Pre-Pulse Inhibition of an escape response in adult fruit fly, Drosophila melanogaster.

Pre-Pulse Inhibition (PPI) is a neural process where suppression of a startle response is elicited by preceding the startling stimulus (Pulse) with a weak, non-startling one (Pre-Pulse). Defective PPI is widely employed as a behavioural endophenotype in humans and mammalian disorder-relevant models for neuropsychiatric disorders. We have developed a user-friendly, semi-automated, high-throughput-compatible Drosophila light-off jump response PPI paradigm, with which we demonstrate that PPI, with similar parameters measured in mammals, exists in adults of this model organism. We report that Drosophila PPI is affected by reduced expression of Dysbindin and both reduced and increased expression of Nmdar1 (N-methyl-D-aspartate receptor 1), perturbations associated with schizophrenia. Studying the biology of PPI in an organism that offers a plethora of genetic tools and a complex and well characterized connectome will greatly facilitate our efforts to gain deeper insight into the aetiology of human mental disorders, while reducing the need for mammalian models.

GATAD2B loss-of-function mutations cause a recognisable syndrome with intellectual disability and are associated with learning deficits and synaptic undergrowth in Drosophila.

BACKGROUND: GATA zinc finger domain containing 2B (GATAD2B) encodes a subunit of the MeCP1-Mi-2/nucleosome remodelling and deacetylase complex involved in chromatin modification and regulation of transcription. We recently identified two de novo loss-of-function mutations in GATAD2B by whole exome sequencing in two unrelated individuals with severe intellectual disability. METHODS: To identify additional individuals with GATAD2B aberrations, we searched for microdeletions overlapping with GATAD2B in inhouse and international databases, and performed targeted Sanger sequencing of the GATAD2B locus in a selected cohort of 80 individuals based on an overlap with the clinical features in the two index cases. To address whether GATAD2B is required directly in neurones for cognition and neuronal development, we investigated the role of Drosophila GATAD2B orthologue simjang (simj) in learning and synaptic connectivity. RESULTS: We identified a third individual with a 240 kb microdeletion encompassing GATAD2B and a fourth unrelated individual with GATAD2B loss-of-function mutation. Detailed clinical description showed that all four individuals with a GATAD2B aberration had a distinctive phenotype with childhood hypotonia, severe intellectual disability, limited speech, tubular shaped nose with broad nasal tip, short philtrum, sparse hair and strabismus. Neuronal knockdown of Drosophila GATAD2B orthologue, simj, resulted in impaired learning and altered synapse morphology. CONCLUSIONS: We hereby define a novel clinically recognisable intellectual disability syndrome caused by loss-of-function of GATAD2B. Our results in Drosophila suggest that GATAD2B is required directly in neurones for normal cognitive performance and synapse development.

CEP89 is required for mitochondrial metabolism and neuronal function in man and fly.

It is estimated that the human mitochondrial proteome consists of 1000-1500 distinct proteins. The majority of these support the various biochemical pathways that are active in these organelles. Individuals with an oxidative phosphorylation disorder of unknown cause provide a unique opportunity to identify novel genes implicated in mitochondrial biology. We identified a homozygous deletion of CEP89 in a patient with isolated complex IV deficiency, intellectual disability and multisystemic problems. CEP89 is a ubiquitously expressed and highly conserved gene of unknown function. Immunocytochemistry and cellular fractionation experiments showed that CEP89 is present both in the cytosol and in the mitochondrial intermembrane space. Furthermore, we ascertained in vitro that downregulation of CEP89 resulted in a severe decrease in complex IV in-gel activity and altered mobility, suggesting that the complex is aberrantly formed. Two-dimensional BN-SDS gel analysis revealed that CEP89 associates with a high-molecular weight complex. Together, these data confirm a role for CEP89 in mitochondrial metabolism. In addition, we modeled CEP89 loss of function in Drosophila. Ubiquitous knockdown of fly Cep89 decreased complex IV activity and resulted in complete lethality. Furthermore, Cep89 is required for mitochondrial integrity, membrane depolarization and synaptic transmission of photoreceptor neurons, and for (sub)synaptic organization of the larval neuromuscular junction. Finally, we tested neuronal Cep89 knockdown flies in the light-off jump reflex habituation assay, which revealed its role in learning. We conclude that CEP89 proteins play an important role in mitochondrial metabolism, especially complex IV activity, and are required for neuronal and cognitive function across evolution.

Specific Cooperation Between Imp-α2 and Imp-ß/Ketel in Spindle Assembly During Drosophila Early Nuclear Divisions.

