PI 3‐kinase delta enhances axonal PIP 3 to support axon regeneration in the adult CNS
2020; Springer Nature; Volume: 12; Issue: 8 Linguagem: Inglês
10.15252/emmm.201911674
ISSN1757-4684
AutoresBart Nieuwenhuis, Amanda C. Barber, Rachel S Evans, Craig S. Pearson, Joachim Fuchs, Amy R. MacQueen, Susan van Erp, Barbara Haenzi, Lianne A. Hulshof, Andrew Osborne, Raquel Conceição, Tasneem Khatib, Sarita S. Deshpande, Joshua Cave, Charles ffrench‐Constant, Patrice D. Smith, Klaus Okkenhaug, Britta J. Eickholt, Keith R. Martin, James W. Fawcett, Richard Eva,
Tópico(s)Axon Guidance and Neuronal Signaling
ResumoArticle17 June 2020Open Access Transparent process PI 3-kinase delta enhances axonal PIP3 to support axon regeneration in the adult CNS Bart Nieuwenhuis orcid.org/0000-0002-2065-2271 John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Laboratory for Regeneration of Sensorimotor Systems, Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences (KNAW), Amsterdam, The Netherlands Search for more papers by this author Amanda C Barber John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Search for more papers by this author Rachel S Evans John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Search for more papers by this author Craig S Pearson John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Search for more papers by this author Joachim Fuchs Institute of Biochemistry, Charité – Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Amy R MacQueen Laboratory of Lymphocyte Signalling and Development, Babraham Institute, Cambridge, UK Search for more papers by this author Susan van Erp orcid.org/0000-0003-0883-2795 MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Barbara Haenzi John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Search for more papers by this author Lianne A Hulshof John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Search for more papers by this author Andrew Osborne John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Search for more papers by this author Raquel Conceicao John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Search for more papers by this author Tasneem Z Khatib John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Search for more papers by this author Sarita S Deshpande John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Search for more papers by this author Joshua Cave John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Search for more papers by this author Charles Ffrench-Constant MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Patrice D Smith Department of Neuroscience, Carleton University, Ottawa, ON, Canada Search for more papers by this author Klaus Okkenhaug orcid.org/0000-0002-9432-4051 Department of Pathology, University of Cambridge, Cambridge, UK Search for more papers by this author Britta J Eickholt Institute of Biochemistry, Charité – Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Keith R Martin John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, Melbourne, Vic., Australia Ophthalmology, Department of Surgery, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author James W Fawcett orcid.org/0000-0002-7990-4568 John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Centre of Reconstructive Neuroscience, Institute of Experimental Medicine, Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Richard Eva Corresponding Author [email protected] orcid.org/0000-0003-0305-0452 John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Search for more papers by this author Bart Nieuwenhuis orcid.org/0000-0002-2065-2271 John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Laboratory for Regeneration of Sensorimotor Systems, Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences (KNAW), Amsterdam, The Netherlands Search for more papers by this author Amanda C Barber John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Search for more papers by this author Rachel S Evans John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Search for more papers by this author Craig S Pearson John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Search for more papers by this author Joachim Fuchs Institute of Biochemistry, Charité – Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Amy R MacQueen Laboratory of Lymphocyte Signalling and Development, Babraham Institute, Cambridge, UK Search for more papers by this author Susan van Erp orcid.org/0000-0003-0883-2795 MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Barbara Haenzi John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Search for more papers by