Analysis of asymptomatic Drosophila models for ALS and SMA reveals convergent impact on functional protein complexes linked to neuro-muscular degeneration.

BACKGROUND: Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS) share phenotypic and molecular commonalities, including the fact that they can be caused by mutations in ubiquitous proteins involved in RNA metabolism, namely SMN, TDP-43 and FUS. Although this suggests the existence of common disease mechanisms, there is currently no model to explain the resulting motor neuron dysfunction. In this work we generated a parallel set of Drosophila models for adult-onset RNAi and tagged neuronal expression of the fly orthologues of the three human proteins, named Smn, TBPH and Caz, respectively. We profiled nuclear and cytoplasmic bound mRNAs using a RIP-seq approach and characterized the transcriptome of the RNAi models by RNA-seq. To unravel the mechanisms underlying the common functional impact of these proteins on neuronal cells, we devised a computational approach based on the construction of a tissue-specific library of protein functional modules, selected by an overall impact score measuring the estimated extent of perturbation caused by each gene knockdown. RESULTS: Transcriptome analysis revealed that the three proteins do not bind to the same RNA molecules and that only a limited set of functionally unrelated transcripts is commonly affected by their knock-down. However, through our integrative approach we were able to identify a concerted effect on protein functional modules, albeit acting through distinct targets. Most strikingly, functional annotation revealed that these modules are involved in critical cellular pathways for motor neurons, including neuromuscular junction function. Furthermore, selected modules were found to be significantly enriched in orthologues of human neuronal disease genes. CONCLUSIONS: The results presented here show that SMA and ALS disease-associated genes linked to RNA metabolism functionally converge on neuronal protein complexes, providing a new hypothesis to explain the common motor neuron phenotype. The functional modules identified represent promising biomarkers and therapeutic targets, namely given their alteration in asymptomatic settings.

Impact of topical emollient, steroids alone or combined with calcipotriol, on the immune infiltrate and clinical outcome in psoriasis.

Psoriasis is a chronic inflammatory disease whereby long-term disease control remains a challenge for the patients. Latest evidence suggests that combined topical treatment with steroids and vitamin D analogue foam (Calcipotriol/Betamethasone) is efficient in long-term management of the disease and reducing the number of relapses. Its effects on cellular inflammation and cytokine production remain to be explored. We set out to examine the effect of topical therapies on cellular infiltrate and cytokine profile in the lesional skin of psoriasis patients. This was a monocentric, double-blind, randomized trial with 30 patients. Patients were treated with the combined Calcipotriol/Betamethasone foam, Betamethasone foam alone, Clobetasol Propionate ointment or placebo. 4 mm skin biopsies from lesional and non-lesional sites were taken before and 4 weeks after treatment. Cellular infiltrate, IFNγ and IL-17 were studied by immunofluorescence. Each patient was their own control. Evolution in skin inflammation was studied in parallel with changes in patient's epidermal thickness and their tPASI clinical score. Lesional skin was characterized by increased epidermal thickness, increased number of IL-17 and IFNγ producing CD8+ T cells, NK cells and neutrophils. All treatment reduced epidermal thickness and improved patients tPASI scores. Only the combined Calcipotriol/Betamethasone foam completely abolished epidermal and dermal influx of CD8+ T cells, reduced number of CD8 + IFNγ+ cells (but not CD8 + IL-17+ cells) and significantly reduced the number of MPO+ neutrophils which were predominantly IL-17+. None of the treatments had effect on NK cells. We have shown the combined topical treatment with Calcipotriol/Betamethasone foam to be effective in reducing cellular influx into lesional skin of psoriasis patients and this effect to be superior to emollient or Betamethasone alone. Its previously described efficacy in the clinic may be attributed to its unique and rapid ability to inhibit both adaptive CD8+ T cell and innate immune neutrophilia influx into the skin, which was not observed for the other treatments.

An RNA-immunoprecipitation protocol to identify RNAs associated with RNA-binding proteins in cytoplasmic and nuclear Drosophila head fractions.

RNA-binding proteins (RBPs) are multifunctional proteins that shuttle between the nucleus and the cytoplasm where they assemble with target RNAs to form multi-molecular complexes. Here, we describe a protocol to selectively identify RNAs associated with RBPs of interest in the cytoplasmic and nuclear compartments of adult Drosophila brain cells. Cytoplasmic and nuclear fractions are differentially collected and used for immunoprecipitation-based purification of GFP-tagged RBPs. This protocol can be applied to samples expressing ectopic or endogenous tagged RBPs.

CLEC12B Decreases Melanoma Proliferation by Repressing Signal Transducer and Activator of Transcription 3.

