Fibroblast Growth Factor Receptor signaling and Src Family Kinase activity gate homeostatic synaptic plasticity
Synapses undergo many stresses and plastic changes throughout the life of an organism. Homeostatic mechanisms respond to these stresses and maintain synaptic activity within a physiologically favorable range. When faced with a reduction in postsynaptic glutamate receptor activity, the Drosophila neuromuscular junction (NMJ) homeostatically compensates by sending a retrograde signal to the presynaptic nerve. This signal triggers an increase in the number of synaptic vesicles released from the presynaptic terminal during an action potential. One of the least well understood aspects of this process is how postsynaptic systems drive production of homeostatic retrograde signals. We have identified several factors that regulate homeostatic synaptic plasticity in the postsynaptic muscle through an RNAi- and electrophysiology-based screen. This screen revealed that C-terminal Src Kinase (Csk) and the fibroblast growth factor receptor (FGFR) Heartless (Htl) are required for homeostatic compensation at the NMJ.
Work with Csk mutant alleles shows that Csk is required for the long-term maintenance of synaptic homeostasis, but not the rapid induction of this process. Csk phosphorylates and inactivates Src Family Kinases (SFKs), of which there are two in Drosophila: Src64B and Src42A. Overexpression and suppression experiments indicate that the homeostatic defects of Csk mutants are due to elevated SFK activity in the postsynaptic muscle. Immunostaining reveals that Csk mutants have altered NMJ localization of the neural cell adhesion molecule (NCAM) ortholog Fasciclin II (FasII). We examined a potential role for FasII in homeostatic plasticity and found that increasing FasII levels partially impairs this process. Additionally, reducing FasII in a Csk mutant background restores homeostatic compensation, suggesting that Csk and FasII may regulate homeostatic compensation through a common pathway.
We show that Htl is required in the postsynaptic muscle for the long term maintenance, but not the rapid induction, of homeostatic signaling. Htl is known to activate Src64B, and we show that Src64B is required for homeostasis in the postsynaptic muscle and link Src64B and Htl/FGFR signaling in the context of homeostatic compensation. FasII has been implicated as a regulator of Htl activity in Drosophila, which is supported by our observation that FasII genetically interacts with Htl during homeostatic compensation. Collectively, these data shed light on several postsynaptic factors that may work in concert to regulate the production of a homeostatic retrograde signal.
Nicotinamide Riboside is Uniquely Bioavailable In Vivo
Nicotinamide riboside is a recently discovered NAD precursor vitamin with unique activities in protection against metabolic and neurodegenerative conditions. Though nicotinamide riboside has been administered through multiple routes, it has not been established whether it achieves different or superior bioavailability in any target tissue with respect to the other NAD precursor vitamins, nicotinic acid and nicotinamide. Moreover, because enzymatic digestion of nicotinamide riboside can produce the other two NAD precursor vitamins, it is not clear whether nicotinamide riboside acts as a unique chemical entity or whether there are nicotinamide riboside-specific biomarkers. Here we show that nicotinamide riboside exhibits superior oral availability in mouse despite its metabolism to nicotinamide prior to absorption.
|Thursday 6/11/2015||Johnny Cruz Corchado||12:00-1:00 pm||106 BBE|
|Thursday 6/25/2015||Katie Weihbrecht||12:00-1:00 pm||106 BBE|
|Thursday 7/9/2015||Danielle Herrig||12:00-1:00 pm||B20 BB|
|Thursday 7/23/2015||Melissa Marchal||12:00-1:00 pm||106 BBE|
|Thursday 8/6/2015||Changya Chen||12:00-1:00 pm||106 BBE|
DM-MYB REGULATION OF CELL CYCLE GENES IS INDEPENDENT OF NURF
J F Santana1, M Parida2, A Long3, J Birdsall3, K Rogers3, M Aguilera3, S McDermott3, and J R Manak1,2,3,4
1Interdisciplinary Graduate Program in Genetics, University of Iowa, Iowa City, IA.
2Interdisciplinary Graduate Program in Informatics, University of Iowa, Iowa City, IA.
3Department of Biology, University of Iowa, Iowa City, IA.