this author Lianne A Hulshof John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Search for more papers by this author Andrew Osborne John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Search for more papers by this author Raquel Conceicao John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Search for more papers by this author Tasneem Z Khatib John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Search for more papers by this author Sarita S Deshpande John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Search for more papers by this author Joshua Cave John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Search for more papers by this author Charles Ffrench-Constant MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Patrice D Smith Department of Neuroscience, Carleton University, Ottawa, ON, Canada Search for more papers by this author Klaus Okkenhaug orcid.org/0000-0002-9432-4051 Department of Pathology, University of Cambridge, Cambridge, UK Search for more papers by this author Britta J Eickholt Institute of Biochemistry, Charité – Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Keith R Martin John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, Melbourne, Vic., Australia Ophthalmology, Department of Surgery, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author James W Fawcett orcid.org/0000-0002-7990-4568 John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Centre of Reconstructive Neuroscience, Institute of Experimental Medicine, Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Richard Eva Corresponding Author [email protected]ac.uk orcid.org/0000-0003-0305-0452 John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Search for more papers by this author Author Information Bart Nieuwenhuis1,2,‡, Amanda C Barber1,‡, Rachel S Evans1,‡, Craig S Pearson1, Joachim Fuchs3, Amy R MacQueen4, Susan Erp5, Barbara Haenzi1, Lianne A Hulshof1, Andrew Osborne1, Raquel Conceicao1, Tasneem Z Khatib1, Sarita S Deshpande1, Joshua Cave1, Charles Ffrench-Constant5, Patrice D Smith6, Klaus Okkenhaug7, Britta J Eickholt3, Keith R Martin1,8,9, James W Fawcett1,10 and Richard Eva *,1 1John Van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK 2Laboratory for Regeneration of Sensorimotor Systems, Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences (KNAW), Amsterdam, The Netherlands 3Institute of Biochemistry, Charité – Universitätsmedizin Berlin, Berlin, Germany 4Laboratory of Lymphocyte Signalling and Development, Babraham Institute, Cambridge, UK 5MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK 6Department of Neuroscience, Carleton University, Ottawa, ON, Canada 7Department of Pathology, University of Cambridge, Cambridge, UK 8Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, Melbourne, Vic., Australia 9Ophthalmology, Department of Surgery, University of Melbourne, Melbourne, Vic., Australia 10Centre of Reconstructive Neuroscience, Institute of Experimental Medicine, Czech Academy of Sciences, Prague, Czech Republic ‡These authors contributed equally to this work *Corresponding author. Tel: +44 1223 331188; E-mail: [email protected] EMBO Mol Med (2020)12:e11674https://doi.org/10.15252/emmm.201911674 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Peripheral nervous system (PNS) neurons support axon regeneration into adulthood, whereas central nervous system (CNS) neurons lose regenerative ability after development. To better understand this decline whilst aiming to improve regeneration, we focused on phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol (3,4,5)-trisphosphate (PIP3). We demonstrate that adult PNS neurons utilise two catalytic subunits of PI3K for axon regeneration: p110α and p110δ. However, in the CNS, axonal PIP3 decreases with development at the time when axon transport declines and regenerative competence is lost. Overexpressing p110α in CNS neurons had no effect; however, expression of p110δ restored axonal PIP3 and increased regenerative axon transport. p110δ expression enhanced CNS regeneration in both rat and human neurons and in transgenic mice, functioning in the same way as the hyperactivating H1047R mutation of p110α. Furthermore, viral delivery of p110δ promoted robust regeneration after optic nerve injury. These findings establish a deficit of axonal PIP3 as a key reason for intrinsic regeneration failure and demonstrate that native p110δ facilitates axon regeneration by functioning in a hyperactive fashion. Synopsis CNS axons lose the ability to regenerate with maturity, whilst PNS axons do not. This study shows that PIP3 