Endosomal phosphatidylinositol 3‐phosphate controls synaptic vesicle cycling and neurotransmission
2022; Springer Nature; Volume: 41; Issue: 9 Linguagem: Inglês
10.15252/embj.2021109352
ISSN1460-2075
AutoresGuan‐Ting Liu, Gaga Kochlamazashvili, Dmytro Puchkov, Rainer Müller, Carsten Schultz, Albert Ian Mackintosh, Dennis Vollweiter, Volker Haucke, Tolga Soykan,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoArticle22 March 2022Open Access Source DataTransparent process Endosomal phosphatidylinositol 3-phosphate controls synaptic vesicle cycling and neurotransmission Guan-Ting Liu Guan-Ting Liu Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Contribution: Investigation, Methodology Search for more papers by this author Gaga Kochlamazashvili Gaga Kochlamazashvili Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Contribution: Data curation, Formal analysis, Investigation, Methodology Search for more papers by this author Dmytro Puchkov Dmytro Puchkov orcid.org/0000-0001-8341-4847 Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Contribution: Conceptualization, Data curation, Investigation, Methodology Search for more papers by this author Rainer Müller Rainer Müller orcid.org/0000-0003-3464-494X European Molecular Biology Laboratory (EMBL), Cell Biology and Biophysics Unit, Heidelberg, Germany Search for more papers by this author Carsten Schultz Carsten Schultz orcid.org/0000-0002-5824-2171 European Molecular Biology Laboratory (EMBL), Cell Biology and Biophysics Unit, Heidelberg, Germany Department of Chemical Physiology & Biochemistry, Oregon Health & Science University (OHSU), Portland, OR, USA Contribution: Supervision, Funding acquisition, Methodology Search for more papers by this author Albert I Mackintosh Albert I Mackintosh Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Contribution: Investigation Search for more papers by this author Dennis Vollweiter Dennis Vollweiter Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Contribution: Investigation, Methodology Search for more papers by this author Volker Haucke Corresponding Author Volker Haucke [email protected] orcid.org/0000-0003-3119-6993 Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Faculty of Biology, Chemistry, Pharmacy, Freie Universität Berlin, Berlin, Germany NeuroCure Cluster of Excellence, Charité Universitätsmedizin Berlin, Berlin, Germany Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Tolga Soykan Corresponding Author Tolga Soykan [email protected] orcid.org/0000-0002-1324-9601 Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Contribution: Conceptualization, Supervision, Investigation, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Guan-Ting Liu Guan-Ting Liu Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Contribution: Investigation, Methodology Search for more papers by this author Gaga Kochlamazashvili Gaga Kochlamazashvili Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Contribution: Data curation, Formal analysis, Investigation, Methodology Search for more papers by this author Dmytro Puchkov Dmytro Puchkov orcid.org/0000-0001-8341-4847 Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Contribution: Conceptualization, Data curation, Investigation, Methodology Search for more papers by this author Rainer Müller Rainer Müller orcid.org/0000-0003-3464-494X European Molecular Biology Laboratory (EMBL), Cell Biology and Biophysics Unit, Heidelberg, Germany Search for more papers by this author Carsten Schultz Carsten Schultz orcid.org/0000-0002-5824-2171 European Molecular Biology Laboratory (EMBL), Cell Biology and Biophysics Unit, Heidelberg, Germany Department of Chemical Physiology & Biochemistry, Oregon Health & Science University (OHSU), Portland, OR, USA Contribution: Supervision, Funding acquisition, Methodology Search for more papers by this author Albert I Mackintosh Albert I Mackintosh Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Contribution: Investigation Search for more papers by this author Dennis Vollweiter Dennis Vollweiter Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Contribution: Investigation, Methodology Search for more papers by this author Volker Haucke Corresponding Author Volker Haucke [email protected] orcid.org/0000-0003-3119-6993 Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Faculty of Biology, Chemistry, Pharmacy, Freie Universität Berlin, Berlin, Germany NeuroCure Cluster of Excellence, Charité Universitätsmedizin Berlin, Berlin, Germany Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Tolga Soykan Corresponding Author Tolga Soykan [email protected] orcid.org/0000-0002-1324-9601 Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany Contribution: