Author(s): Meigen Yu, Baylor College of Medicine; Meigen Yu, Baylor College of Medicine; Ismael Al-Ramahi, Baylor College of Medicine; Juan Botas, Baylor College of Medicine; Joshua Shulman, Baylor College of Medicine // ABSTRACT: Parkinson’s disease (PD) is a common and incurable neurodegenerative disorder with strong evidence for heritability. GBA is one of the most common genetic risk factors for PD, with carriers of loss-of-function variants having a 5- to 10-fold increased risk of PD. Complete loss of GBA causes a metabolic disorder called Gaucher’s disease, one in a group of over 50 rare diseases called lysosomal storage disorders (LSDs). In an exome-wide association study including 1156 subjects with PD, we discovered a significant variant burden among 53 LSD gene loci in addition to GBA, suggesting that other LSD genes also contribute to PD risk. To investigate further, I have utilized a Drosophila transgenic model to experimentally confirm which LSD genes likely modify PD pathogenesis. In flies, pan-neuronal over-expression of human α-synuclein induces Lewy body-like PD pathology along with progressive, age-dependent neurodegeneration and locomotor impairment. Using more than 300 independent RNA-interference strains, I knocked down 94 conserved LSD genes and screened for enhancers or suppressors of α-synuclein-mediated neurodegeneration. My screen has identified 15 genetic modifiers whose knockdown strongly enhances the α-synuclein-associated locomotor phenotype, including homologs of well-established PD risk genes (e.g. GBA, SCARB2, and SMPD1), as well as many novel gene candidates (ARSB, IDS, IDUA, LIPA, and NPC1). I have also identified that loss-of-function alleles for homologs of DNAJC5 and NPC1…

Author(s): Abby L. Olsen, Brigham and Women’s Hospital, Harvard Medical School; Mel B. Feany, Brigham and Women’s Hospital, Harvard Medical School // ABSTRACT: Idiopathic Parkinson’s disease is the second most common neurodegenerative disease and is estimated to be approximately 30% heritable. Genome wide association studies have revealed numerous loci associated with risk of development of Parkinson’s disease. The majority of genes identified in these studies are expressed in glia at either similar or greater levels than their expression in neurons, suggesting that glia may play a role in Parkinson’s disease pathogenesis. The role of individual glial risk genes in Parkinson’s disease development or progression is unknown, however. We hypothesized that some Parkinson’s disease risk genes exert their effects through glia. We developed a Drosophila model of alpha-synucleinopathy in which we can independently manipulate gene expression in neurons and glia. Human wild type alpha-synuclein is expressed in all neurons, and these flies develop the hallmarks of Parkinson’s disease, including motor impairment, death of dopaminergic and other neurons, and alpha-synuclein aggregation. In these flies, we performed a candidate genetic screen, using RNAi to knockdown 14 well-validated Parkinson’s disease risk genes in glia and measuring the effect on locomotion in order to identify glial modifiers of the alpha-synuclein phenotype. We identified 4 modifiers: aux, Lrrk, Ric, and Vps13, orthologs of the human genes GAK, LRRK2, RIT2, and VPS13C, respectively. Knockdown of each gene exacerbated neurodegeneration as measured by total and dopaminergic neuron loss. Knockdown of each modifier also increased alpha-synuclein oligomerization. These results…

Author(s): George C. Murray, The Jackson Laboratory & The University of Maine; Tim Hines, The Jackson Laboratory; Abby L.D. Tadenev, The Jackson Laboratory; Leeza Kopaeva, The Jackson Laboratory; Robert W. Burgess, The Jackson Laboratory // ABSTRACT: The SOX10/EGR2 transcription factors regulate expression of multiple genes involved in Schwann cell development and myelination, including the CMT1A-causal gene peripheral myelin protein 22 (PMP22). Downregulation of genes in the SOX10/EGR2 network to mitigate the effect of elevated PMP22 gene dosage is a compelling therapeutic strategy for CMT1A (SRINIVASAN et al. 2012). One gene in this expression network, signal induced proliferation associated 1 like 2 (SIPA1L2), became a target for CMT1A treatments when a patient GWAS identified a significant association between intronic SNPs in SIPA1L2 and severity of CMT1A symptoms (TAO et al. 2019). We evaluated the efficacy of Sipa1l2 knockout for improvement of neuromuscular dysfunction in a mouse model of CMT1A. We found that Sipa1l2 knockout causes a modest, non-significant improvement in nerve conduction velocity at 6 months in vivo. To determine whether CMT1A mice model the transcriptomic shifts exhibited by patient Schwann cells, and therefore are good models for testing interventions targeting the SOX10/EGR2 network, we reinterrogated publicly accessible RNASeq datasets from CMT1A mice and patient Schwann cell lines (ZHAO et al. 2018; MUKHERJEE-CLAVIN et al. 2019). This analysis revealed relatively consistent differential expression of SOX10/EGR2 network genes between mouse sciatic nerves and patient cell lines, findings that support model validity. Analysis of shared gene sets involved in disease-relevant processes like neuron…