levels decline in CNS neurons at the time when regenerative ability is lost. CNS overexpression of one isoform of PI3K, p110δ, enhances axonal PIP3, axon transport, and regenerative ability. p110α and p110δ were found to be required for axon regeneration in adult PNS neurons, however PI3K and PIP3 declined in CNS neurons as they developed to maturity. p110α or p110δ were overexpressed in mature CNS neurons, but only p110δ restored PIP3 and regeneration, whilst the activating H1047R mutation was required in p110α to promote regeneration similarly. p110δ mediated regeneration through multiple downstream pathways, including mTOR, pS6, CRMP2, ARF6, and increased axonal transport of integrins and Rab11-positive endosomes. Transgenic expression of p110δ or hyperactive p110αH1047R stimulated axon regeneration after optic nerve injury and increased RGC survival, whilst viral delivery of p110δ led to further enhanced axon regeneration. The paper explained Problem Young neurons in the central nervous system (CNS) can regrow their axons after injury, but this ability is lost as they mature. Axonal injury or disease in the adult brain, eyes and spinal cord therefore has devastating consequences, and can result in neurological impairment, vision loss or paralysis. Conversely, neurons of the peripheral nervous system (PNS) maintain the ability to regenerate their axons into adulthood. Comparing PNS and CNS neurons is one approach to identifying new ways of promoting injured CNS axons to regenerate after injury. Results Our study found that adult PNS neurons use two versions of the enzyme PI 3-kinase to regenerate their axons, p110δ and p110α. These enzymes generate the phospholipid PIP3. Visualisation of PIP3 in maturing CNS neurons revealed that PIP3 is strongly downregulated at the time when these neurons lose their regenerative ability. Overexpression of p110δ elevated axonal PIP3 and regeneration after laser injury, but p110α required the activating H1047R mutation to behave similarly, demonstrating that native p110δ functions in a hyperactive fashion. The study found that p110δ functioned through multiple mechanisms, including the enhanced transport of regenerative machinery into axons. Importantly, viral delivery of p110δ to the retina promoted axon regeneration after an optic nerve crush injury and was accompanied by enhanced survival of RGC neurons in the retina. Impact These findings suggest that signalling through PI 3-kinase-linked receptors is limited in adult CNS axons, contributing to their weak regenerative ability. Exogenous expression of the p110δ subunit elevates axonal PI 3-kinase activity by functioning in a hyperactive fashion, leading to enhanced regeneration in human and rodent models of CNS injury, and enhanced neuroprotection and regeneration after a mouse optic nerve crush injury. Introduction Adult central nervous system (CNS) neurons have a weak capacity for axon regeneration, meaning that injuries in the brain, spinal cord and optic nerve have devastating consequences (He & Jin, 2016; Curcio & Bradke, 2018). In most CNS neurons, regenerative capacity is lost as axons mature, both in vitro (Goldberg et al, 2002; Koseki et al, 2017) and in vivo (Kalil & Reh, 1979; Wu et al, 2007). Conversely, peripheral nervous system (PNS) neurons maintain regenerative potential through adult life. This is partly because PNS neurons mount an injury response in the cell body (Smith & Skene, 1997; Ylera et al, 2009; Puttagunta et al, 2014), and also because PNS axons support efficient transport of growth-promoting receptors, whilst many of these are selectively excluded from mature CNS axons (Hollis et al, 2009a,b; Franssen et al, 2015; Andrews et al, 2016). Studies into intrinsic regenerative capacity have implicated signalling molecules, genetic factors and axon transport pathways as critical regeneration determinants (Park et al, 2008; Blackmore et al, 2012; Fagoe et al, 2015; Eva et al, 2017; Weng et al, 2018; Hervera et al, 2019). This leads to a model where axon growth capacity is controlled by genetic and signalling events in the cell body, and by the selective transport of growth machinery into the axon to re-establish a growth cone after injury. To better understand the mechanisms regulating axon regeneration, we focused on the class I phosphoinositide 3-kinases (PI3Ks). These enzymes mediate signalling through integrins, growth factor and cytokine receptors, by producing the membrane phospholipid PIP3 from PIP2 (phosphatidylinositol (3,4,5)-trisphosphate from phosphatidylinositol(4,5)-bisphosphate). Class 1 PI3Ks