Abnormal Social Interactions in a Drosophila Mutant of an Autism Candidate Gene: Neuroligin 3.

Social interactions are typically impaired in neuropsychiatric disorders such as autism, for which the genetic underpinnings are very complex. Social interactions can be modeled by analysis of behaviors, including social spacing, sociability, and aggression, in simpler organisms such as Drosophila melanogaster. Here, we examined the effects of mutants of the autism-related gene neuroligin 3 (nlg3) on fly social and non-social behaviors. Startled-induced negative geotaxis is affected by a loss of function nlg3 mutation. Social space and aggression are also altered in a sex- and social-experience-specific manner in nlg3 mutant flies. In light of the conserved roles that neuroligins play in social behavior, our results offer insight into the regulation of social behavior in other organisms, including humans.

Drosophila neuroligin 1 regulates synaptic growth and function in response to activity and phosphoinositide-3-kinase.

Neuroligins are postsynaptic neural cell adhesion molecules that mediate synaptic maturation and function in vertebrates and invertebrates, but their mechanisms of action and regulation are not well understood. At the Drosophila larval neuromuscular junction (NMJ), previous analysis demonstrated a requirement for Drosophila neuroligin 1 (dnlg1) in synaptic growth and maturation. The goal of the present study was to better understand the effects and mechanisms of loss-of-function and overexpression of dnlg1 on synapse size and function, and to identify signaling pathways that control dnlg1 expression. Consistent with reduced synapse size, evoked excitatory junctional currents (EJCs) were diminished in dnlg1 mutants but displayed normal Ca(2+) sensitivity and short-term plasticity. However, postsynaptic function was also perturbed, in that glutamate receptor staining and the distribution of amplitudes of miniature excitatory junctional currents (mEJCs) were abnormal in mutants. All the above phenotypes were rescued by a genomic transgene. Overexpression of dnlg1 in muscle resulted in synaptic overgrowth, but reduced the amplitudes of EJCs and mEJCs. Overgrowth and reduced EJC amplitude required Drosophila neurexin 1 (dnrx1) function, suggesting that increased DNlg1/DNrx1 signaling attenuates synaptic transmission and regulates growth through a retrograde mechanism. In contrast, reduced mEJC amplitude was independent of dnrx1. Synaptic overgrowth, triggered by neuronal hyperactivity, absence of the E3 ubiquitin ligase highwire, and increased phosphoinositide-3-kinase (PI3K) signaling in motor neurons reduced synaptic DNlg1 levels. Likewise, postsynaptic attenuation of PI3K, which increases synaptic strength, was associated with reduced DNlg1 levels. These observations suggest that activity and PI3K signaling pathways modulate growth and synaptic transmission through dnlg1-dependent mechanisms.

RNA Trans-Splicing Targeting Endogenous ß-Globin Pre-Messenger RNA in Human Erythroid Cells.

Sickle cell disease results from a point mutation in exon 1 of the ß-globin gene (total 3 exons). Replacing sickle ß-globin exon 1 (and exon 2) with a normal sequence by trans-splicing is a potential therapeutic strategy. Therefore, this study sought to develop trans-splicing targeting ß-globin pre-messenger RNA among human erythroid cells. Binding domains from random ß-globin sequences were comprehensively screened. Six candidates had optimal binding, and all targeted intron 2. Next, lentiviral vectors encoding RNA trans-splicing molecules were constructed incorporating a unique binding domain from these candidates, artificial 5' splice site, and γ-globin cDNA, and trans-splicing was evaluated in CD34+ cell-derived erythroid cells from healthy individuals. Lentiviral transduction was efficient, with vector copy numbers of 9.7 to 15.3. The intended trans-spliced RNA product, including exon 3 of endogenous ß-globin and γ-globin, was detected at the molecular level. Trans-splicing efficiency was improved to 0.07-0.09% by longer binding domains, including the 5' splice site of intron 2. In summary, screening was performed to select efficient binding domains for trans-splicing. Detectable levels of trans-splicing were obtained for endogenous ß-globin RNA in human erythroid cells. These methods provide the basis for future trans-splicing directed gene therapy.

Identification of Hedgehog pathway components by RNAi in Drosophila cultured cells.

Classical genetic screens can be limited by the selectivity of mutational targeting, the complexities of anatomically based phenotypic analysis, or difficulties in subsequent gene identification. Focusing on signaling response to the secreted morphogen Hedgehog (Hh), we used RNA interference (RNAi) and a quantitative cultured cell assay to systematically screen functional roles of all kinases and phosphatases, and subsequently 43% of predicted Drosophila genes. Two gene products reported to function in Wingless (Wg) signaling were identified as Hh pathway components: a cell surface protein (Dally-like protein) required for Hh signal reception, and casein kinase 1alpha, a candidate tumor suppressor that regulates basal activities of both Hh and Wg pathways. This type of cultured cell-based functional genomics approach may be useful in the systematic analysis of other biological processes.

Genes required for Drosophila nervous system development identified by RNA interference.

RNA interference was used to screen 3,314 Drosophila double-stranded RNAs, corresponding to approximately 25% of Drosophila genes, for genes that affect the development of the embryonic nervous system. RNA-interference-mediated gene silencing in Drosophila embryos resulted in loss-of-function mutant phenotypes for 43 genes, which is 1.3% of the genes that were screened. We found 18 genes that were not known previously to affect the development of the nervous system. The functions of some of the genes are unknown. Other genes encode protein kinases, transcription factors, and signaling proteins, as well as proteins with other functions.