Author(s): Sheila Marte, University of Michigan; Anthony Antonellis, University of Michigan // ABSTRACT: Aminoacyl-tRNA synthetases (ARSs) are essential enzymes required to charge tRNA molecules to cognate amino acids in the cytoplasm and mitochondria. Although ARSs are essential and ubiquitously expressed, loss-of-function (LOF) missense mutations in five dimeric ARS enzymes have been associated with dominant peripheral neuropathy (also known as Charcot-Marie-Tooth disease ). CMT is a genetically and clinically heterogeneous inherited peripheral neuropathy characterized by the progressive loss of motor and sensory function. Mutations in glycyl-tRNA synthetase (GARS1) have been associated with distinct clinical phenotypes where individuals can present with later-onset CMT or infantile spinal muscular atrophy. The mechanism by which mutations in GARS1 lead to distinct clinical phenotypes is currently unclear. Since all five implicated ARSs function as dimers and since CMT-associated ARS variants all cause a loss-of-function effect, we propose the possibility of a dominant-negative mechanism. To test dominant-negative effects of pathogenic glycyl-tRNA synthetase (GARS1) variants, we will develop a humanized yeast model and test GARS1 mutations for the ability to repress a wild-type copy of GARS1. To better understand the distinct clinical phenotypes, we will assess the dominant toxicity of a series of pathogenic GARS1 alleles to determine if toxicity in our yeast model correlates with disease severity. Finally, we will identify pathways that, when manipulated, improve GARS1 function by performing experimental evolution and gain-of-function studies using a hypomorphic allele and yeast growth assays. Here, we will present preliminary data on development of our humanized model and on…

Author(s): Grant Mangleburg, Baylor College of Medicine; Omar El Fadel, Baylor College of Medicine; Anh Le, Baylor College of Medicine; Ismael Al-Ramahi, Baylor College of Medicine; Ying-Wooi Wan, Baylor College of Medicine; Juan Botas, Baylor College of Medicine; Joshua Shulman, Baylor College of Medicine // ABSTRACT: Alzheimer’s disease (AD) is the most prevalent cause of dementia. Large RNA-Sequencing data sets have provided detailed molecular profiles of AD, but they do not give insight into the mechanistic underpinnings of disease process or differentiate between causal and downstream changes in gene expression. In this study, we combined bioinformatic prioritization and genetic screens in Drosophila melanogaster to identify human brain transcriptional changes that exacerbate or ameliorate neurotoxicity induced by Tau or amyloid-β (Aβ), the proteins which form the hallmark pathologic aggregates that define AD. First, we used human brain RNA-Seq data to nominate candidate drivers of large, AD-associated coexpression networks based on multiple bioinformatic criteria. We then tested nominated drivers for modification of Tau or Aβ neurotoxicity in vivo using transgenic Drosophila in a locomotor screen. Through our screen, we determined that modulating expression of 144 of 357 (40.3%) nominated human driver genes modified Tau or Aβ neurotoxicity. We additionally found that combining multiple bioinformatic prioritization criteria performed better than individual criteria for identification of genetic modifiers. We identified a module with 51.9% of its prioritized genes modifying Tau/Aβ neurotoxicity in our screen. We used this module with our screen data to identify a subnetwork containing genes involved in synaptic function including GRIN2A,…

Author(s): Michael J. Molumby, Howard Hughes Medical Institute, Department of Cellular and Molecular Medicine, University of California San Diego, San Diego, United States; Michael J. Molumby, Howard Hughes Medical Institute, Department of Cellular and Molecular Medicine, University of California San Diego, San Diego, United States; Mridu Kapur, Howard Hughes Medical Institute, Department of Cellular and Molecular Medicine, University of California San Diego, San Diego, United States; Susan L. Ackerman, Howard Hughes Medical Institute, Department of Cellular and Molecular Medicine, University of California San Diego, San Diego, United States // ABSTRACT: Many genetic loci contribute to complex polygenic disease etiology. Genome-wide association studies have established that most complex disease risk loci reside in non-coding regions containing single nucleotide polymorphisms (SNPs) within regulatory elements. Sequence variation at these non-coding risk loci may contribute to complex disease manifestation via epistatic interactions with other risk loci to alter gene expression of coding and non-coding genes. Transfer RNAs (tRNAs) are highly conserved non-coding adapter molecules critical for protein synthesis and maintenance of cellular homeostasis. Strong purifying selection acts on mutations that occur within the mature tRNA sequence, however, the regions flanking highly expressed tRNA genes exhibit elevated mutation rates as a result of transcription-associated mutagenesis. To date, the impact of sequence changes in these regions on tRNA expression and function is unknown. Here, we present evidence that SNPs upstream of a tRNA gene differentially alter its expression by regulating RNA Polymerase III recruitment. These regulatory SNPs were identified during a forward genetic screen that utilized…

Author(s): Megan L. Mair, Baylor College of Medicine; Emma McCormick, Baylor College of Medicine; Bismark Amoh, Baylor College of Medicine; Cole Deisseroth, Baylor College of Medicine; Jiayang Li, Baylor College of Medicine; Justin Moore, Baylor College of Medicine; Juan Botas, Baylor College of Medicine // ABSTRACT: Research objectives: Aging is the single largest risk factor for the development of neurodegenerative disease. An interesting feature of this link between aging and neurodegeneration is that populations predisposed to long lifespan appear to be able to delay or avoid the development of neurodegenerative disease entirely. The objective of this study is to systemically analyze which neurodegenerative disease modifiers also have an impact on lifespan and healthspan of the central nervous system by using bioinformatics techniques and the Drosophila melanogaster animal model.    Methods: Utilizing a unique, high-throughput robotic behavioral assay system in tandem with the genetic tractability of the Drosophila animal model, our lab has identified over 1,000 genes that act as modifiers in Drosophila models of Alzheimer’s Disease, Parkinson’s Disease, and Huntington’s Disease when expression is manipulated in neurons and/or glia by the GAL4-UAS system. In addition, we have leveraged data from gene perturbation studies in model organisms documented in GeneAge and the literature in order to construct a biological network of lifespan extending genes that are conserved across evolution. Utilizing these methodologies to select gene candidates, we have screened genetic variants for their ability to protect against neurodegenerative disease pathology and confer longevity in Drosophila disease and aging models. Additionally, we have…