comprise 4 catalytic isoforms called p110α, β, γ and δ, with distinct roles for some of these emerging in specific cell populations (Bilanges et al, 2019). The p110α and p110β isoforms are ubiquitously expressed, whilst p110δ and p110γ are highly enriched in leucocytes (Hawkins & Stephens, 2015). p110β and p110γ have not been studied in neurons, but p110α mediates axon growth during chick development (Hu et al, 2013) whilst p110δ is required for axon regeneration of the PNS (Eickholt et al, 2007) and localises to the Golgi in mature cortical neurons, where it controls the trafficking of the amyloid precursor protein (APP) (Low et al, 2014; Martinez-Marmol et al, 2019). The PI3K pathway is strongly implicated in the regulation of regenerative ability because transgenic deletion of PTEN promotes CNS regeneration (Park et al, 2008; Liu et al, 2010; Geoffroy et al, 2015). PTEN opposes PI3K by converting PIP3 back to PIP2. Additionally, inhibiting negative feedback of this pathway similarly enhances regrowth (Al-Ali et al, 2017). These findings indicate a pro-regenerative role for PIP3; however, this molecule has not been directly studied in adult CNS axons. Axonal PI3K contributes to polarity in developing hippocampal axons (Shi et al, 2003), and PNS axons segregate PI3K at the growth cone to elicit rapid growth (Zhou et al, 2004); however, the neuronal distribution of PIP3 is not known. We reasoned that CNS regenerative failure might be associated with a developmental decline in PIP3 specifically within the axon, and wondered whether individual PI3K isoforms were required to yield sufficient axonal PIP3. We investigated the class I PI3K isoforms and found that both p110α and p110δ are required for PNS axon regeneration and that p110δ is specifically required within the axon. In CNS neurons, we found PIP3 was sharply downregulated with development, diminishing in the axon at the time when axon transport and regeneration also decline. We attempted to restore PIP3 through overexpression of either p110α or p110δ; however, only p110δ led to elevated axonal PIP3. Importantly, by introducing the hyperactivating H1047R mutation into p110α we found that it could mimic the effect of p110δ, with the expression of either p110δ or p110αH1047R facilitating axon regeneration. This suggests that regeneration is hindered by low activation of PI3K in mature CNS axons. We found that transgenic expression of p110δ or p110αH1047R led to enhanced retinal ganglion cell (RGC) survival and axon regeneration after optic nerve crush, whilst viral expression of p110δ led to stronger regeneration. Importantly, overexpression of p110δ had both somatic and axonal effects, enhancing the axonal transport of growth machinery and signalling through ribosomal S6 in the cell body. These findings demonstrate a deficit of axonal PIP3 as a novel reason for intrinsic regenerative failure, whilst establishing that native p110δ functions in a hyperactive fashion to enable CNS axon regeneration. Our results emphasise the importance of elevating growth-promoting pathways in the axon as well as the cell body to stimulate axon regeneration. Results Gene expression of p110 isoforms in the nervous system Because localised axonal activation of PI3K is essential for rapid PNS axon growth (Zhou et al, 2004), we reasoned that there may be specific PI3K isoforms that support regeneration within PNS axons and that these might be under-represented in CNS neurons. p110δ plays a role in PNS axon regeneration (Eickholt et al, 2007); however, the contribution of other isoforms has not been examined. We investigated the RNA expression of the individual p110 subunits (p110α, β, γ and δ) in four published neuronal RNAseq datasets (Fig EV1), examining expression in dorsal root ganglion (DRG) neurons during axon growth and regeneration (Tedeschi et al, 2016), in developing cortical neurons (Koseki et al, 2017) and in the Brain-RNAseq databases (http://www.brainrnaseq.org/) of individual cell types in mouse and human brain (Zhang et al, 2014, 2016). In DRG neurons, p110α, β and δ are expressed at all stages in vitro, whilst p110γ is expressed at very low levels. p110α is present at the highest levels, whilst p110δ is upregulated in development and further upregulated upon peripheral nerve lesion (Fig EV1A–C). In developing cortical neurons, p110α is again expressed at the highest levels, p110β and δ are present but not abundant, and p110γ is at very low levels. Remarkably, p110α, β and δ are all downregulated as cortical neurons mature (Fig EV1D and E). The Brain-RNAseq database indicates