An in vivo drug repurposing screen and transcriptional analyses reveals the serotonin pathway and GSK3 as major therapeutic targets for NGLY1 deficiency.

NGLY1 deficiency, a rare disease with no effective treatment, is caused by autosomal recessive, loss-of-function mutations in the N-glycanase 1 (NGLY1) gene and is characterized by global developmental delay, hypotonia, alacrima, and seizures. We used a Drosophila model of NGLY1 deficiency to conduct an in vivo, unbiased, small molecule, repurposing screen of FDA-approved drugs to identify therapeutic compounds. Seventeen molecules partially rescued lethality in a patient-specific NGLY1 deficiency model, including multiple serotonin and dopamine modulators. Exclusive dNGLY1 expression in serotonin and dopamine neurons, in an otherwise dNGLY1 deficient fly, was sufficient to partially rescue lethality. Further, genetic modifier and transcriptomic data supports the importance of serotonin signaling in NGLY1 deficiency. Connectivity Map analysis identified glycogen synthase kinase 3 (GSK3) inhibition as a potential therapeutic mechanism for NGLY1 deficiency, which we experimentally validated with TWS119, lithium, and GSK3 knockdown. Strikingly, GSK3 inhibitors and a serotonin modulator rescued size defects in dNGLY1 deficient larvae upon proteasome inhibition, suggesting that these compounds act through NRF1, a transcription factor that is regulated by NGLY1 and regulates proteasome expression. This study reveals the importance of the serotonin pathway in NGLY1 deficiency, and serotonin modulators or GSK3 inhibitors may be effective therapeutics for this rare disease.

A Drosophila screen identifies NKCC1 as a modifier of NGLY1 deficiency.

N-Glycanase 1 (NGLY1) is a cytoplasmic deglycosylating enzyme. Loss-of-function mutations in the NGLY1 gene cause NGLY1 deficiency, which is characterized by developmental delay, seizures, and a lack of sweat and tears. To model the phenotypic variability observed among patients, we crossed a Drosophila model of NGLY1 deficiency onto a panel of genetically diverse strains. The resulting progeny showed a phenotypic spectrum from 0 to 100% lethality. Association analysis on the lethality phenotype, as well as an evolutionary rate covariation analysis, generated lists of modifying genes, providing insight into NGLY1 function and disease. The top association hit was Ncc69 (human NKCC1/2), a conserved ion transporter. Analyses in NGLY1-/- mouse cells demonstrated that NKCC1 has an altered average molecular weight and reduced function. The misregulation of this ion transporter may explain the observed defects in secretory epithelium function in NGLY1 deficiency patients.

An Unbiased Drug Screen for Seizure Suppressors in Duplication 15q Syndrome Reveals 5-HT1A and Dopamine Pathway Activation as Potential Therapies.

BACKGROUND: Duplication 15q (Dup15q) syndrome is a rare neurogenetic disorder characterized by autism and pharmacoresistant epilepsy. Most individuals with isodicentric duplications have been on multiple medications to control seizures. We recently developed a model of Dup15q in Drosophila by elevating levels of fly Dube3a in glial cells using repo-GAL4, not neurons. In contrast to other Dup15q models, these flies develop seizures that worsen with age. METHODS: We screened repo>Dube3a flies for approved compounds that can suppress seizures. Flies 3 to 5 days old were exposed to compounds in the fly food during development. Flies were tested using a bang sensitivity assay for seizure recovery time. At least 40 animals were tested per experiment, with separate testing for male and female flies. Studies of K+ content in glial cells of the fly brain were also performed using a fluorescent K+ indicator. RESULTS: We identified 17 of 1280 compounds in the Prestwick Chemical Library that could suppress seizures. Eight compounds were validated in secondary screening. Four of these compounds regulated either serotonergic or dopaminergic signaling, and subsequent experiments confirmed that seizure suppression occurred primarily through stimulation of serotonin receptor 5-HT1A. Additional studies of K+ levels showed that Dube3a regulation of the Na+/K+ exchanger ATPα (adenosine triphosphatase α) in glia may be modulated by serotonin/dopamine signaling, causing seizure suppression. CONCLUSIONS: Based on these pharmacological and genetic studies, we present an argument for the use of 5-HT1A agonists in the treatment of Dup15q epilepsy.

Transcriptomic and proteomic profiling of glial versus neuronal Dube3a overexpression reveals common molecular changes in gliopathic epilepsies.

