Cell-Specific Alternative Splicing of Drosophila Dscam2 Is Crucial for Proper Neuronal Wiring

SummaryHow a finite number of genes specify a seemingly infinite number of neuronal connections is a central question in neurobiology. Alternative splicing has been proposed to increase proteome diversity in the brain. Here we show that cell-specific alternative splicing of a cell-surface protein is crucial for neuronal wiring. Down syndrome cell adhesion molecule 2 (Dscam2) is a conserved homophilic binding protein that can induce repulsion between opposing neurons. In the fly visual system, L1 and L2 neurons both require Dscam2 repulsion, but paradoxically, they also physically contact each other. We found that the cell-specific expression of two biochemically distinct alternative isoforms of Dscam2 prevents these cells from repelling each other. Phenotypes were observed in the axon terminals of L1 and L2 when they expressed the incorrect isoform, demonstrating a requirement for distinct isoforms. We conclude that cell-specific alternative splicing is a mechanism for achieving proper connectivity between neurons

Enhanced ribosomal association of p27Kip1 mRNA is a mechanism contributing to accumulation during growth arrest

p27(Kip1) regulates the decision to enter into S-phase or withdraw from the cell cycle by establishing an inhibitory threshold above which G(1) cyclin-dependent kinases accumulate before activation. We have used the HL-60 cell line to study regulation of p27 as cells withdraw from the cell cycle following treatment with 12-O-tetra-decanoylphorbol-13-acetate (TPA). We found that the amount of p27 is maximal in G(0) cells, lower in G(1) cells, and undetectable in S-phase cells, In contrast to the protein, the amount of p27 mRNA was the same in these populations, suggesting tliat accumulation of p27 during the cell cycle and as cells withdraw hom the cell cycle is controlled by post-transcriptional mechanisms, In S-phase cells, the degradation of p27 appears to predominate as a regulatory mechanism, In G(0) cells, there was an increase in the synthesis rate of p27, Our data demonstrate that, in G(0) cells, accumulation of p27 is due to an increase in the amount of p27 mRNA in polyribosomes

Informing the development of an online self-management program for men living with HIV: a needs assessment

Background: The aim of this mixed methods study was to conduct a multifaceted needs assessment to inform the development of an online self-management program for men living with HIV. The objectives were to describe the health-related quality of life for men living with HIV, the impact of living with HIV, and the perceived problem areas and service and support needs of these men. The needs assessment was conducted in accordance with the PRECEDE model for health promotion program planning.Methods: A survey assessing the quality of life of men living with HIV (n = 72) was conducted and results were compared to Australian normative data. Focus groups were also undertaken with men living with HIV (n = 11) and a multidisciplinary team of service providers working in the area of HIV (n = 11). Focus groups enabled an in-depth description of the impact of HIV on quality of life and perceived problem areas in daily life.Results: HIV-positive men experience significantly lower quality of life when compared with Australian normative data, particularly in those domains concerned with social and emotional aspects of quality of life. Qualitative focus groups yielded an overarching theme ‘The psychosocial impact of HIV’ which contained three sub-themes; (1) Life before and after HIV – a changed identity and its repercussions; (2) Resilience and the importance of social support; (3) Negotiating the practicalities – intimate relationships and disclosure.Conclusions: The findings from this needs assessment highlight the need to target socio-emotional contexts of HIV positive men’s daily lives to improve quality of life and well-being. Intervention priorities for the proposed online self-management program include: (1) managing the emotional impact of HIV; (2) disclosing HIV status to family and friends; (3) maintaining social connectedness; (4) managing HIV within intimate relationships; and (5) disclosure of HIV status to intimate partners

The positive outlook study- a randomised controlled trial evaluating the effectiveness of an online self-management program targeting psychosocial issues for men living with HIV: a study protocol