p110α and β are the principal neuronal PI3K isoforms in both the mouse and human adult brain, with p110α again expressed at the highest levels, whilst p110δ and γ are enriched in microglia and macrophages (Fig EV1F–M). These datasets indicate that p110α, β and δ are expressed in regenerative PNS neurons, but are almost absent or downregulated with maturation in CNS neurons. We therefore chose to investigate the contribution of the p110α, β and δ isoforms of PI3K to axon regeneration of DRG neurons. Click here to expand this figure. Figure EV1. Gene expression profile of p110 isoforms in the nervous system from previously published RNAseq datasetsPanels A to C show data from Tedeschi et al (2016), Panels D and E from Koseki et al (2017), and Panels F to M from Brain-RNAseq databases (http://www.brainrnaseq.org/), Zhang et al (2014) and (2016). A. Normalised mean expression values of p110 genes in adult mouse DRG neurons isolated after sciatic nerve lesion compared to a sham control. B. Normalised mean expression values of p110 genes in cultured mouse DRG neurons 6-, 12, 24- and 36 h post-plating (representing the shift from arborising to elongating axon growth). C. Normalised mean expression values of p110 genes in cultured mouse DRG neurons from at embryonic days 12.5 and 17.5. D. Relative abundance of p110 mRNA levels in cortical neurons cultured from E18 rat embryos at increasing periods of time in vitro. E. Relative abundance of p110 mRNA levels in cortical neurons cultured from E18 rat embryos at increasing periods of time in vitro, also showing expression levels other regeneration-associated genes. F–M. Relative abundance (FPKM) of p110 genes in various mouse and human brain cell types (astrocytes, neurons oligodendrocyte precursor cells, newly formed oligodendrocytes, myelinating oligodendrocytes, microglia/macrophages and endothelial cells). Each replicate consists of pooled cortices from 3 to 12 mice. For human samples, n = 6–12 for each cell type. Data are shown as the mean ± SEM. See Zhang et al (2014) and (2016) for full details. Download figure Download PowerPoint p110α and δ are required for DRG axon regeneration We used laser axotomy to sever the axons of adult DRG neurons and measured the effect of specific inhibitors of p110α, β and δ on growth cone regeneration (Fig 1). Inhibiting either p110α or δ reduced the percentage of axons regenerating, as did pan-PI3K inhibition (α, β and δ) or targeting p110α and δ together. Inhibition of p110β had no effect, but inhibiting all of the isoforms increased the time taken to develop a new growth cone (Fig 1A and B and Movies EV1 and EV2). Some inhibitors also caused uncut axons to stop growing, so we measured the extension rate of uncut axons. Treatment with p110α inhibitors, pan-PI3K or dual α/δ inhibitors led to a dramatic reduction in the percentage of uncut axons extending in 2 h, whilst they continued to extend in the presence of specific p110δ inhibitors (Fig 1C and D and Movies EV3 and EV4). We next examined the effect of PI3K inhibition using microfluidic compartmentalised chambers, in which axons extend through microchannels into a separate compartment from the cell bodies (Fig 1E). Inhibition of p110α or p110δ showed different effects depending on localisation. Inhibition of p110δ in the axonal compartment reduced the percentage of regenerating axons, but had no effect in the somatic chamber. In contrast, the p110α inhibitor A66 reduced regeneration when applied either to the axonal or somatic compartment, and also increased the time taken to generate a new growth cone (Fig 1F). These data show that DRG neurons require p110α and δ for efficient regeneration. Axon growth and regeneration require p110α activity in both the cell body and axon, whilst axon regeneration further relies on p110δ activity specifically within the axon. Figure 1. p110α and δ are required for rat DRG axon regeneration; p110δ functions in the axonal compartment A. Laser-injured DRG axons showing growth cone regeneration (upper panels) and regenerative failure (lower panels). Arrows as indicated. B. Upper graph shows percentage of regenerating axons of DIV 1–2 adult DRG neurons in the presence of p110 inhibitors, 2 h after injury. Lower graph shows time taken to regenerate a new growth cone. Inhibitors were added 1 h before axons were injured and maintained throughout: LY294002, 20 μM; A66, 5 μM; XL-147, 5 μM; TGX221, 500 nM; IC-87114, 10 μM; idelalisib, 500 nM. Upper graph data are shown as the mean ± SEM. P-values indicate statistical significance analysed by Fisher's exact (upper