Spatiotemporally controlled genetic perturbation for efficient large-scale studies of cell non-autonomous effects.

Studies in genetic model organisms have revealed much about the development and pathology of complex tissues. Most have focused on cell-intrinsic gene functions and mechanisms. Much less is known about how transformed, or otherwise functionally disrupted, cells interact with healthy ones toward a favorable or pathological outcome. This is largely due to technical limitations. We developed new genetic tools in Drosophila melanogaster that permit efficient multiplexed gain- and loss-of-function genetic perturbations with separable spatial and temporal control. Importantly, our novel tool-set is independent of the commonly used GAL4/UAS system, freeing the latter for additional, non-autonomous, genetic manipulations; and is built into a single strain, allowing one-generation interrogation of non-autonomous effects. Altogether, our design opens up efficient genome-wide screens on any deleterious phenotype, once plasmid or genome engineering is used to place the desired miRNA(s) or ORF(s) into our genotype. Specifically, we developed tools to study extrinsic effects on neural tumor growth but the strategy presented has endless applications within and beyond neurobiology, and in other model organisms.

Orientation information in encoding facial expressions.

Previous research showed that we use different regions of a face to categorize different facial expressions, e.g. mouth region for identifying happy faces; eyebrows, eyes and upper part of nose for identifying angry faces. These findings imply that the spatial information along or close to the horizontal orientation might be more useful than others for facial expression recognition. In this study, we examined how the performance for recognizing facial expression depends on the spatial information along different orientations, and whether the pixel-level differences in the face images could account for subjects' performance. Four facial expressions-angry, fearful, happy and sad-were tested. An orientation filter (bandwidth = 23°) was applied to restrict information within the face images, with the center of the filter ranged from 0° (horizontal) to 150° in steps of 30°. Accuracy for recognizing facial expression was measured for an unfiltered and the six filtered conditions. For all four facial expressions, recognition performance (normalized d') was virtually identical for filter orientations of -30°, horizontal and 30°, and declined systematically as the filter orientation approached vertical. The information contained in mouth and eye regions is a significant predictor for subject's response (based on the confusion patterns). We conclude that young adults with normal vision categorizes facial expression most effectively based on the spatial information around the horizontal orientation which captures primary changes of facial features across expressions. Across all spatial orientations, the information contained in mouth and eye regions contributes significantly to facial expression categorization.

Signal Integration by the IκB Protein Pickle Shapes Drosophila Innate Host Defense.

Pattern recognition receptors are activated following infection and trigger transcriptional programs important for host defense. Tight regulation of NF-κB activation is critical to avoid detrimental and misbalanced responses. We describe Pickle, a Drosophila nuclear IκB that integrates signaling inputs from both the Imd and Toll pathways by skewing the transcriptional output of the NF-κB dimer repertoire. Pickle interacts with the NF-κB protein Relish and the histone deacetylase dHDAC1, selectively repressing Relish homodimers while leaving other NF-κB dimer combinations unscathed. Pickle's ability to selectively inhibit Relish homodimer activity contributes to proper host immunity and organismal health. Although loss of pickle results in hyper-induction of Relish target genes and improved host resistance to pathogenic bacteria in the short term, chronic inactivation of pickle causes loss of immune tolerance and shortened lifespan. Pickle therefore allows balanced immune responses that protect from pathogenic microbes while permitting the establishment of beneficial commensal host-microbe relationships.

Insights into ALS pathomechanisms: from flies to humans.

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease causing the death of motor neurons with consequent muscle atrophy and paralysis. Several neurodegenerative diseases have been modeled in Drosophila and genetic studies on this model organism led to the elucidation of crucial aspects of disease mechanisms. ALS, however, has lagged somewhat behind possibly because of the lack of a suitable genetic model. We were the first to develop a fly model for ALS and over the last few years, we have implemented and used this model for a large scale, unbiased modifier screen. We also report an extensive bioinformatic analysis of the genetic modifiers and we show that most of them are associated in a network of interacting genes controlling known as well as novel cellular processes involved in ALS pathogenesis. A similar analysis for the human homologues of the Drosophila modifiers and the validation of a subset of them in human tissues confirm and expand the significance of the data for the human disease. Finally, we analyze a possible application of the model in the process of therapeutic discovery in ALS and we discuss the importance of novel "non-obvious" models for the disease.

Network analyses reveal novel aspects of ALS pathogenesis.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective loss of motor neurons, muscle atrophy and paralysis. Mutations in the human VAMP-associated protein B (hVAPB) cause a heterogeneous group of motor neuron diseases including ALS8. Despite extensive research, the molecular mechanisms underlying ALS pathogenesis remain largely unknown. Genetic screens for key interactors of hVAPB activity in the intact nervous system, however, represent a fundamental approach towards understanding the in vivo function of hVAPB and its role in ALS pathogenesis. Targeted expression of the disease-causing allele leads to neurodegeneration and progressive decline in motor performance when expressed in the adult Drosophila, eye or in its entire nervous system, respectively. By using these two phenotypic readouts, we carried out a systematic survey of the Drosophila genome to identify modifiers of hVAPB-induced neurotoxicity. Modifiers cluster in a diverse array of biological functions including processes and genes that have been previously linked to hVAPB function, such as proteolysis and vesicular trafficking. In addition to established mechanisms, the screen identified endocytic trafficking and genes controlling proliferation and apoptosis as potent modifiers of ALS8-mediated defects. Surprisingly, the list of modifiers was mostly enriched for proteins linked to lipid droplet biogenesis and dynamics. Computational analysis reveals that most modifiers can be linked into a complex network of interacting genes, and that the human genes homologous to the Drosophila modifiers can be assembled into an interacting network largely overlapping with that in flies. Identity markers of the endocytic process were also found to abnormally accumulate in ALS patients, further supporting the relevance of the fly data for human biology. Collectively, these results not only lead to a better understanding of hVAPB function but also point to potentially relevant targets for therapeutic intervention.

