Molecular control of local translation in axon development and maintenance.

The tips of axons are often far away from the cell soma where most proteins are synthesized. Recent work has revealed that axonal mRNA transport and localised translation are key regulatory mechanisms that allow these distant outposts of the cell to respond rapidly to extrinsic factors and maintain axonal homeostasis. Here, we review recent evidence pointing to an increasingly broad role for local protein synthesis in controlling axon shape, synaptogenesis and axon survival by regulating diverse cellular processes such as vesicle trafficking, cytoskeletal remodelling and mitochondrial integrity. We further highlight current research on the regulatory mechanisms that coordinate the localization and translation of functionally linked mRNAs in axons

Prescribing as affective clinical practice: Transformations in sexual health consultations through HIV pre-exposure prophylaxis

New medicines can transform routines and priorities in clinical practice, but how do clinicians think and feel about these changes, and how does it affect their work? In Australia, the HIV prevention regimen pre-exposure prophylaxis (PrEP) has been rapidly rolled out, transforming the sexual cultures and practices of users, but less attention has been given to the ways PrEP has reconfigured clinical practice. This paper draws on 28 qualitative semi-structured interviews conducted between 2019 and 2020 with PrEP-providing doctors and nurses in Australia to consider how they have affectively engaged with PrEP and put it into practice. Through a reflexive thematic analysis, we explore how clinicians adapted to PrEP, how the field of HIV prevention has been transformed, and how these developments have changed how clinicians approach patients. While the introduction of PrEP was initially received with uncertainty and shock, clinicians described PrEP as enjoyable to prescribe, and better aligned with the moral duties of sexual health consultations than existing HIV prevention strategies like condoms. Through approaching clinical work as an ‘affective practice’, we argue for attending not only to how new interventions change expectations and practices, but also how these changes are felt and valued by clinicians

Growth cone Tctp is dynamically regulated by guidance cues

Tctp contributes to retinal circuitry formation by promoting axon growth and guidance, but it remains unknown to what extent axonal Tctp specifically influences axon development programs. Various genome-wide profiling studies have ranked tctp transcripts among the most enriched in the axonal compartment of distinct neuronal populations, including embryonic retinal ganglion cells (RGCs), suggesting its expression can be regulated locally and that this may be important during development. Here, we report that growth cone Tctp levels change rapidly in response to Netrin-1 and Ephrin-A1, two guidance cues encountered by navigating RGC growth cones. This regulation is opposite in effect, as we observed protein synthesis- and mTORC1-dependent increases in growth cone Tctp levels after acute treatment with Netrin-1, but a decline upon exposure to Ephrin-A1, an inhibitor of mTORC1. Live imaging with translation reporters further showed that Netrin-1-induced synthesis of Tctp in growth cones is driven by a short 3’ untranslated region (3’UTR) tctp mRNA isoform. However, acute inhibition of de novo Tctp synthesis in axons did not perturb the advance of retinal projections through the optic tract in vivo, indicating that locally produced Tctp is not necessary for normal axon growth and guidance.This work was supported by Fundação para a Ciência e Tecnologia (CR; fellowship SFRH/BD/33891/2009), a Wellcome Trust Programme Grant (085314/Z/08/Z) and a ERC Advanced Grant (322817; CH)

Essential role of heparan sulfates in axon navigation and targeting in the developing visual system

Heparan sulfate (HS) is abundant in the developing brain and is a required co-factor for many types of fibroblast growth factor (FGF) signaling in vitro. We report that some HSs, when added exogenously to the developing Xenopus optic pathway, severely disrupt target recognition causing axons from the retina to bypass their primary target, the optic tectum. Significantly, HS sidechains from a neuroepithelial perlecan variant that preferentially bind FGF-2, HS(FGF-2), cause aberrant targeting, whereas those that preferentially bind FGF-1 do not. Charge-matched fragments of HS(FGF-2) show that the mistargeting activity associates with the FGP-binding fragments. Heparitinase removal of native HSs at the beginning of optic tract formation retards retinal axon elongation; addition of FGF-2 restores axon extension but axons lose directionality. Late HS removal, after axons have extended through the tract, elicits a tectal bypass phenotype indicating a growth promoting and guidance function for native HSs. Our results demonstrate that different HS sidechains from the same care protein differentially affect axon growth in vivo, possibly due to their distinct FGF-binding preferences, and suggest that growth factors and HSs are important partners in regulating axon growth and guidance in the developing visual system