4Department of Pediatrics, University of Iowa Carver College of Medicine, Iowa City, IA.
c-Myb is a proto-oncogene which when mutated causes leukemia and lymphoma in birds and mammals. Vertebrates contain three representatives of the Myb gene family consisting of A-, B- and c-Myb, all of which encode DNA-binding factors that are important for the proper expression of large numbers of genes including those that regulate cell cycle progression. Drosophila melanogaster contains a single Myb gene (Dm-Myb), mutants of which die before reaching adulthood. Dm-Myb protein is present in a complex which includes the nucleosome remodeling factor NURF. Through yeast two-hybrid experiments and genetic screens, we have shown that Dm-Myb is directly interacting with the major subunit of NURF (NURF301). In light of these results, we performed gene expression analyses in wing discs of Dm-Myb and Nurf301 mutant animals and found that there is a strong overlap of the genes regulated by these two proteins. We show that in vivo, as previously reported in cell lines, Dm-Myb is necessary for the activation of cell cycle genes, specifically those involved in the G2/M transition. However, despite the strong overlap of genes co-regulated by Dm-Myb and NURF, the latter is not required for the regulation of this class of genes, suggesting that Dm-Myb and NURF function together in some contexts but independently in others. Consistent with these data, Dm-Myb, but not Nurf301, mutant wing discs have an increased mitotic index, with only Dm-Myb mutant animals showing a significant developmental delay presumably due to the increased time required for mitotic cells to progress through G2/M.
The Genetics Social Activities Committee would like to congratulate Dr. Ralph Hazelwood on the successful defense of his thesis and completion of his Doctorate!
Ralph is from the Fingert lab in the Visual Sciences department. His thesis seminar, entitled “Molecular genetics of optic nerve disease using patients with cavitary optic disc anomaly”, was on Monday, March 9th, 2015. Ralph has accepted a postdoctoral appointment at Vanderbilt, where we expect he will continue to excel.
We asked Ralph a few questions about his experience in the Genetics program here at Iowa:
Q. What is the most valuable/favorite thing that you have learned/experienced here at Iowa?
A. Building networks outside of my department and field was invaluable because it exposed me to different areas of research and allowed me to make great contacts and actually build friendships with well-known and senior investigators at great institutions and markets.
Q. What was your favorite class to TA?
A. Human Molecular Genetics
Q. Do you have any advice about preparing for defense?
A. Tons of advice but the most important ones are: don’t wait until the last minute to write your thesis even if your committee hasn’t given the ok. It will help you gather your thoughts and help you to start making a story which will help in the long run. The Grad college template for submission is no easy task so the earlier your start the better sanity you will have. Also, the defense is literally just that, you have to defend the work you did so be prepared to talk about why you did the particular experiment(s) and why you came to that particular conclusion(s) and the caveats based on the data. But most importantly, you pretty much know more about your project than anyone else and so your thesis committee wouldn’t allow you to schedule a defense if they didn’t feel you were ready. Finally, think about big picture impacts and/or similar genetics mechanisms or disease processes to your project which should show you have a grasp of the field and genetics in general
Again, congratulations Ralph and good luck at Vanderbilt!
Genome-wide analysis of differential DNA methylation due to social defeat stress mouse model
Early life stress is a significant risk factor for anxiety and depressive disorders, and DNA methylation (DNAm) plays an important role in the intervening pathways. The extent to which DNAm is altered across the genome in these disorders is unknown. We sought to identify the genes and regulatory sequences altered by stress in a mouse model of the disorders. Using a social defeat stress paradigm, we assessed genome-wide DNAm changes in mouse brain. We used two cohorts of stressed mice (N=7 and 7) and non-stressed controls (N=8 and 12), for discovery and validation, respectively. DNA was extracted from the dentate gyrus using a punch technique. DNA from the first cohort was examined in the discovery phase using the target enrichment system Methyl-Seq, which captures ~220,000 CpG-rich regulatory regions of the genome. DNA from the second cohort was used for validation with bisulfite pyrosequencing. Analysis of the Methyl-Seq data was done using GENESPRING and MethylKit. Thirty-three regions were significant in the discovery phase. Of these, 31 did not validate. In the remaining two regions, we detected differentially methylated loci between stressed and control groups. These loci represent an intronic region of Drosha (encoding a microRNA processing protein) and an intergenic region on chromosome X (IntchX). However when these two regions were further interrogated with a new cohort of mice (N=16 stressed and non-stressed), only IntchX replicated. IntchX included eight differentially methylated CpGs over 150 bps within an evolutionary conserved region. Because only one differentially methylated region due to social defeat stress region was replicated, further analysis of the Methyl-Seq data with follow-up bisulfite pyrosequencing will be performed to elucidate additional regions. Moreover, the nucleus accumbens, the brain region essential to reward, will be investigated for DNAm differences induced from the social defeat stress paradigm.