The potential role of CLEC12B, a gene predominantly expressed by skin melanocytes discovered through transcriptomic analysis, in melanoma is unknown. In this study, we show that CLEC12B expression is lower in melanoma and melanoma metastases than in melanocytes and benign melanocytic lesions and that its decrease correlates with poor prognosis. We further show that CLEC12B recruits SHP2 phosphatase through its immunoreceptor tyrosine-based inhibition motif domain, inactivates signal transducer and activator of transcription 1/3/5, increases p53/p21/p27 expression/activity, and modulates melanoma cell proliferation. The growth of human melanoma cells overexpressing CLEC12B in nude mice after subcutaneous injection is significantly decreased compared with that in the vehicle control group and is associated with decreased signal transducer and activator of transcription 3 phosphorylation and increased p53 levels in the tumors. Reducing the level of CLEC12B had the opposite effect. We show that CLEC12B represses the activation of the signal transducer and activator of transcription pathway and negatively regulates the cell cycle, providing a proliferative asset to melanoma cells.

CLEC12B Is a Melanocytic Gene Regulating the Color of the Skin.

Pigmentation of the human skin is a complex process regulated by many genes. However, only a few have a profound impact on melanogenesis. Transcriptome analysis of pigmented skin compared with analysis of vitiligo skin devoid of melanocytes allowed us to unravel CLEC12B as a melanocytic gene. We showed that CLEC12B, a C-type lectin receptor, is highly expressed in melanocytes and that its expression is decreased in dark skin compared with that in white skin. CLEC12B directly recruits and activates SHP1 and SHP2 through its immunoreceptor tyrosine-based inhibitory motif domain and promotes CRE-binding protein degradation, leading to the downregulation of the downstream MITF pathway. CLEC12B ultimately controls melanin production and pigmentation in vitro and in a model of reconstructed human epidermis. The identification of CLEC12B in melanocytes shows that C-type lectin receptors exert function beyond immunity and inflammation. It also provides insights into the understanding of melanocyte biology and regulation of melanogenesis.

Tyramine induces dynamic RNP granule remodeling and translation activation in the Drosophila brain.

Ribonucleoprotein (RNP) granules are dynamic condensates enriched in regulatory RNA binding proteins (RBPs) and RNAs under tight spatiotemporal control. Extensive recent work has investigated the molecular principles underlying RNP granule assembly, unraveling that they form through the self-association of RNP components into dynamic networks of interactions. How endogenous RNP granules respond to external stimuli to regulate RNA fate is still largely unknown. Here, we demonstrate through high-resolution imaging of intact Drosophila brains that Tyramine induces a reversible remodeling of somatic RNP granules characterized by the decondensation of granule-enriched RBPs (e.g. Imp/ZBP1/IGF2BP) and helicases (e.g. Me31B/DDX-6/Rck). Furthermore, our functional analysis reveals that Tyramine signals both through its receptor TyrR and through the calcium-activated kinase CamkII to trigger RNP component decondensation. Finally, we uncover that RNP granule remodeling is accompanied by the rapid and specific translational activation of associated mRNAs. Thus, this work sheds new light on the mechanisms controlling cue-induced rearrangement of physiological RNP condensates.

The prion-like domain of Drosophila Imp promotes axonal transport of RNP granules in vivo.

Prion-like domains (PLDs), defined by their low sequence complexity and intrinsic disorder, are present in hundreds of human proteins. Although gain-of-function mutations in the PLDs of neuronal RNA-binding proteins have been linked to neurodegenerative disease progression, the physiological role of PLDs and their range of molecular functions are still largely unknown. Here, we show that the PLD of Drosophila Imp, a conserved component of neuronal ribonucleoprotein (RNP) granules, is essential for the developmentally-controlled localization of Imp RNP granules to axons and regulates in vivo axonal remodeling. Furthermore, we demonstrate that Imp PLD restricts, rather than promotes, granule assembly, revealing a novel modulatory function for PLDs in RNP granule homeostasis. Swapping the position of Imp PLD compromises RNP granule dynamic assembly but not transport, suggesting that these two functions are uncoupled. Together, our study uncovers a physiological function for PLDs in the spatio-temporal control of neuronal RNP assemblies.

Inhibitory activities of short linear motifs underlie Hox interactome specificity in vivo.

Hox proteins are well-established developmental regulators that coordinate cell fate and morphogenesis throughout embryogenesis. In contrast, our knowledge of their specific molecular modes of action is limited to the interaction with few cofactors. Here, we show that Hox proteins are able to interact with a wide range of transcription factors in the live Drosophila embryo. In this context, specificity relies on a versatile usage of conserved short linear motifs (SLiMs), which, surprisingly, often restrains the interaction potential of Hox proteins. This novel buffering activity of SLiMs was observed in different tissues and found in Hox proteins from cnidarian to mouse species. Although these interactions remain to be analysed in the context of endogenous Hox regulatory activities, our observations challenge the traditional role assigned to SLiMs and provide an alternative concept to explain how Hox interactome specificity could be achieved during the embryonic development.