Author(s): Jonathan Merritt, Vanderbilt University Medical Center; Jonathan Merritt, PhD, Vanderbilt University Medical Center; Chetan Immanneni, Vanderbilt University; Alan Percy, MD, University of Alabama at Birmingham; Jeffrey Neul, MD, PhD, Vanderbilt Kennedy Center, Vanderbilt University Medical Center // ABSTRACT: Rett Syndrome (RTT) is a severe neurodevelopmental disorder primarily caused by mutations in the transcriptional regulator Methyl-CpG Binding Protein 2. Although the features of RTT are distinctive, there is variation in clinical severity and clear genotype-phenotype relationships can be observed on a group level. Despite this clear mutation group level genotype-phenotype relationship in RTT, there are individual “outliers” who are either less severely or more severely affected than the specific MECP2 mutation they carry would indicate. One source for this phenotypic variation is the presence of second site genetic modifiers with some genetic variants acting in a protective fashion and others in a detrimental manner to the clinical phenotypes of RTT. In order to identify genetic modifiers of RTT, we performed next generation sequencing on individuals who shared MECP2 mutations but had divergent clinical presentation (“severe” or “mild”). At this time, exome sequences have been obtained for 26 severe and 29 mild individuals, and whole genome sequencing has been performed on 55 severe and 57 mild individuals. To classify candidate modifiers, we performed association testing and pathway analysis to identify genes with differential frequency of variation between these phenotypic extremes. Among top-level results, we found damaging variants in genes related to isoprenoid biosynthesis associate with RTT severity. Understanding the biological role…

Author(s): Caroline Aragon, University of Minnesota; Divya Alley, University of Minnesota; Lihsia Chen, University of Minnesota // ABSTRACT: Primary congenital hydrocephalus (CH) is a progressive, life-threatening condition resulting from an imbalance in production, flow, and absorption of cerebrospinal fluid (CSF) in the brain. If untreated, subsequent ventricular dilation and increased intracranial pressure resulting from CSF buildup can cause brain malformations, progressive macrocephaly, neurocognitive deficits, and death. While the pathophysiology underlying CH is undefined, the most common cause is loss or impaired function of L1, a transmembrane immunoglobulin cell adhesion molecule with well-defined roles in neuronal migration, synaptic regulation, and axon guidance. While these roles suggest neurodevelopmental defects as the underlying cause, the precise mechanism for how loss of L1 causes CH remains largely unknown. A major factor contributing to our lack of knowledge is the incomplete penetrance and variable expressivity seen within families harboring L1 mutations. This variability in phenotypic severity, also observed in L1 knockout mice, points to the influence of genetic background on L1-associated CH. Uncovering L1 mechanisms of action is key to developing novel therapies and preventative measures for CH.   To facilitate the identification of genes interacting with L1, we use C. elegans, which has been a premier model  system to investigate clinically significant human genetic variation. The C. elegans model provides simplicity and ease for genetic manipulation while preserving access to complex genetic interactions. Importantly, C. elegans has a single canonical L1 gene, sax-7, with conserved neurodevelopmental roles including axon branching and dendrite morphogenesis and maintenance…

Author(s): Wenxi Yu, University of Michigan; James Xenakis, University of North Carolina at Chapel Hill; Jacy L. Wagnon, Ohio State University; Megan K. Mulligan, University of Tennessee Health Science Center; Robert W. Williams, University of Tennessee Health Science Center; Fernando Pardo-Manuel de Villena, University of North Carolina at Chapel Hill; Miriam H. Meisler, University of Michigan // ABSTRACT: SCN8A encephalopathy is a developmental epileptic encephalopathy that can result from de novo gain-of-function mutations in Nav 1.6. Affected individuals exhibit refractory seizures, developmental delay, cognitive disabilities, movement disorders, and elevated risk of sudden death. Patients with the identical SCN8A variant can differ in clinical course, suggesting a role for modifier genes in determining disease severity. To identify genetic modifiers of DEE, we generated F1 and F2 crosses between inbred mouse strains and mice carrying the human pathogenic variants SCN8A-R1872W and SCN8A-N1768D. Quantitative trait locus (QTL) analysis of seizure-related phenotypes was used for chromosomal mapping of modifier loci. In an F2 cross between strain SJL/J and C57BL/6J mice carrying the patient mutation R1872W, we identified a major QTL on chromosome 5 containing the Gabra2 gene. Strain C57BL/6J carries a splice site deletion mutation that results in a 75% reduction in expression of Gabra2, which encodes the α2 subunit of the aminobutyric acid type A (GABAA) receptor. Homozygosity for the hypomorphic Gabra2 mutation was associated with early seizure onset and shortened life span in the F2 mice. We confirmed Gabra2 as the modifier gene in crosses with a mouse knock-in allele in which…

Author(s): Alexis R. Demonbreun, Northwestern University; Katherine S. Fallon, Northwestern University; Mattia Quattrocelli, Northwestern University; Patrick G.T. Page, Northwestern University; Michele Hadhazy, Northwestern University; Carl Morris, Solid Biosciences; Elizabeth M. McNally, Northwestern University // ABSTRACT: Background and Methods:    Duchenne Muscular Dystrophy (DMD) is an X-linked recessive neuromuscular disorder caused by dystrophin mutations.  DMD is treated with glucocorticoid steroids to delay disease progression. Antisense oligonucleotides have now been approved to treat certain DMD mutations, and gene replacement therapy is being evaluated in clinical trials.  A genomewide scan was conducted in a mouse model of muscular dystrophy and identified a powerful modifier locus, which was ultimately identified as Ltbp4, encoding Latent TGF- Binding Protein 4.  LTBP4 protein localizes to the myofiber exterior where it binds and sequesters all three forms of TGF-beta, regulating latent TGF-beta release and activation.  Excess TGF-beta activation is a pathological finding in many forms of neuromuscular disease, especially DMD, the limb girdle muscular dystrophies and the congenital muscular dystrophies.  In the muscular dystrophies, excess or hyper-activated TGF-beta is linked to fibrotic infiltration of muscle and impaired muscle regeneration. The genetic data from mice identified an insertion/deletion polymorphism in the hinge region of LTBP4 as critical to latent TGF-beta release and activation. LTBP4’s hinge region can be proteolytically cleaved, and this cleavage promotes release of latent TGF-beta which is then fully activated by additional steps. The genetically protective form of LTBP4 in mice contains 12 amino acids inserted into the hinge, rendering it less susceptible to protease cleavage, correlating…