Sleep Abnormalities in a Drosophila Model of Human GEFS+
Despite an established link between epilepsy and sleep behavior, it remains unclear exactly how specific epileptogenic mutations affect sleep and how sleep influences epileptic seizures. Drosophila is an attractive model for studying the underlying mechanisms of this seizure/sleep relationship as it is routinely used to examine the genetic basis of seizure susceptibility and sleep behavior. Sun et al (2012) recently created a knock-in fly model of human Generalized Epilepsy with Febrile Seizures Plus (GEFS+), a wide spectrum disorder characterized by fever-associated seizing in childhood and lifelong affliction. GEFS+ flies carry a mutation in the voltage-gated sodium channel (Nav) gene, mimicking a disease-causing human Nav mutation (SCN1AK1270T) and display a semidominant heat-induced seizure phenotype as a result of abnormal electrophysiology in inhibitory GABAergic neurons. We found that GEFS+ mutation also dominantly modifies sleep behavior, with mutants exhibiting rapid sleep onset at dusk and increased nighttime sleep as compared to controls. This sleep profile was observed regardless of sex, mating status, and genetic background. Mutants’ exaggerated sleep was more resistant to carbamazepine (CBZ), a drug that reduces Drosophila GABAA receptor activity, and could be suppressed by either constant or acute scotophase light. We further observed that GEFS+ flies have normal circadian rhythm in free-running dark conditions, but significantly lack homeostatic rebound following sleep deprivation. Intriguingly, sleep deprivation treatment increased the heat-induced seizure susceptibility of control flies, but reduced the seizure severity of GEFS+ mutants. Ongoing experiments are addressing the potential significance of GABAergic inhibition on wake-promoting PDF+ neurons in GEFS+ mutant sleep and the impact of seizing on subsequent sleep behavior. Our findings thus far have characterized the sleep architecture of Drosophila harboring a human GEFS+ mutation and provided unique insight into the relationship between sleep and epilepsy.
DISCOVERY OF DRUGS TO RESCUE ΔF508-CFTR USING A GENOMIC SIGNATURE APPROACH
Matthew D. Strub and Paul B. McCray, Jr.
Background: Cystic fibrosis (CF) is a lethal autosomal recessive disease caused by mutations in the CFTR gene. The most common CFTR mutation, ΔF508, causes protein misfolding, resulting in proteosomal degradation. However, if ΔF508-CFTR is allowed to traffick to the cell membrane, anion channel function may be partially restored. The McCray Lab previously reported that transfection with a miR-138 mimic or knockdown of SIN3A in primary CF airway epithelia increases ΔF508-CFTR mRNA and protein levels, and partially restores cAMP-stimulated Cl- conductance.
Objective: We hypothesized that a genomic signature approach can be used to identify new bioactive molecules affecting ΔF508-CFTR rescue.
The Connectivity Map (CMAP): CMAP is a catalog of gene expression profiles from cells treated with a variety of bioactive molecules and has pattern-matching software to mine data. CMAP queries using miR-138 mimic and SIN3A DsiRNA gene expression signatures identified 27 molecules that mimicked miR-138 and SIN3A DsiRNA treatments. The molecules were screened in vitro for efficacy in improving ΔF508-CFTR trafficking, maturation, and Cl- current. The McCray Lab reported the identification of 4 molecules that partially restored ΔF508-CFTR, highlighting the utility of a genomic signature approach in drug discovery.
LINCS: CMAP has greatly expanded into the Library of Integrated Network-based Cellular Signatures (LINCS). Previously generated gene sets were used to iteratively query LINCS and 125 candidate molecules were selected for further testing. Functional screens performed in CFBE-(ΔF508/ΔF508) cells identified 7/125 compounds that partially rescued ΔF508.