graph) or Kruskal–Wallis test (lower graph). C. Examples of static vs. growing uninjured axons treated with p110α or δ inhibitors as indicated. Both arrows in the bottom right image indicate growing axons. D. Percentage of uninjured, growing axons in the presence of p110 inhibitors. Inhibitors were added as for (B). Lower graph shows length grown in 2 h. Data are shown as the mean ± SEM. P-values indicate significance measured by Fisher's exact test (upper graph) or ANOVA with Tukey's post-hoc analysis (lower graph). E. Adult DRG neurons in microfluidic compartmental chambers. Cell bodies on the left side, axons extending through microchannels on the right. Lower panels are schematics. F. Upper graph shows percentage of regenerating axons in microfluidic chambers. p110α or δ inhibitors were applied to either soma or axons. Lower graph shows time taken to regenerate a new growth cone. Inhibitors were added 1hr before axons were injured and maintained throughout: A66, 5 μM; idelalisib, 500 nM. Upper graph data are shown as the mean ± SEM. P-values indicate statistical significance as measured by Fisher's exact test (upper graph) or by Kruskal–Wallis test (lower graph). Data information: Numbers on bars are the total numbers of axons cut, from n = 4 experiments for (B, D and F). Download figure Download PowerPoint PIP3 is developmentally downregulated in cortical neurons, but present in adult DRG axons The RNAseq data described above (Fig EV1) suggest that PI3K expression increases with maturation in DRG neurons, whilst it is downregulated as cortical neurons mature. CNS regenerative failure might therefore be due to insufficient PI3K activity within the axon and insufficient PIP3. PIP3 is implicated in axon growth but its developmental distribution has not been examined. PIP3 has previously been localised using fluorescently tagged pleckstrin homology (PH)-domain reporters, such as AKT-PH-GFP; however, the principle readout of these reporters is translocation to the surface membrane, and they do not report on abundance or "steady-state" distribution. To accurately measure PIP3 in neurons developing in vitro, we optimised a fixation technique (Hammond et al, 2009) for antibody-based PIP3 detection on immobilised membrane phospholipids utilising an antibody widely used for biochemical assays. To validate this in neurons, we isolated DRG neurons from transgenic mice expressing AKT-PH-GFP at low levels (Nishio et al, 2007) to avoid inhibition of downstream signalling (Varnai et al, 2005). Live imaging of adult DRG neurons from these mice revealed dynamic hotpots of PIP3 at the axon growth cone (Fig EV2A and Movie EV5), and membrane labelling confirmed these are not regions of membrane enrichment (Fig EV2B and Movie EV6). Phospholipid fixation and labelling with anti-PIP3 revealed colocalisation between AKT-PH-GFP and anti-PIP3 at regions within DRG growth cones (Fig EV2C), as well as at hotspots and signalling platforms in non-neuronal cells from DRG cultures (Fig EV2D). In addition to validating this technique, our data also confirm the presence of dynamic PIP3 in the growth cone and axons of regenerative DRG neurons. To further confirm the specificity of the stain for PIP3, we stimulated N1E cells with insulin and labelled for PIP3 in the presence or absence of the pan-PI3K inhibitor GDC-0941, detecting increased PIP3 staining after insulin stimulation alone, and not in the presence of the PI3K inhibitor (Fig EV2E–G). Click here to expand this figure. Figure EV2. Validation of a protocol for detecting PIP3 in neuronal membranes A. Time-lapse images (single confocal section imaged by spinning disc microscopy) of a DRG growth cone cultured from adult AKT-PH-GFP mice. Arrows point to hotspots/regions of increased fluorescence indicative of AKT-PH recruitment. See also associated Movie EV1. B. Time-lapse images (single confocal section imaged by spinning disc microscopy) of a DRG growth cone cultured from adult AKT-PH-GFP mice, stained with cell-mask orange (magenta colour) to detect the membrane. Arrows indicate a dynamic region of AKT-PH-GFP recruitment that does not label for membrane aggregation. See also associated Movie EV2. C. Adult DRG growth cone cultured from AKT-PH-GFP mice, fixed for PIP3 immobilisation (see methods section) and labelled with an antibody to PIP3 (magenta). Arrows and dotted circles indicate colocalisation. D. Non-neuronal cell from a dissociated DRG culture from AKT-PH-GFP mice, fixed for PIP3 immobilisation (see methods section), and labelle
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