Increased levels of phosphoinositides cause neurodegeneration in a Drosophila model of amyotrophic lateral sclerosis.

The Vesicle-associated membrane protein (VAMP)-Associated Protein B (VAPB) is the causative gene of amyotrophic lateral sclerosis 8 (ALS8) in humans. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective death of motor neurons leading to spasticity, muscle atrophy and paralysis. VAP proteins have been implicated in various cellular processes, including intercellular signalling, synaptic remodelling, lipid transport and membrane trafficking and yet, the molecular mechanisms underlying ALS8 pathogenesis remain poorly understood. We identified the conserved phosphoinositide phosphatase Sac1 as a Drosophila VAP (DVAP)-binding partner and showed that DVAP is required to maintain normal levels of phosphoinositides. Downregulating either Sac1 or DVAP disrupts axonal transport, synaptic growth, synaptic microtubule integrity and the localization of several postsynaptic components. Expression of the disease-causing allele (DVAP-P58S) in a fly model for ALS8 induces neurodegeneration, elicits synaptic defects similar to those of DVAP or Sac1 downregulation and increases phosphoinositide levels. Consistent with a role for Sac1-mediated increase of phosphoinositide levels in ALS8 pathogenesis, we found that Sac1 downregulation induces neurodegeneration in a dosage-dependent manner. In addition, we report that Sac1 is sequestered into the DVAP-P58S-induced aggregates and that reducing phosphoinositide levels rescues the neurodegeneration and suppresses the synaptic phenotypes associated with DVAP-P58S transgenic expression. These data underscore the importance of DVAP-Sac1 interaction in controlling phosphoinositide metabolism and provide mechanistic evidence for a crucial role of phosphoinositide levels in VAP-induced ALS.

Characterization of the properties of a novel mutation in VAPB in familial amyotrophic lateral sclerosis.

Following the mutation screening of genes known to cause amyotrophic lateral sclerosis (ALS) in index cases from 107 familial ALS (FALS) kindred, a point mutation was identified in vesicle-associated membrane protein-associated protein B (VAPB), or VAMP-associated protein B, causing an amino acid change from threonine to isoleucine at codon 46 (T46I) in one FALS case but not in 257 controls. This is an important finding because it is only the second mutation identified in this gene that causes ALS. In order to investigate the pathogenic effects of this mutation, we have used a motor neuron cell line and tissue-specific expression of the mutant protein in Drosophila. We provide substantial evidence for the pathogenic effects of this mutation in abolishing the effect of wild type VAPB in the unfolded protein response, promoting ubiquitin aggregate formation, and activating neuronal cell death. We also report that expression of the mutant protein in the Drosophila motor system induces aggregate deposition, endoplasmic reticulum disorganization, and chaperone up-regulation both in neurons and in muscles. Our integrated analysis of the pathogenic effect of the T46I mutation and the previously identified P56S mutation indicate extensive commonalities in the disease mechanism for these two mutations. In summary, we show that this newly identified mutation in human FALS has a pathogenic effect, supporting and reinforcing the role of VAPB as a causative gene of ALS.

hVAPB, the causative gene of a heterogeneous group of motor neuron diseases in humans, is functionally interchangeable with its Drosophila homologue DVAP-33A at the neuromuscular junction.

Motor neuron diseases (MNDs) are progressive neurodegenerative disorders characterized by selective death of motor neurons leading to spasticity, muscle wasting and paralysis. Human VAMP-associated protein B (hVAPB) is the causative gene of a clinically diverse group of MNDs including amyotrophic lateral sclerosis (ALS), atypical ALS and late-onset spinal muscular atrophy. The pathogenic mutation is inherited in a dominant manner. Drosophila VAMP-associated protein of 33 kDa A (DVAP-33A) is the structural homologue of hVAPB and regulates synaptic remodeling by affecting the size and number of boutons at neuromuscular junctions. Associated with these structural alterations are compensatory changes in the physiology and ultrastructure of synapses, which maintain evoked responses within normal boundaries. DVAP-33A and hVAPB are functionally interchangeable and transgenic expression of mutant DVAP-33A in neurons recapitulates major hallmarks of the human diseases including locomotion defects, neuronal death and aggregate formation. Aggregate accumulation is accompanied by a depletion of the endogenous protein from its normal localization. These findings pinpoint to a possible role of hVAPB in synaptic homeostasis and emphasize the relevance of our fly model in elucidating the patho-physiology underlying motor neuron degeneration in humans.

The impact of skin banking and the use of its cadaveric skin allografts for severe burn victims in Singapore.

The skin banking programme was set-up in Singapore in 1998 to provide a ready source of allografts for patients with severe burns. The process and problems in establishing a local skin bank will be described together with a retrospective review of skin allograft recipients to determine the efficacy of the programme. For the skin bank set-up, pertinent issues related to legislation, methods, logistics, quality assurance and donation rate are discussed. In this retrospective review, a comparison between patients who had early complete excision with skin allograft transplantation and those who received conventional staged excision and coverage, was analysed in terms of clinical profile and outcome using statistical methods. The former group presented a significant reduction of mortality rate and hospital stay by 29% and 10 days, respectively. The establishment of the skin bank has helped in the management of severe burn patients by facilitating early excision and allografting. In a Burn Centre, therefore, it is essential to have an ample supply of skin allograft for burn victims in readiness for mass disaster situations.