Author(s): Sophie Hill, University of Michigan; Sophie Hill, University of Michigan; Paymaan Jafar-Nejad, Ionis Pharmaceuticals; Frank Rigo, Ionis Pharmaceuticals; Miriam Meisler, University of Michigan // ABSTRACT: The neuronal voltage-gated sodium channel Nav1.6 is concentrated at the axon initial segment (AIS), where it plays a key role in initiation of action potentials. Gain-of-function mutations in SCN8A are a cause of developmental and epileptic encephalopathy (DEE). Administration of an antisense oligonucleotide (ASO) that reduces expression of Scn8a results in delayed seizure onset in a mouse model of Scn8a DEE. We previously showed interaction of Scn8a expression with the Scn1a gene (Lenk et al, Ann. Neurol. 87:339-346, 2020).  We then asked whether reduction of Scn8a expression could ameliorate the seizure phenotypes of mice with inactivation of Kcna1, which encodes a voltage-gated potassium channel that is also localized at the AIS. Kcna1 null mice have spontaneous seizures and premature death (Smart et al, 1998 Neuron). Homozygous Kcna1 null mice were treated on postnatal day 2 by intracerebroventricular injection of the Scn8a ASO. Surviving mice were given repeated doses of the ASO at four week intervals. Treatment with the Scn8a ASO rescued premature death in the Kcna1 null mice. This work demonstrates a novel genetic interaction between two ion channels expressed at the axon initial segment. Source of Funding: NINDS R01 NS34509

Author(s): Rebecca A. MacPherson, Clemson University; Rebecca A. MacPherson, Clemson University; Robert R. H. Anholt, Clemson University; Trudy F. C. Mackay, Clemson University // ABSTRACT: Coffin-Siris syndrome (CSS) and Nicolaides-Baraitser syndrome (NCBRS) are rare disorders of chromatin modification associated with alterations of subunits within the highly conserved mammalian SWI/SNF complex. CSS and NCBRS patients typically present with intellectual disability, facial and digit abnormalities, seizures, and hypotonia. However, phenotypic presentation and disease severity varies within and across CSS- and NCBRS-associated mutations; individuals with identical genetic alterations do not necessarily present with identical phenotypes. We hypothesize that there are naturally occurring genetic variants segregating in the human population that serve as modifiers of disease severity. Identification of candidate genetic modifiers may lead to insights on the pathogenesis and treatment of CSS, NCBRS, and related disorders, but genome wide association analyses that are used to map genetic variants associated with common human diseases require large sample sizes that are not possible for rare diseases.  Using a bipartite UAS-GAL4 RNA interference system in the model organism Drosophila melanogaster, we have developed a CSS/NCBRS fly model and show that flies with reduced expression of CSS-associated fly orthologs exhibit changes in sleep, activity, and sensorimotor integration. We have identified genes co-regulated with CSS-associated fly orthologs that exhibit similar changes in behavior to our CSS fly models, suggesting that these co-regulated genes may serve as plausible candidate genetic modifiers for CSS and NCBRS. We are currently working to identify epistatic genetic interactions between CSS-associated fly orthologs and…

Author(s): Paul C. Marcogliese, Baylor College of Medicine; Debdeep Dutta, Baylor College of Medicine; Shrestha Sinha-Ray, The Research Institute at Nationwide Children’s Hospital; Shinya Yamamoto, Baylor College of Medicine; Kathrin C Meyer, The Research Institute at Nationwide Children’s Hospital; Nan Cher Yeo, University of Alabama, Birmingham; Hugo J. Bellen, Baylor College of Medicine // ABSTRACT: De novo truncations in Interferon Regulatory Factor 2 Binding Protein Like (IRF2BPL) lead to severe childhood-onset neurodegenerative disorders. To determine how loss of IRF2BPL causes neural dysfunction, we examined its function in Drosophila and zebrafish. Overexpression of either IRF2BPL or Pits, the Drosophila ortholog, impairs Wnt signaling in flies. In contrast, neuronal depletion of Pits leads to increased Wingless (Wg) levels in the brain and is associated with axonal loss whereas inhibition of Wg is neuroprotective. Moreover, increased neuronal expression of wg in flies is sufficient to cause age-dependent axonal loss, similar to reduction of Pits. Loss of irf2bpl in zebrafish also causes neurological defects and increased Wnt signaling. WNT1 is also upregulated in patient-derived astrocytes, and pharmacological inhibition of Wnt suppresses the neurological phenotypes. Finally, IRF2BPL and the Wnt antagonist, CKIα, physically and genetically interact, showing that IRF2BPL and CkIα antagonize Wnt signaling. Source of Funding: This work was primarily supported by the Stand by Eli foundation (www.standbyeli.org) via The Giving Back Fund (www.givingback.org). Research was also supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health under Award Number P50HD103555 for use of…