Conceptualization, Supervision, Investigation, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Author Information Guan-Ting Liu1, Gaga Kochlamazashvili1, Dmytro Puchkov1, Rainer Müller2, Carsten Schultz2,3, Albert I Mackintosh1, Dennis Vollweiter1, Volker Haucke *,1,4,5 and Tolga Soykan *,1 1Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany 2European Molecular Biology Laboratory (EMBL), Cell Biology and Biophysics Unit, Heidelberg, Germany 3Department of Chemical Physiology & Biochemistry, Oregon Health & Science University (OHSU), Portland, OR, USA 4Faculty of Biology, Chemistry, Pharmacy, Freie Universität Berlin, Berlin, Germany 5NeuroCure Cluster of Excellence, Charité Universitätsmedizin Berlin, Berlin, Germany *Corresponding author. Tel: +49 30 94793 101; E-mail: [email protected] *Corresponding author. Tel: +49 30 94793 208; E-mail: [email protected] The EMBO Journal (2022)41:e109352https://doi.org/10.15252/embj.2021109352 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 Neural circuit function requires mechanisms for controlling neurotransmitter release and the activity of neuronal networks, including modulation by synaptic contacts, synaptic plasticity, and homeostatic scaling. However, how neurons intrinsically monitor and feedback control presynaptic neurotransmitter release and synaptic vesicle (SV) recycling to restrict neuronal network activity remains poorly understood at the molecular level. Here, we investigated the reciprocal interplay between neuronal endosomes, organelles of central importance for the function of synapses, and synaptic activity. We show that elevated neuronal activity represses the synthesis of endosomal lipid phosphatidylinositol 3-phosphate [PI(3)P] by the lipid kinase VPS34. Neuronal activity in turn is regulated by endosomal PI(3)P, the depletion of which reduces neurotransmission as a consequence of perturbed SV endocytosis. We find that this mechanism involves Calpain 2-mediated hyperactivation of Cdk5 downstream of receptor- and activity-dependent calcium influx. Our results unravel an unexpected function for PI(3)P-containing neuronal endosomes in the control of presynaptic vesicle cycling and neurotransmission, which may explain the involvement of the PI(3)P-producing VPS34 kinase in neurological disease and neurodegeneration. Synopsis Neural circuit function requires mechanisms for the control of neurotransmitter release. We Here, presynaptic vesicle cycling and neurotransmission are found to be feedback-regulated by the endosomal lipid phosphatidylinositol 3-phosphate (PI(3)P). Neuronal activity inhibits endosomal PI(3)P synthesis in neurons. Reduced PI(3)P levels repress neurotransmission and impair vesicle recycling. Loss of PI(3)P triggers calpain and Cdk5 activation, a key negative control switch for neurotransmission. PI(3)P loss prevents hyperactivity of neuronal networks independent of synaptic inhibition. Introduction Neurotransmission is mediated by the stochastic calcium-triggered fusion of presynaptic vesicles with the plasma membrane to release neurotransmitters (Sudhof & Rothman, 2009), which bind to and activate postsynaptic receptors, for example, ionotropic glutamate receptors. Neurotransmission is fine-tuned in a synapse-specific manner by short- and long-term synaptic plasticity (Katz & Shatz, 1996; Abbott & Regehr, 2004), while the robustness of synaptic transmission is ensured by homeostatic adaptations such as presynaptic homeostatic plasticity that stabilize excitatory neurotransmission in response to alterations in postsynaptic function (Davis & Muller, 2015). For example, perturbation of postsynaptic excitability in Drosophila neuromuscular junctions (Davis & Goodman, 1998) is counteracted by a homeostatic facilitation of presynaptic neurotransmitter release. In addition to these pathways, the proper functioning of neuronal networks also requires mechanisms that limit excitatory neurotransmission. On the circuit level, glutamate-induced postsynaptic depolarization is thought to be counteracted mainly by inhibitory synaptic inputs (Katz & Shatz, 1996; Abbott & Regehr, 2004; Marder & Goaillard, 2006; Hu et al, 2014). Consistently, impairment of inhibitory synapse function is associated with hyperexcitation and epileptiform activity in animal models and in humans (Nelson & Valakh, 2015). In parallel to circuit level organization, neurons may intrinsically monitor and adjust their excitability and neurotransmission via mechanisms that mediate crosstalk between synaptic activity and intracellular signaling. How such cell-intrinsic control of neurotransmission is achieved at the molecular-mechanistic level remains poorly understood, but could possibly involve alterations in the exo-endocytosis of SVs, that is, the cell biological processes that