Epilepsy affects millions of individuals worldwide and many cases are pharmacoresistant. Duplication 15q syndrome (Dup15q) is a genetic disorder caused by duplications of the 15q11.2-q13.1 region. Phenotypes include a high rate of pharmacoresistant epilepsy. We developed a Dup15q model in Drosophila melanogaster that recapitulates seizures in Dup15q by over-expressing fly Dube3a or human UBE3A in glial cells, but not neurons, implicating glia in the Dup15q epilepsy phenotype. We compared Dube3a overexpression in glia (repo>Dube3a) versus neurons (elav>Dube3a) using transcriptomics and proteomics of whole fly head extracts. We identified 851 transcripts differentially regulated in repo>Dube3a, including an upregulation of glutathione S-transferase (GST) genes that occurred cell autonomously within glial cells. We reliably measured approximately 2,500 proteins by proteomics, most of which were also quantified at the transcript level. Combined transcriptomic and proteomic analysis revealed an enrichment of 21 synaptic transmission genes downregulated at the transcript and protein in repo>Dube3a indicating synaptic proteins change in a cell non-autonomous manner in repo>Dube3a flies. We identified 6 additional glia originating bang-sensitive seizure lines and found upregulation of GSTs in 4 out of these 6 lines. These data suggest GST upregulation is common among gliopathic seizures and may ultimately provide insight for treating epilepsy.

The Drosophila Gene Sulfateless Modulates Autism-Like Behaviors.

Major challenges to identifying genes that contribute to autism spectrum disorder (ASD) risk include the availability of large ASD cohorts, the contribution of many genes overall, and small effect sizes attributable to common gene variants. An alternative approach is to use a model organism to detect alleles that impact ASD-relevant behaviors and ask whether homologous human genes infer ASD risk. Here we utilized the Drosophila genetic reference panel (DGRP) as a tool to probe for perturbation in naturally occurring behaviors in Drosophila melanogaster that are analogous to three behavior domains: impaired social communication, social reciprocity and repetitive behaviors or restricted interests. Using 40 of the available DGRP lines, we identified single nucleotide polymorphisms (SNPs) in or near genes controlling these behavior domains, including ASD gene orthologs (neurexin 4 and neuroligin 2), an intellectual disability (ID) gene homolog (kirre), and a gene encoding a heparan sulfate (HS) modifying enzyme called sulfateless (sfl). SNPs in sfl were associated with all three ASD-like behaviors. Using RNAi knock-down of neuronal sfl expression, we observed significant changes in expressive and receptive communication during mating, decreased grooming behavior, and increased social spacing. These results suggest a role for HS proteoglycan synthesis and/or modification in normal social communication, repetitive behavior, and social interaction in flies. Finally, using the DGRP to directly identify genetic effects relevant to a neuropsychiatric disorder further demonstrates the utility of the Drosophila system in the discovery of genes relevant to human disease.

A genome-wide enhancer/suppressor screen for Dube3a interacting genes in Drosophila melanogaster.

The genetics underlying autism spectrum disorder (ASD) are complex. Approximately 3-5% of ASD cases arise from maternally inherited duplications of 15q11.2-q13.1, termed Duplication 15q syndrome (Dup15q). 15q11.2-q13.1 includes the gene UBE3A which is believed to underlie ASD observed in Dup15q syndrome. UBE3A is an E3 ubiquitin ligase that targets proteins for degradation and trafficking, so finding UBE3A substrates and interacting partners is critical to understanding Dup15q ASD. In this study, we take an unbiased genetics approach to identify genes that genetically interact with Dube3a, the Drosophila melanogaster homolog of UBE3A. We conducted an enhancer/suppressor screen using a rough eye phenotype produced by Dube3a overexpression with GMR-GAL4. Using the DrosDel deficiency kit, we identified 3 out of 346 deficiency lines that enhanced rough eyes when crossed to two separate Dube3a overexpression lines, and subsequently identified IA2, GABA-B-R3, and lola as single genes responsible for rough eye enhancement. Using the FlyLight GAL4 lines to express uas-Dube3a + uas-GFP in the endogenous lola pattern, we observed an increase in the GFP signal compared to uas-GFP alone, suggesting a transcriptional co-activation effect of Dube3a on the lola promoter region. These findings extend the role of Dube3a/UBE3A as a transcriptional co-activator, and reveal new Dube3a interacting genes.

A recurrent de novo missense mutation in UBTF causes developmental neuroregression.