Author(s): Jamy Peng, St. Jude Children’s Research Hospital; Yurika Matsui, St. Jude Children’s Research Hospital; Hongfeng Chen, St. Jude Children’s Research Hospital; Beisi Xu, St. Jude Children’s Research Hospital // ABSTRACT: Mutations in enzymes that modify lysine 27 in histone H3 (H3K27) are strongly associated with developmental disorders including Weaver syndrome, Kabuki syndrome, and Rubinstein-Taybi syndrome.  These enzymes deposit methylation (PRC2; Weaver), remove methylation (UTX; Kabuki), or deposit acetylation (CREBBP or EP300; Rubinstein-Taybi) on H3K27.  H3K27 modifications are linked to suppressed or active expression of developmental regulators, suggesting that dysfunction in H3K27 modifiers leads to deregulated genetic programs that cause these developmental disorders.  As the disorders present striking brain phenotypes, my group studies H3K27 modifiers in human brain organoids and the mouse embryonic brain.  We have been focused on identifying new players in PRC2 and UTX networks in neural stem cells.  We recently showed that the nucleoprotein Ybx1 binds to PRC2, influencing PRC2 localization and reducing H3K27 trimethylation levels genome-wide in embryonic neural stem cells.  The PRC2–Ybx1 interaction regulates spatiotemporal genetic programs in the developing brain.  In follow-up studies, we found that Ybx1 binds neurogenic enhancers and is required for maintaining H3K27 acetylation levels and expression of the enhancer target genes.  CUT&RUN followed by immunoprecipitation revealed that Ybx1 proteins co-occupy chromatin regions with either H3K27 methylation or acetylation.  We are using mouse knockout of Ybx1, PRC2, Utx, Cbp, and p300 to interrogate a synergistic or antagonistic interaction of Ybx1 with H3K27 modifiers in regulating embryonic brain development.  We hypothesize that…

Author(s): Sabrina G. Clemens, Brigham and Women’s Hospital; Abby L. Olsen, Brigham and Women’s Hospital // ABSTRACT: OBJECTIVE: To develop a Drosophila model for testing gene-environment interactions in Parkinson’s disease.   BACKGROUND: Parkinson’s disease (PD) is a neurodegenerative disease characterized by α-synuclein aggregation and the progressive loss of dopamine (DA) neurons in the substantia nigra. Risk of PD arises due to a combination of genetic and environmental factors, which may interact with one another, termed gene-environment (GxE) interactions. An inverse association between smoking cigarettes and risk of developing PD is well-established, but trials of nicotine as a therapeutic option for PD have yielded mixed results. A previous genome-wide GxE interaction study identified genetic variation in the synaptic-vesicle glycoprotein 2C (SV2C) locus as an important mediator of the degree to which smoking is inversely associated with PD. We sought to determine the mechanism of the smoking-SV2C interaction in a Drosophila model of PD.   DESIGN/METHODS: In this model, human α-synuclein is expressed in all neurons, and flies develop the hallmarks of PD, including motor dysfunction, loss of DA neurons, and formation of α-synuclein inclusions. We assessed the effects of increasing doses of nicotine on these parameters of neurodegeneration, in the presence or absence of SV2C knockdown.   RESULTS: We demonstrate that α-synuclein-expressing flies treated with nicotine (the presumed active ingredient in tobacco) have significant improvement in locomotion, in total number of brain cells and in DA neuron counts, and in α-synuclein aggregation and oligomerization. However, in α-synuclein-expressing flies in which Drosophila homologs of SV2C…

Author(s): Bin Gu, The Ohio State University; Bin Gu, The Ohio State University; John Shorter, the University of North Carolina, Chapel Hill; Timothy Bell, the University of North Carolina, Chapel Hill; Pablo Hock, the University of North Carolina, Chapel Hill; Benjamin Philpot, the University of North Carolina, Chapel Hill; Fernando Pardo-Manuel de Villena, the University of North Carolina, Chapel Hill // ABSTRACT: Objective: Epilepsies are a spectrum of clinically heterogeneous neurological disorders characterized by recurrent seizures and have complex etiology and genetic architecture. According to CDC, 1.2% of the US population have active epileptic seizures, which adversely affect patients’ life quality and increase the risk of mental comorbidities, hospitalization and mortality. Animal models play a fundamental role in understanding the genetic basis of epilepsy and identifying therapeutic interventions. However, most existing animal models of epilepsy suffer from limitations such as an inability to model genetically complex diseases. The Collaborative Cross (CC) mice is an innovative recombinant inbred panel of mice. The CC offers large genetic diversity and powerful genomic tools including whole genome sequence to facilitate identification of candidate genes and genetic variants. This study leverages the resource of CC to define the genetic basis of seizure related outcomes. Method: We measured multiple epilepsy related traits in 35 CC strains using flurothyl kindling model. We created an F2 mapping population from CC strains with extreme seizure susceptibility. We then tested their seizure related outcomes and genotyped them using Mini Mouse Universal Genotyping Array. We performed quantitative trait locus (QTL) mapping…

Author(s): Nicholas Johnson, MD, Virginia Commonwealth University; Melissa Hale, PhD, Virginia Commonwealth University; Kameron Bates, MS, Virginia Commonwealth University; Nicholas Johnson, MD, Virginia Commonwealth University // ABSTRACT: Research Objective: To identify genetic modifiers of DM1 in a large cohort of myotonic dystrophy type 1.    Background: Myotonic dystrophy type 1 is caused by a CTG repeat in the 3’UTR of the DMPK gene.  Genome-wide association studies (GWAS) have recently identified common trans-acting variants that associate with age of onset for Huntington disease (HD).  HD, like DM1, also results from germline and somatic expansion of CAG:CTG repeats, and it appears that the major trans-modifiers of HD act mainly at the level of somatic instability of repeats.  DM1 may be subject to similar influences and previous investigators have used a candidate gene approach in small DM1 cohorts; these studies reported nominal associations with common SNPs in DNA repair genes.    Methods: We will perform a GWAS in 700 individuals with DM1.  A PCR based method will determine length of the CTG repeat and test for CGG or CCG interruptions within the repeat tract.  We will use age of onset as the primary phenotype, incorporating a statistical model to control for the effects of repeat length.  Secondary phenotypes will include mean change on 10-meter walk (to assess the trajectory of motor function decline), grip strength, video hand opening time, and pulmonary or cardiac complications.  On all samples, whole exome genotyping will be performed with Illumina Omni2.5-Exome SNP arrays providing comprehensive coverage with ~2.5 million common,…