The multifunctional factors Imp-α and Imp-ß are involved in nuclear protein import, mitotic spindle dynamics, and nuclear membrane formation. Furthermore, each of the three members of the Imp-α family exerts distinct tasks during development. In Drosophila melanogaster, the imp-α2 gene is critical during oogenesis for ring canal assembly; specific mutations, which allow oogenesis to proceed normally, were found to block early embryonic mitosis. Here, we show that imp-α2 and imp-ß genetically interact during early embryonic development, and we characterize the pattern of defects affecting mitosis in embryos laid by heterozygous imp-α2(D14) and imp-ß(KetRE34) females. Embryonic development is arrested in these embryos but is unaffected in combinations between imp-ß(KetRE34) and null mutations in imp-α1 or imp-α3. Furthermore, the imp-α2(D14)/imp-ß(KetRE34) interaction could only be rescued by an imp-α2 transgene, albeit not imp-α1 or imp-α3, showing the exclusive imp-α2 function with imp-ß. Use of transgenes carrying modifications in the major Imp-α2 domains showed the critical requirement of the nuclear localization signal binding (NLSB) site in this process. In the mutant embryos, we found metaphase-arrested mitoses made of enlarged spindles, suggesting an unrestrained activity of factors promoting spindle assembly. In accordance with this, we found that Imp-ß(KetRE34) and Imp-ß(KetD) bind a high level of RanGTP/GDP, and a deletion decreasing RanGTP level suppresses the imp-ß(KetRE34) phenotype. These data suggest that a fine balance among Imp-α2, Imp-ß, RanGTP, and the NLS cargos is critical for mitotic progression during early embryonic development.

High-resolution, high-throughput SNP mapping in Drosophila melanogaster.

Single nucleotide polymorphisms (SNPs) are useful markers for genetic mapping experiments in model organisms. Here we report the establishment of a high-density SNP map and high-throughput genotyping assays for Drosophila melanogaster. Our map comprises 27,367 SNPs in common laboratory Drosophila stocks. These SNPs were clustered within 2,238 amplifiable markers at an average density of 1 marker every 50.3 kb, or 6.3 genes. We have also constructed a set of 62 Drosophila stocks, each of which facilitates the generation of recombinants within a defined genetic interval of 1-2 Mb. For flexible, high-throughput SNP genotyping, we used fluorescent tag-array mini-sequencing (TAMS) assays. We designed and validated TAMS assays for 293 SNPs at an average resolution of 391.3 kb, and demonstrated the utility of these tools by rapidly mapping 14 mutations that disrupt embryonic muscle patterning. These resources enable high-resolution high-throughput genetic mapping in Drosophila.

Dscam diversity is essential for neuronal wiring and self-recognition.

Neurons are thought to use diverse families of cell-surface molecules for cell recognition during circuit assembly. In Drosophila, alternative splicing of the Down syndrome cell adhesion molecule (Dscam) gene potentially generates 38,016 closely related transmembrane proteins of the immunoglobulin superfamily, each comprising one of 19,008 alternative ectodomains linked to one of two alternative transmembrane segments. These ectodomains show isoform-specific homophilic binding, leading to speculation that Dscam proteins mediate cell recognition. Genetic studies have established that Dscam is required for neural circuit assembly, but the extent to which isoform diversity contributes to this process is not known. Here we provide conclusive evidence that Dscam diversity is essential for circuit assembly. Using homologous recombination, we reduced the entire repertoire of Dscam ectodomains to just a single isoform. Neural circuits in these mutants are severely disorganized. Furthermore, we show that it is crucial for neighbouring neurons to express distinct isoforms, but that the specific identity of the isoforms expressed in an individual neuron is unimportant. We conclude that Dscam diversity provides each neuron with a unique identity by which it can distinguish its own processes from those of other neurons, and that this self-recognition is essential for wiring the Drosophila brain.

Deletion of proteasomal subunit S5a/Rpn10/p54 causes lethality, multiple mitotic defects and overexpression of proteasomal genes in Drosophila melanogaster.

The regulatory complex of the 26S proteasome is responsible for the selective recognition and binding of multiubiquitinated proteins. It was earlier shown that the subunit S5a/Rpn10/p54 of the regulatory complex is the only cellular protein capable of binding multiubiquitin chains in an in vitro overlay assay. The role of this subunit in substrate selection, however, is a subject of debate, following the observation that its deletion in Saccharomyces cerevisiae is not lethal and instead causes only a mild phenotype. To study the function of this subunit in higher eukaryotes, a mutant Drosophila strain was constructed by deleting the single copy gene encoding subunit S5a/Rpn10/p54. This deletion caused larval-pupal polyphasic lethality, multiple mitotic defects, the accumulation of higher multimers of ubiquitinated proteins and a huge accumulation of defective 26S proteasome particles. Deletion of the subunit S5a/Rpn10/p54 does not destabilise the regulatory complex and does not disturb the assembly of the regulatory complex and the catalytic core. The pupal lethality is a consequence of the depletion of the maternally provided 26S proteasome during the larval stages and a sudden increase in the proteasomal activity demands during the first few hours of pupal development. The huge accumulation of the fully assembled 26S proteasome in the deletion mutant and the lack of free subunits or partially assembled particles indicate that there is a highly coordinated accumulation of all the subunits of the 26S proteasome. This suggests that in higher eukaryotes, as with yeast, a feedback circuit coordinately regulates the expression of the proteasomal genes, and this adjusts the actual proteasome concentration in the cells according to the temporal and/or spatial proteolytic demands.