UBTF (upstream binding transcription factor) exists as two isoforms; UBTF1 regulates rRNA transcription by RNA polymerase 1, whereas UBTF2 regulates mRNA transcription by RNA polymerase 2. Herein, we describe 4 patients with very similar patterns of neuroregression due to recurrent de novo mutations in UBTF (GRCh37/hg19, NC_000017.10: g.42290219C > T, NM_014233.3: c.628G > A) resulting in the same amino acid change in both UBTF1 and UBTF2 (p.Glu210Lys [p.E210K]). Disease onset in our cohort was at 2.5 to 3 years and characterized by slow progression of global motor, cognitive and behavioral dysfunction. Notable early features included hypotonia with a floppy gait, high-pitched dysarthria and hyperactivity. Later features included aphasia, dystonia, and spasticity. Speech and ambulatory ability were lost by the early teens. Magnetic resonance imaging showed progressive generalized cerebral atrophy (supratentorial > infratentorial) with involvement of both gray and white matter. Patient fibroblasts showed normal levels of UBTF transcripts, increased expression of pre-rRNA and 18S rRNA, nucleolar abnormalities, markedly increased numbers of DNA breaks, defective cell-cycle progression, and apoptosis. Expression of mutant human UBTF1 in Drosophila neurons was lethal. Although no loss-of-function variants are reported in the Exome Aggregation Consortium (ExAC) database and Ubtf-/- is early embryonic lethal in mice, Ubtf+/- mice displayed only mild motor and behavioral dysfunction in adulthood. Our data underscore the importance of including UBTF E210K in the differential diagnosis of neuroregression and suggest that mainly gain-of-function mechanisms contribute to the pathogenesis of the UBTF E210K neuroregression syndrome.

Cell-Type-Specific Changes in Intrinsic Excitability in the Subiculum following Learning and Exposure to Novel Environmental Contexts.

The subiculum is the main target of the hippocampal region CA1 and is the principle output region of the hippocampus. The subiculum is critical to learning and memory, although it has been relatively understudied. There are two functional types of principle neurons within the subiculum: regular spiking (RS) and burst spiking (BS) neurons. To determine whether these cell types are differentially modified by learning-related experience, we performed whole-cell patch clamp recordings from male mouse brain slices following contextual fear conditioning (FC) and memory retrieval relative to a number of control behavioral paradigms. RS cells, but not BS cells, displayed a greater degree of experience-related plasticity in intrinsic excitability measures [afterhyperpolarization (AHP), input resistance (Rinput), current required to elicit a spike], with fear conditioned animals having generally more excitable RS cells compared to naïve controls. Furthermore, we found that the relative proportion of RS to BS neurons is modified by the type of exposure, with the lowest proportion of BS subicular cells occurring in animals that underwent contextual FC followed by a retrieval test. These studies indicate that pyramidal neurons in the subiculum undergo experience- and learning-related plasticity in intrinsic properties in a cell-type-specific manner. As BS and RS cells are thought to convey distinct types of information, this plasticity may be particularly important in encoding, consolidating, and recalling spatial information by modulating information flow from the hippocampus to cortical regions.

Glial overexpression of Dube3a causes seizures and synaptic impairments in Drosophila concomitant with down regulation of the Na+/K+ pump ATPα.

Duplication 15q syndrome (Dup15q) is an autism-associated disorder co-incident with high rates of pediatric epilepsy. Additional copies of the E3 ubiquitin ligase UBE3A are thought to cause Dup15q phenotypes, yet models overexpressing UBE3A in neurons have not recapitulated the epilepsy phenotype. We show that Drosophila endogenously expresses Dube3a (fly UBE3A homolog) in glial cells and neurons, prompting an investigation into the consequences of glial Dube3a overexpression. Here we expand on previous work showing that the Na+/K+ pump ATPα is a direct ubiquitin ligase substrate of Dube3a. A robust seizure-like phenotype was observed in flies overexpressing Dube3a in glial cells, but not neurons. Glial-specific knockdown of ATPα also produced seizure-like behavior, and this phenotype was rescued by simultaneously overexpressing ATPα and Dube3a in glia. Our data provides the basis of a paradigm shift in Dup15q research given that clinical phenotypes have long been assumed to be due to neuronal UBE3A overexpression.

The Drosophila melanogaster homolog of UBE3A is not imprinted in neurons.

In mammals, expression of UBE3A is epigenetically regulated in neurons and expression is restricted to the maternal copy of UBE3A. A recent report claimed that Drosophila melanogaster UBE3A homolog (Dube3a) is preferentially expressed from the maternal allele in fly brain, inferring an imprinting mechanism. However, complex epigenetic regulatory features of the mammalian imprinting center are not present in Drosophila, and allele specific expression of Dube3a has not been documented. We used behavioral and electrophysiological analysis of the Dube3a loss-of-function allele (Dube3a15b) to investigate Dube3a imprinting in fly neurons. We found that motor impairment (climbing ability) and a newly-characterized defect in synaptic transmission are independent of parental inheritance of the Dube3a15b allele. Furthermore, expression analysis of coding single nucleotide polymorphisms (SNPs) in Dube3a did not reveal allele specific expression differences among reciprocal crosses. These data indicate that Dube3a is neither imprinted nor preferentially expressed from the maternal allele in fly neurons.