Conclusion: Querying LINCS with relevant genomic signatures offers a novel method to identify new candidates for rescuing ΔF508-CFTR. Further analysis of these molecules and their derivatives are ongoing. We are also generating additional genomic signatures representing ΔF508 rescue for use in LINCS queries. These results represent an important step forward from our proof-of-concept CMAP studies and highlight the utility of LINCS in drug discovery for CF.
|1/14/2015||Xue Xiao||2-501 BSB|
|2/11/2015||Matthew Strub||2-501 BSB|
|3/11/2015||Patricia Braun||2-501 BSB|
|4/8/2015||Johnny Cruz Corchado||2-501 BSB|
|5/13/2015||Ralph Hazlewood||2-501 BSB|
THE ROLE OF COPY NUMBER VARIATION IN NON-SYNDROMIC CLEFT LIP AND PALATE
LA Harney1,3, BW Darbro1,3, A Long2, J Standley1, JC Murray1,3, JR Manak1,2,3
1Department of Pediatrics, The University of Iowa
2Department of Biology, The University of Iowa
3Interdiciplinary Genetics Program, The University of Iowa
Orofacial clefting is a common congenital abnormality with clefts of the lip and/or palate (CL/P) affecting approximately 1 in 700 live births. About 70% of CL/P cases are estimated to be non-syndromic (NS) and do not exhibit cognitive or multiple congenital abnormalities. Although numerous genetic studies have been performed, no large-scale studies have examined the contribution of amplified and deleted regions of the genome, known as copy number variations (CNVs), to CL/P. We performed array-based genomic hybridization on a NSCL/P cohort from the Philippines to identify CNVs associated with clefting. After using bioinformatic quality controls to minimize false-positives, we analyzed 84 NSCL/P cases and processed a replication cohort of 854 NSCL/P cases for further analysis. We used an analysis pipeline to identify CNVs that overlapped with exons of genes in regions sharing 50% or less overlap with segmental duplications and common CNVs annotated in the Database of Genomic Variants. Analysis of CNVs in the cohort of 84 NSCL/P cases identified 358 genes in amplified regions and 36 genes in deleted regions. 21 of these genes have been previously linked to clefting including SKI, CDH1, CHD7, PAX6, OFD1 and TGFBR3. We are conducting a trio study using the losses identified in the small cohort to determine if the CNVs are de novo or familial. CNV analysis of the replication cohort is currently underway, and we will perform expression analysis of genes within the altered copy number regions and alter their dose in zebrafish to determine their role in CL/P. In the future we plan to extend this analysis to intronic and intergenic CNVs in hopes to define how CNVs contribute to NSCL/P and identify novel, causative variants for the disease.
Understanding the mechanism of nutritional therapies for inherited seizure disorders in Drosophila
Epilepsy is one of the most common neurologic problems in the world. A significant portion of epileptic individuals are diagnosed with refractory epilepsy, and will not respond to anti-epileptic drugs. Nutritional therapies, such as the high-fat, low-carbohydrate ketogenic diet, show great promise to prevent or treat refractory epilepsy inexpensively and without serious side effects. However, the mechanism behind the beneficial effects of certain diets remains unknown. In the Kitamoto lab, we use the fruit fly Drosophila melanogaster as a model organism to study the effects of diet on neurological phenotypes displayed by mutants for the voltage-gated sodium (Nav) channel. Nav channels have been implicated in various human seizure disorders. In particular, mutations in the human SCN1A gene encoding a Nav channel have been associated with multiple seizure disorders including Generalized Epilepsy with Febrile Seizures Plus (GEFS+) and Dravet Syndrome. Shudderer (Shu), a gain-of-function mutant for the Drosophila Nav channel gene, is characterized by seizure-like behavioral defects such as spontaneous leg jerking and twitching. We have recently shown that food containing milk whey drastically suppresses these neurological phenotypes of Shu.
Here we find that the same dietary therapy which improved Shu’s phenotypes can significantly improve the seizure-like phenotypes of Drosophila Nav channel mutants, bang senseless (bss1, bss2) and a Drosophila knock-in model of human GEFS+ (dGEFS+). Further, we found that the rescue effect of milk whey can be extended to the seizure-prone mutants, easily shocked (eas) and slamdance (sda) which lack altered Nav channel function. These results suggest milk whey has a broad effect among Drosophila seizure-prone mutants. Ongoing research aims to further characterize the effects of milk whey on these and other seizure-prone mutants and identify the specific component in milk whey that improves their seizure-like phenotypes.