Author(s): Kristin C. Bussey, Clemson University; Kristin C. Bussey, Clemson University; Robert R. H. Anholt, Clemson University; Trudy F. C. Mackay, Clemson University // ABSTRACT: Mediator subunit 12 (MED12) is a component of the mediator complex, which acts as a co-activator/co-repressor of RNA polymerase II. Mutations in different domains of the MED12 protein are associated with rare genetic disorders, including FG Syndrome, Ohdo Syndrome, and Lujan Syndrome, which are characterized by a range of neurodevelopmental, physical, and behavioral anomalies. It is increasingly recognized that these disorders overlap in symptoms forming a spectrum of MED12-related phenotypes with variable penetrance. However, identifying genetic modifiers of rare diseases is impossible in human populations. To investigate the pathogenicity of MED12 mutations and to identify possible epistatic modifiers, we generated a Drosophila model centered on the fly ortholog of MED12, kohtalo (kto). First, we used RNAi-mediated knockdown of kto and measured locomotion and sleep parameters and startle response as a proxy for sensory motor integration in 3-5 day old flies and 14-16 day old flies, sexes separately.   Using two independent RNAi lines we found that knockdown of kto resulted in reduced nighttime sleep at both ages and in both sexes. Reduction in expression of kto also resulted in increased startle behavior in young flies and a reduction in startle response in 14-16 day old females. After we verified that impairment of kto expression results in phenotypic effects that are analogous to symptoms of MED12 disorders, we generated a CRISPR/Cas9 mediated excision line, which contains an…

Author(s): Jenna Levy, Scripps Research; Jenna Levy, Scripps Research; Christy LaFlamme, Florida Atlantic University; George Tsaprailis, Scripps Research; Gogce Crynen, Scripps Research; Damon T. Page, Scripps Research // ABSTRACT: Mutations in DYRK1A are a cause of microcephaly, autism spectrum disorder (ASD), and intellectual disability (ID); however, the underlying cellular and molecular mechanisms are not well understood. We generated a conditional mouse model using Emx1-cre, including conditional heterozygous and homozygous knockouts, to investigate the necessity of Dyrk1a in the cortex during development. We employed unbiased, high throughput phospho-proteomics to identify dysregulated signaling mechanisms in the developing Dyrk1a mutant cortex as well as classic genetic modifier approaches and pharmacological therapeutic intervention to rescue microcephaly and neuronal undergrowth caused by Dyrk1a mutations. We found that cortical deletion of Dyrk1a in mice causes decreased brain mass and neuronal size, structural hypoconnectivity, and autism-relevant behaviors. Using phospho-proteomic screening, we identified growth-associated signaling cascades dysregulated upon Dyrk1a deletion, including TrkB/BDNF, an important regulator of ERK/MAPK and mTOR signaling. Genetic suppression of Pten or pharmacological treatment with IGF-1, both of which impinge on these signaling cascades, rescued microcephaly and neuronal undergrowth in neonatal mutants. Altogether, these findings identify a previously unknown mechanism through which Dyrk1a mutations disrupt growth factor signaling in the developing brain, thus influencing neuronal growth and connectivity. Our results place Dyrk1a as a critical regulator of a biological pathway known to be dysregulated in humans with autism spectrum disorder and intellectual disability. Additionally, these data position Dyrk1a within a larger group of ASD/ID risk…

Author(s): Sheryl Anne Vermudez, Vanderbilt University; Aditi Buch, Vanderbilt University; Kelly Weiss, Vanderbilt University; Yuta Moxley, Vanderbilt University; Hemangi Rajpal, Vanderbilt University; Rocco G. Gogliotti, Loyola University Chicago; Colleen M. Niswender, Vanderbilt University // ABSTRACT: Pitt-Hopkins syndrome (PTHS) is a rare neurodevelopmental disorder caused by loss-of-function (LOF) mutations in the Transcription Factor 4 (TCF4) gene. Interestingly, PTHS closely resembles Rett syndrome (RTT), another neurodevelopmental disorder caused by LOF mutations in a related gene, Methyl-CpG-Binding Protein 2 (MECP2). These two disorders share molecular and behavioral phenotypes, including cognitive and motor dysfunction, in both the preclinical and clinical settings. Based upon this phenotypic/symptom overlap, and the potential clinical application of normalizing MECP2 gene dosage for RTT, we investigated whether such a treatment strategy could be leveraged in a PTHS mouse model. Specifically, we tested the hypothesis that genetic supplementation of MeCP2 would reverse the abnormal phenotypes in PTHS model mice that are Tcf4 haplosufficient, Tcf4+/-.    We interrogated this hypothesis at the preclinical level by introducing a wild-type MECP2 transgene from MeCP2-overexpressing mice (MECP2Tg1/o) to Tcf4+/- mice. Behavioral characterization illustrated that the MECP2 transgene reversed the phenotypes of Tcf4+/- mice, specifically hyperlocomotion, attenuated anxiety and contextual fear learning and memory deficit. Molecular studies suggest that MeCP2 does not impact Tcf4 expression, suggesting a more complex relationship between these two proteins. Subsequent RNA-sequencing studies have identified a subset of genes and pathways in the hippocampus and striatum that are disrupted in Tcf4+/- mice and normalized with the MECP2 transgene. Of note are genes involved…