Subcellular profiling reveals distinct and developmentally regulated repertoire of growth cone mRNAs

Cue-directed axon guidance depends partly on local translation in growth cones. Many mRNA transcripts are known to reside in developing axons, yet little is known about their subcellular distribution or, specifically, which transcripts are in growth cones. Here laser capture microdissection (LCM) was used to isolate the growth cones of retinal ganglion cell (RGC) axons of two vertebrate species, mouse and Xenopus, coupled with unbiased genomewide microarray profiling. An unexpectedly large pool of mRNAs defined predominant pathways in protein synthesis, oxidative phosphorylation, cancer, neurological disease, and signaling. Comparative profiling of "young" (pathfinding) versus "old" (target-arriving) Xenopus growth cones revealed that the number and complexity of transcripts increases dramatically with age. Many presynaptic protein mRNAs are present exclusively in old growth cones, suggesting that functionally related sets of mRNAs are targeted to growth cones in a developmentally regulated way. Remarkably, a subset of mRNAs was significantly enriched in the growth cone compared with the axon compartment, indicating that mechanisms exist to localize mRNAs selectively to the growth cone. Furthermore, some receptor transcripts (e.g., EphB4), present exclusively in old growth cones, were equally abundant in young and old cell bodies, indicating that RNA trafficking from the soma is developmentally regulated. Our findings show that them RNA repertoire in growth cones is regulated dynamically with age and suggest that mRNA localization is tailored to match the functional demands of the growing axon tip as it transforms into the presynaptic terminal. Copyright © 2010 the authors

Total internal reflection fluorescence anisotropy imaging microscopy: setup, calibration, and data processing for protein polymerization measurements in living cells

Fluorescence anisotropy imaging microscopy (FAIM) measures the depolarization properties of fluorophores to deduce molecular changes in their environment. For successful FAIM, several design principles have to be considered and a thorough system-specific calibration protocol is paramount. One important calibration parameter is the G factor, which describes the system-induced errors for different polarization states of light. The determination and calibration of the G factor is discussed in detail in this article. We present a novel measurement strategy, which is particularly suitable for FAIM with high numerical aperture objectives operating in TIRF illumination mode. The method makes use of evanescent fields that excite the sample with a polarization direction perpendicular to the image plane. Furthermore, we have developed an ImageJ/Fiji plugin, AniCalc, for FAIM data processing. We demonstrate the capabilities of our TIRF-FAIM system by measuring β-actin polymerization in human embryonic kidney cells and in retinal neurons.This work was funded by grants from the Medical Research Council UK (MR/K015850/1 and MR/K02292X/1), the EPSRC (EP/L015889/1 and EP/H018301/1), theWellcome Trust (3-3249/Z/16/Z and 089703/Z/09/Z), and In nitus, China, Ltd (CFK); the Cambridge Trust, Croucher Foundation, Sir Edward Youde Memorial Fund (HHWW); a Wellcome Trust Programme Grant (085314/Z/08/Z) and an ERC Advanced Grant (322817) (CEH)

The neuronal architecture of Xenopus retinal ganglion cells is sculpted by rho-family GTPases in vivo

Dendritogenesis, axonogenesis, pathfinding, and target recognition are all affected in distinct ways when Xenopus retinal ganglion cells (RGCs) are transfected with constitutively active (ca), wild-type (wt), and dominant negative (dn) Rho-family GTPases in vivo. Dendritogenesis required Rac1 and Cdc42 activity. Moreover, ca-Rac1 caused dendrite hyperproliferation. Axonogenesis, in contrast, was inhibited by ca-Rac1. This phenotype was partially rescued by the coexpression of dn cyclin-dependent kinase (Cdk5), a proposed effector of Rac1, suggesting that Rac1 activity must be regulated tightly for normal axonogenesis. Growth cone morphology was particularly sensitive to dn-RhoA and wt-Cdc42 constructs. These also caused targeting errors, such as tectal bypass, suggesting that cytoskeletal rearrangements are involved in target recognition and are transduced by these pathways

Engaging Stigmatised Communities in Australia with Digital Health Systems: Towards Data Justice in Public Health

