metabolism. Using a Drosophila model of ALS we have previously identified a role for TDP-43 in the localization and translation of futsch/MAP1B mRNA, and have shown that remodeling of TDP-43 complexes by Fragile X Protein (FMRP) is neuroprotective by restoring the translation of target mRNAs. Here we use a combination of genetic, molecular, and imaging approaches to show that disease associated TDP-43G298S but not TDP-43WT regulates the translation of hsc70-4 mRNA, a molecular chaperone that functions at multiple steps in the synaptic vesicle cycle. FM1-43 dye uptake experiments reveal defects in endocytosis and a reduction in the size of the readily releasable and recycling vesicle pools in both TDP-43WT and TDP-43G298S variants. However, upon overexpression of Hsc70-4, endocytosis is restored specifically only in the disease associated mutant. Genetic interaction experiments with cysteine string protein (CSP), dynamin, clathrin, lap, and auxilin suggest that synaptic vesicle cycling defects caused by TDP-43 occur during early endocytic events at the presynaptic membrane. In addition, overexpression of Hsc70-4 but not Hsc70-1 or Hsp83 mitigates multiple aspects of TDP-43 toxicity including locomotor dysfunction and reduced lifespan. Notably, this rescue is dependent on both the chaperone and membrane bending activities of Hsc70-4 as evidenced by genetic interactions with ATPase or membrane binding mutants, respectively. Thus, both chaperone and membrane bending activities, both of which contribute to the maintenance of synaptic protein pools, are affected upon TDP-43 overexpression and contribute to ALS phenotypes and toxicity. These results highlight mechanistic differences between TDPWT and TDPG298S, and provide the first evidence for synaptic dysfunction via translation inhibition caused by ALS associated mutant TDP-43. Furthermore, the discovery of Hsc70-4, a molecular chaperone, as a translation target of mutant TDP-43 helps integrate the ribostasis and proteostasis models of neurodegeneration.
- Zarnescu, D. C. (2015, Summer). TDP-43 dependent translation dysregulation in motor neurons. EMBO Mechanisms of neurodegeneration.
- Zarnescu, D. C. (2014, August). Futsch/MAP1B is a translational target of TDP-43 and is neuroprotective in a Drosophila model of ALS. GRC Neurobiology of brain disorders.
- Zarnescu, D. C. (2013, November). FMRP regulates TDP-43 toxicity in a Drosophila model of ALS. RNA binding proteins in neurological disease.
- Zarnescu, D. C. (2012, October). Decipering the role and therapeutic potential of the insulin signaling pathway in ALS. Northeast ALS Consortium. Clearwater.
- Coyne, A. N., & Zarnescu, D. C. (2016. A Helping Hand: RNA-Binding Proteins Guide Gene-Binding Choices by Cohesin Complexes(pp e1006419).