Author(s): Emmy Li, University of California, San Francisco; Emmy Li, University of California, San Francisco; Mark Koontz, University of California, San Francisco; Nina Draeger, University of California, San Francisco; Steven Boggess, University of California, San Francisco; Erik Ullian, University of California, San Francisco; Martin Kampmann, University of California, San Francisco // ABSTRACT: The interactions between glial and neuronal cells play a large role in disease. Both astrocytes and microglia, for example, can become reactive in neurodegenerative diseases, leading to a response that may be detrimental to neuronal survival. Though many studies have investigated how certain glial cell types may independently contribute to disease, how interactions between neurons and glia may make neurons more vulnerable is less well understood.    Thus, we generated 3D iPSC-derived neuron-astrocyte-microglia assembloids (iAssembloids) to model how neuronal and glial cells interact. Making use of transcription factor-based neuronal and microglia differentiation protocols, iAssembloids are reproducible and easy to generate in a high-throughput manner, making them suitable for large-scale screening applications. In addition, transcriptional profiling and functional studies have shown that iPSC-derived neurons cultured in iAssembloids have higher expression of axon guidance-related genes and are more electrophysiologically active than standard monoculture iPSC-derived Ngn2 neurons. Therefore, iAssembloids can provide a more physiologically relevant handle on probing glia-neuron interactions.     With these iAssembloids, we performed pooled CRISPRi-based functional genomics screens in the iPSC-derived neurons and identified pathways that can promote or hinder neuronal survival specifically in the iAssembloid context, but not in monoculture. Pathways that we have identified include those important for…

Author(s): Kyle A. Sullivan, Oak Ridge National Laboratory, Computational and Predictive Biology, Oak Ridge, TN; David Kainer, Oak Ridge National Laboratory, Computational and Predictive Biology, Oak Ridge, TN; Xuejun Qin, Duke University School of Medicine, Duke University, Durham, NC; Durham Veterans Affairs Medical Center, Durham, NC; Jennifer Lindquist, Duke University School of Medicine, Duke University, Durham, NC; Durham Veterans Affairs Medical Center, Durham, NC; Nathan A. Kimbrel, Duke University School of Medicine, Duke University, Durham, NC; Durham Veterans Affairs Medical Center, Durham, NC; Daniel Jacobson, Oak Ridge National Laboratory, Computational and Predictive Biology, Oak Ridge, TN // ABSTRACT: Suicidal behavior comprises a complex psychiatric disorder and little evidence to date has identified significant genetic risk factors for suicidality. We therefore used a cross-ancestry, genome-wide association study (GWAS) to identify the causative genetic loci underlying suicide attempts using data from the Veterans’ Affairs (VA) Million Veteran Program (MVP). We identified 14,829 cases of non-fatal and fatal suicide attempts using a combination of electronic health record data from the VA Corporate Data Warehouse (CDW), Suicide Prevention Application Network (SPAN) database, and CDW Mental Health Domain; 410,464 subjects with no history of suicidal behavior were used as controls. Using genetic variants identified by GWAS, we assigned these variants to genes using H-MAGMA, a cutting-edge genomics approach used for assigning variants to genes based on Hi-C chromatin conformation data. We then used a novel systems biology, graph-driven approach (Random Walk with Restart Filter; RWR-Filter) to determine which variants, genes and associated biological pathways were…

Author(s): Diana J Zajac, University of Kentucky; Diana J Zajac, University of Kentucky; James Simpson, University of Kentucky; Josh Morganti, University of Kentucky; Steve Estus, University of Kentucky // ABSTRACT: INPP5D (Phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase 1), the gene encoding SHIP1 protein, contains single nucleotide polymorphisms (SNPs) that are strongly associated with Alzheimer’s Disease (AD) risk. In the brain and mostly in microglia, INPP5D is expressed as several isoforms. Full-length INPP5D encodes 27 exons, including an amino-terminal SH2 binding domain followed by the phosphatase domain that modulates downstream receptor signaling. Truncated isoforms lacking the SH2 domain begin from internal transcription start sites. First, we investigated INPP5D isoform expression as a function of AD status, and AD-associated SNP status to better understand the function of INPP5D in the human brain. The expression of microglial INPP5D isoforms was analyzed using quantitative polymerase chain reaction used on RNA from AD and non-AD anterior cingulate human brain samples. The AD-associated SNPs rs35349669 and rs10933431 were identified using TaqMan SNP kits (ABI). Isoform expression results were analyzed as a function of microglial gene expression (ITGAM and AIF1), total INPP5D expression, AD status, and SNP status. Total INPP5D expression is increased in AD vs non-AD samples, but there remains to be seen a significant association with SNP status. Second, single cell RNAseq was performed on APP/PS1 mice to understand the relationship between INPP5D expression and microglial activation. APP/PS1 mice showed an increased expression of disease-associated microglia compared to controls. INPP5D was expressed at equivalent levels in both homeostatic and…

Author(s): Sanjay Bidichandani, University of Oklahoma Health Sciences Center; Layne N. Rodden, University of Oklahoma Health Sciences Center; Kaitlyn M. Gilliam, University of Oklahoma Health Sciences Center; Christina Lam, University of Oklahoma Health Sciences Center; David R. Lynch, The Children’s Hospital of Philadelphia // ABSTRACT: Research objective: Friedreich ataxia (FRDA) is typically caused by homozygosity for an expanded GAA triplet-repeat in intron 1 of the FXN gene. The expanded repeat induces repressive histone changes and DNA hypermethylation, which result in epigenetic silencing and FXN transcriptional deficiency. A class I histone deacetylase inhibitor (HDACi-109) reactivates the silenced FXN gene, although with considerable inter-individual variability, which remains etiologically unexplained. Because HDAC inhibitors work by reversing epigenetic silencing, we hypothesized that epigenetic heterogeneity among patients may help to explain this inter-individual variability.  Methods: Fresh PBMCs were isolated from a prospective cohort of 50 FRDA patients. Somatic epigenetic heterogeneity was assessed via bisulfite deep sequencing, a highly quantitative measurement of DNA hypermethylation with single molecule resolution. Analysis of 1000 individual molecules per patient revealed the prevalence of unmethylated, partially methylated, and fully methylated FXN epialleles. PBMCs from this cohort were treated with HDACi-109, and gene reactivation was measured by RT-qPCR.  Results: HDACi-109 significantly increased FXN transcript to levels seen in asymptomatic heterozygous carriers, albeit with the expected inter-individual variability. Response to HDACi-109 correlated significantly with the prevalence of unmethylated and partially methylated FXN molecules, supporting the model that FXN reactivation involves a proportion of genes that are amenable to correction in non-dividing somatic cells,…