Introduction: In 2018, following government policy changes to Australia’s national electronic health record system, ‘My Health Record’, consumer advocates—including organisations representing people living with HIV, people who use drugs and sex workers—raised concerns about privacy and data security. Responding to these controversies, this study explores the practical, ethical and political complexities of engaging stigmatised communities with digital health systems. Methods: We conducted 16 qualitative semi-structured interviews in 2020 with key informants representing communities who experience stigma, discrimination and marginalisation in Australia. These communities included people living with HIV, sex workers, people who inject drugs, gay and bisexual men and transgender and gender diverse people. We conducted a reflexive thematic analysis. Results: Key informants were sceptical of proposed benefits of electronic health records for their communities, and concerned about privacy risks and the potential for discrimination. Meaningful consultation, consent mechanisms and tackling structural stigma were raised as solutions for engaging communities. Conclusions: Although communities could benefit from being included in digital health systems, significant cultural, legal and social reforms from government were believed to be necessary to build trust in digital health systems. We argue that these forms of data justice are necessary for effective future systems. Policy Implications: Engaging stigmatised communities—including in relation to gender, sexuality, sex work, drug use, HIV—requires a commitment to data justice. The design and implementation of digital health systems requires investment in ongoing and meaningful consultation with communities and representative organisations

RNA Docking and Local Translation Regulate Site-Specific Axon Remodeling In Vivo

Nascent proteins can be positioned rapidly at precise subcellular locations by local protein synthesis (LPS) to facilitate localized growth responses. Axon arbor architecture, a major determinant of synaptic connectivity, is shaped by localized growth responses, but it is unknown whether LPS influences these responses in vivo. Using high-resolution live imaging, we examined the spatiotemporal dynamics of RNA and LPS in retinal axons during arborization in vivo. Endogenous RNA tracking reveals that RNA granules dock at sites of branch emergence and invade stabilized branches. Live translation reporter analysis reveals that de novo ß-actin hotspots colocalize with docked RNA granules at the bases and tips of new branches. Inhibition of axonal ß-actin mRNA translation disrupts arbor dynamics primarily by reducing new branch emergence and leads to impoverished terminal arbors. The results demonstrate a requirement for LPS in building arbor complexity and suggest a key role for pre-synaptic LPS in assembling neural circuits.This work was supported by Cambridge Trust, Croucher Foundation, Sir Edward Youde Memorial Fund (H.H.-W.W.), Gates Cambridge (J.Q.L.), Fundac¸ a˜ o para a Cieˆ ncia e Tecnologia (C.M.R.), Wellcome Trust Senior Investigator Award (100329/Z/ 12/Z) (W.A.H.), EPSRC Grant (EP/H018301/1), MRC Grant (MR/K015850/1 and MR/K02292X/1), Wellcome Trust (089703/Z/09/Z) (C.F.K.), Wellcome Trust Programme Grant (085314/Z/08/Z), and ERC Advanced Grant (322817) (C.E.H.)

ESCRT-II controls retinal axon growth by regulating DCC receptor levels and local protein synthesis.

Endocytosis and local protein synthesis (LPS) act coordinately to mediate the chemotropic responses of axons, but the link between these two processes is poorly understood. The endosomal sorting complex required for transport (ESCRT) is a key regulator of cargo sorting in the endocytic pathway, and here we have investigated the role of ESCRT-II, a critical ESCRT component, in Xenopus retinal ganglion cell (RGC) axons. We show that ESCRT-II is present in RGC axonal growth cones (GCs) where it co-localizes with endocytic vesicle GTPases and, unexpectedly, with the Netrin-1 receptor, deleted in colorectal cancer (DCC). ESCRT-II knockdown (KD) decreases endocytosis and, strikingly, reduces DCC in GCs and leads to axon growth and guidance defects. ESCRT-II-depleted axons fail to turn in response to a Netrin-1 gradient in vitro and many axons fail to exit the eye in vivo These defects, similar to Netrin-1/DCC loss-of-function phenotypes, can be rescued in whole (in vitro) or in part (in vivo) by expressing DCC. In addition, ESCRT-II KD impairs LPS in GCs and live imaging reveals that ESCRT-II transports mRNAs in axons. Collectively, our results show that the ESCRT-II-mediated endocytic pathway regulates both DCC and LPS in the axonal compartment and suggest that ESCRT-II aids gradient sensing in GCs by coupling endocytosis to LPS.This work was supported by EMBO LTF (AB), Sir Edward Youde Memorial Fund, Croucher Foundation, Cambridge Trust (HHW), Wellcome Trust Programme Grant (085314) and ERC Advanced Grant (322817)