Assessment of the Tumorigenic Potential of Spontaneously Immortalized and hTERT-Immortalized Cultured Dental Pulp Stem Cells.

Dental pulp stem cells (DPSCs) provide an exciting new avenue to study neurogenetic disorders. DPSCs are neural crest-derived cells with the ability to differentiate into numerous tissues including neurons. The therapeutic potential of stem cell-derived lines exposed to culturing ex vivo before reintroduction into patients could be limited if the cultured cells acquired tumorigenic potential. We tested whether DPSCs that spontaneously immortalized in culture acquired features of transformed cells. We analyzed immortalized DPSCs for anchorage-independent growth, genomic instability, and ability to differentiate into neurons. Finally, we tested both spontaneously immortalized and human telomerase reverse transcriptase (hTERT)-immortalized DPSC lines for the ability to form tumors in immunocompromised animals. Although we observed increased colony-forming potential in soft agar for the spontaneously immortalized and hTERT-immortalized DPSC lines relative to low-passage DPSC, no tumors were detected from any of the DPSC lines tested. We noticed some genomic instability in hTERT-immortalized DPSCs but not in the spontaneously immortalized lines tested. We determined that immortalized DPSC lines generated in our laboratory, whether spontaneously or induced, have not acquired the potential to form tumors in mice. These data suggest cultured DPSC lines that can be differentiated into neurons may be safe for future in vivo therapy for neurobiological diseases.

TRPC3 channels critically regulate hippocampal excitability and contextual fear memory.

Memory formation requires de novo protein synthesis, and memory disorders may result from misregulated synthesis of critical proteins that remain largely unidentified. Plasma membrane ion channels and receptors are likely candidates given their role in regulating neuron excitability, a candidate memory mechanism. Here we conduct targeted molecular monitoring and quantitation of hippocampal plasma membrane proteins from mice with intact or impaired contextual fear memory to identify putative candidates. Here we report contextual fear memory deficits correspond to increased Trpc3 gene and protein expression, and demonstrate TRPC3 regulates hippocampal neuron excitability associated with memory function. These data provide a mechanistic explanation for enhanced contextual fear memory reported herein following knockdown of TRPC3 in hippocampus. Collectively, TRPC3 modulates memory and may be a feasible target to enhance memory and treat memory disorders.

Parental and larval exposure to nicotine modulate spontaneous activity as well as cholinergic and GABA receptor expression in adult C. elegans.

Early nicotine exposure has been associated with many long-term consequences that include neuroanatomical alterations, as well as behavioral and cognitive deficits. To describe the effects of early nicotine exposure in Caenorhabditis elegans, the current study observed spontaneous locomotor activity (i.e., reversals) either in the presence or absence of nicotine. Expression of acr-16 (a nicotinic receptor subunit) and a ß-like GABA(A) receptor subunit, gab-1, were also examined with RT-PCR. Worms were exposed to nicotine (30 µM) throughout "zygote formation" (period that includes oocyte maturation, ovulation and fertilization), from hatching to adulthood ("larval development") or across both zygote and larval development. Adult larval-exposed worms only showed an increase in spontaneous behavior when tested on nicotine (p<0.001) but levels of activity similar to controls when tested on plain plates (p>0.30). Larval-exposed worms also showed control levels of acr-16 nicotinic receptor expression (p>0.10) but increased gab-1 expression relative to controls (p<0.01). In contrast, zygote-exposed and zygote- plus larval-exposed worms showed a similar increase in spontaneous behavior on plain plates (p<0.001 and p=0.001, respectively) but control levels of responding when tested on nicotine (p>0.90 for each). However, expression of acr-16 and gab-1 was downregulated in zygote-exposed (p<0.01 and p<0.05, respectively) and significantly upregulated in the zygote- plus larval-exposed worms (p<0.000 for each); most surprising was the over five-fold increase in gab-1 expression. These results suggest that spontaneous motor behavior and receptor expression are differentially modulated by nicotine exposure during larval development and/or zygote formation. As well, these findings demonstrate that C. elegans, as a model system, is also sensitive to nicotine exposure during early development and provides the basis for future research to uncover specific mechanisms by which early nicotine exposure modifies neuronal signaling and alters behavior.