Author(s): Alexis R. Demonbreun, Northwestern University; Dominic E. Fullenkamp, Northwestern University; Jodi L. Curtin, Northwestern University; Nina L. Rieser, Northwestern University; Lauren A. Vaught, Northwestern University; Elizabeth M. McNally, Northwestern University // ABSTRACT: Background and Methods:    Many monogenic disorders, including the muscular dystrophies, display phenotypic variability despite the same disease-causing mutation. To identify genetic modifiers of muscular dystrophy as future therapeutic targets, we used quantitative trait locus mapping and whole genome sequencing in a mouse model of muscular dystrophy. This approach uncovered a modifier locus on chromosome 11 associated with increased skeletal and cardiac muscle membrane damage.  Whole genome and RNA sequencing identified Anxa6, encoding annexin A6, as a modifier gene.  Annexin A6 (ANXA6) belongs to the annexin family of calcium-dependent phospholipid binding proteins that facilitate membrane repair, accumulating at sites of damage.  A synonymous variant in exon 11 creates a cryptic splice donor resulting in a truncated annexin A6 protein, referred to as ANXA6N32.  This polymorphism is present in both the C57 and DBA/2J background strains, but not in the 129 strain.  In vivo, the presence of ANXA6N32 was associated with reduced membrane-associated annexin A6 in damaged myofibers and increased dye uptake.  To define the molecular machinery that directs muscle repair, we developed a method to visualize membrane resealing and repair in real-time in live myofibers.  This method uses electroporation to express fluorescently tagged proteins combined with high-resolution confocal microscopy.  In this system, muscle fibers are wounded with a confocal laser and then imaged to observe the resealing process…

Author(s): Dwi U. Kemaladewi, UPMC Children’s Hospital of Pittsburgh; Jia Qi Cheng-Zhang, UPMC Children’s Hospital of Pittsburgh; Annie I. Arockiaraj, UPMC Children’s Hospital of Pittsburgh; Marie Johnson, UPMC Children’s Hospital of Pittsburgh; Anushe Munir, UPMC Children’s Hospital of Pittsburgh; Dwi U. Kemaladewi, UPMC Children’s Hospital of Pittsburgh // ABSTRACT: LAMA2-deficient congenital muscular dystrophy (LAMA2-CMD) is caused by mutations in the LAMA2 gene encoding laminin-α2, an extracellular protein essential for skeletal muscle and Schwann cell functions. Individualized correction of LAMA2 mutations is hampered by the heterogeneity found in patient populations. In contrast, upregulation of compensatory disease modifier gene LAMA1 can serve as a mutation-independent approach and benefit a larger number of patients.     We previously showed that upregulation of Lama1 via AAV-mediated CRISPR activation (CRISPRa) rescues disease phenotypes in mice. Despite the promising outcome, the CRISPRa technology requires the use of dual AAV delivery system, which directly contributes to the high dose, toxicity, and production cost.    In this project, our goal is to develop a miniaturized CRISPRa (mini-CRISPRa) technology suitable for a single AAV9 delivery system.    First, we swapped a commonly used CMV promoter with a novel, 7.5X-shorter synthetic promoter 4XNRF1 to drive dCas9 expression. Subsequently, we substituted previously used 2XVP64 transcriptional activators with miniaturized tripartite VP64, P65, RTA activators (miniVPR). We coupled the 4XNRF1-dCas9-miniVPR with sgRNA targeting either mouse Lama1 or human LAMA1 promoter. We were able to reduce the number of sgRNAs and fit all the miniaturized components within the 4.7 kb-packaging capacity of the AAV9. Importantly, our data show…

Author(s): Hallie Gaitsch, NINDS/NIH (NIH Oxford-Cambridge Scholars Program); Jing-Ping Lin, PhD, NINDS/NIH; Daniel Reich, MD, PhD, NINDS/NIH // ABSTRACT: Cell-free DNA (cfDNA) is present in human plasma during both health and disease states. These DNA fragments are derived from intra- and extracellular nuclease cleavage of DNA in cells undergoing apoptosis or necrosis. Sequence differences in circulating tumor DNA, including known tumorigenic mutations, have been of particular interest in the field of oncology for their potential use as prognostic indicators and early-stage biomarkers for screening. However, the nongenetic characteristics of circulating cfDNA can also provide useful information that is applicable to diseases which do not involve acquired genetic mutations, including many neurologic disorders. Epigenetic signatures such as DNA methylation patterns can be used to identify the tissue- and even cell-type of origin for cfDNA samples. Given that ~28 million unique CpG methylation sites exist within the human genome, the use of methylation array technology and deconvolution algorithms make it possible to identify differentially methylated regions corresponding to various cell types and levels of gene expression. This also opens the possibility of using the cfDNA of cell-types which are minority populations in their tissue, including oligodendrocytes and oligodendrocyte progenitor cells (OPCs) which are central players in multiple sclerosis (MS) pathology, as targets for liquid biopsy development. The low concentration of cfDNA in human blood and high potential for genomic DNA contamination necessitate careful handling during nucleic acid extraction from fluid samples. The objective of this study was to develop an optimized and…