Background: The emergence of HIV as a chronic condition means that people living with HIV are required to takemore responsibility for the self-management of their condition, including making physical, emotional and socialadjustments. This paper describes the design and evaluation of Positive Outlook, an online program aiming toenhance the self-management skills of gay men living with HIV.Methods/design: This study is designed as a randomised controlled trial in which men living with HIV in Australiawill be assigned to either an intervention group or usual care control group. The intervention group willparticipate in the online group program ‘Positive Outlook’. The program is based on self-efficacy theory and uses aself-management approach to enhance skills, confidence and abilities to manage the psychosocial issues associatedwith HIV in daily life. Participants will access the program for a minimum of 90 minutes per week over seven weeks.Primary outcomes are domain specific self-efficacy, HIV related quality of life, and outcomes of health education.Secondary outcomes include: depression, anxiety and stress; general health and quality of life; adjustment to HIV;and social support. Data collection will take place at baseline, completion of the intervention (or eight weeks postrandomisation) and at 12 week follow-up.Discussion: Results of the Positive Outlook study will provide information regarding the effectiveness of onlinegroup programs improving health related outcomes for men living with HIV

Cdc42 Regulates Apical Junction Formation in Human Bronchial Epithelial Cells through PAK4 and Par6B

A systematic screen of Cdc42 targets was carried out in human bronchial epithelial cells. Two kinases, PAK4 and Par6B/aPKC, were identified and are required for maturation of primordial junctions into apical junctions. PAK4 recruitment to primordial junctions is Cdc42-dependent, but maintenance at junctions during maturation is Par6B-dependent

Dscam-mediated repulsion controls tiling and self-avoidance

Recent studies have uncovered the molecular basis of self-avoidance and tiling, two fundamental principles required for the formation of neural circuits. Both of these wiring strategies are established through homophilic repulsion between Dscam proteins expressed on opposing cell surfaces. In Drosophila, Dscam1 mediates self-avoidance, whereas Dscam2 mediates tiling. By contrast, phenotypes in the retina of the DSCAM mutant mouse indicate that DSCAM functions in both self-avoidance and tiling. These findings suggest that homophilic recognition molecules that have classically been defined as adhesive may also function as repulsive cues and that Dscam proteins specialize in this function

Strategies for assembling columns and layers in the Drosophila visual system

A striking feature of neural circuit structure is the arrangement of neurons into regularly spaced ensembles (i.e. columns) and neural connections into parallel layers. These patterns of organization are thought to underlie precise synaptic connectivity and provide a basis for the parallel processing of information. In this article we discuss in detail specific findings that contribute to a framework for understanding how columns and layers are assembled in the Drosophila visual system, and discuss their broader implications

Deterministic splicing of Dscam2 is regulated by Muscleblind

Alternative splicing increases the proteome diversity crucial for establishing the complex circuitry between trillions of neurons. To provide individual cells with different repertoires of protein isoforms, however, this process must be regulated. Previously, we found that the mutually exclusive alternative splicing of Drosophila Dscam2 produces two isoforms (A and B) with unique binding properties. This splicing event is cell type specific, and the transmembrane proteins that it generates are crucial for the development of axons, dendrites, and synapses. Here, we show that Muscleblind (Mbl) controls Dscam2 alternative splicing. Mbl represses isoform A and promotes the selection of isoform B. Mbl mutants exhibit phenotypes also observed in flies engineered to express a single Dscam2 isoform. Consistent with this, mbl expression is cell type specific and correlates with the splicing of isoform B. Our study demonstrates how the regulated expression of a splicing factor is sufficient to provide neurons with unique protein isoforms crucial for development

Dscam2 affects visual perception in Drosophila melanogaster

Dscam2, a cell surface protein that mediates cellular repulsion, plays a crucial role in the development of the Drosophila melanogaster visual system. Dscam2 generates boundaries between neighboring modules in the fly optic lobe; in Dscam2 mutants this visual system modularity is compromised. Although developmental wiring defects have been well described in the Dscam2 mutant, behavioral consequences have not been investigated. To address this, we examined the visual behavior of Dscam2 mutant flies. Using a phototaxis assay, we ascertained that these flies are not blind, but have a reduced phototaxic response. Through population-based and single fly optomotor assays, we found that Dscam2 mutant flies can track motion but that their response is opposite to control flies under defined experimental conditions. In a fixation paradigm, which allows tethered flies to control the angular position of a visual stimulus, mutant flies' responses were diametrically opposed to those seen in control flies. These data suggest that modest changes in the modularity of the fly visual system in the Dscam2 mutant can dramatically change the perception of specific visual cues and modify behavior