underlie chemical neurotransmission. SV cycling is based on the action potential (AP)-triggered calcium-driven exocytic fusion of SVs at active zone release sites (Jahn & Fasshauer, 2012; Sudhof, 2013). Exocytic SV fusion is followed by endocytosis of SV membranes and the endocytic reformation of functional SVs (Saheki & De Camilli, 2012; Rizzoli, 2014; Chanaday et al, 2019) from internal, possibly endosome-like organelles via clathrin- and adaptor-mediated budding (Cheung & Cousin, 2012; Kononenko et al, 2014; Watanabe et al, 2014). The speed and efficacy of SV endocytosis is facilitated by the calcium-dependent phosphatase calcineurin (Saheki & De Camilli, 2012; Cheung & Cousin, 2013; Rizzoli, 2014; Wu et al, 2014; Soykan et al, 2016) and repressed by the synaptic protein kinase Cdk5 (Tan et al, 2003; Ferguson et al, 2007; Armbruster et al, 2013), a master regulatory switch for presynaptic neurotransmission (Kim & Ryan, 2010, 2013). In most cell types, endosomes are marked by high levels of phosphatidylinositol 3-phosphate [PI(3)P], a signaling lipid that is crucial for endosome function (Di Paolo & De Camilli, 2006; Balla, 2013; Raiborg et al, 2013). Hence, endosomal PI(3)P may conceivably regulate SV cycling (see e.g., (Geumann et al, 2010; Uytterhoeven et al, 2011; Rizzoli, 2014; Jahne et al, 2015)) and, thereby, impinge on neuronal network activity, a hypothesis that we have tested in this study. Here, we unravel a mechanism for the control of presynaptic function that involves a reciprocal interplay between synaptic activity and endosomal PI(3)P levels. We demonstrate that PI(3)P synthesis via VPS34 controls neurotransmission and SV cycling via a feedback-regulated mechanism that determines the activity status of Calpain 2 and cyclin-dependent kinase 5 (Cdk5), an enzyme crucial for tuning presynaptic neurotransmitter release and SV recycling (Tan et al, 2003; Kim & Ryan, 2010, 2013; Shah & Lahiri, 2014), to prevent hyperexcitation and epileptiform neuronal activity. Our data have implications for our understanding of neuronal network function and help to explain the role of VPS34 in neurological and neurodegenerative disorders. Results Neuronal activity represses endosomal PI(3)P synthesis mediated by the lipid kinase VPS34 Endosomes and endosome-like organelles have been proposed to play important roles in synaptic function including the recycling of SV membranes, presynaptic protein sorting and quality control, and the exo-endocytic trafficking of postsynaptic receptors during synaptic plasticity (Park et al, 2004; Uytterhoeven et al, 2011; van der Sluijs & Hoogenraad, 2011; Saheki & De Camilli, 2012; Rizzoli, 2014; Watanabe et al, 2014; Wu et al, 2014; Chanaday et al, 2019). Surprisingly little is known about the distribution and dynamics of neuronal endosomes marked by PI(3)P, a lipid of crucial importance for endosome and lysosome function in non-neuronal cells (Simonsen et al, 1998; Di Paolo & De Camilli, 2006; Balla, 2013; Raiborg et al, 2013) and in neurons (Morel et al, 2013; Miranda et al, 2018). To close this knowledge gap, we first monitored the nanoscale distribution of PI(3)P in cultured hippocampal neurons by multicolor time-gated stimulated emission depletion microscopy (gSTED) using the recombinant 2xFYVE domain of Hrs (Gaullier et al, 1998; Balla, 2013) (see also (Ketel et al, 2016) for specificity) as a specific probe. PI(3)P-containing endosomes were detected throughout neuronal somata and neurites including pre- and postsynaptic compartments immunopositive for the SV-associated protein Synapsin and postsynaptic Homer, respectively (Fig 1A and B). Further analysis by confocal microscopy imaging revealed similar levels of PI(3)P at excitatory synapses marked by the vesicular glutamate transporter 1 (vGLUT1) and at inhibitory synapses identified by the vesicular GABA transporter vGAT (Fig 1C and D). These data show that PI(3)P-containing endosomes are present at both excitatory and inhibitory synapses, consistent with their presumed roles in synapse physiology and neurotransmission. Figure 1. The abundance of somatic and synaptic PI(3)P-positive endosomes is regulated by neuronal activity via Cdk5 Representative 3-color STED images of synapses of cultured hippocampal neurons fixed and stained for Synapsin1/2 (presynaptic marker), PI(3)P, and Homer 1 (postsynaptic marker). Scale bar, 0.5 µm. PI(3)P-positive structures appear in both pre- and postsynaptic compartments. Line scan fluorescence intensity profiles of single synapses from the images in A, normalized to maximum value for each channel. Representative 3-color confocal images of cultured hippocampal neurons fixed and stained for VGlut1, VGAT, and PI(3)P. Arrows indicate PI(3)P puncta in VGlut1+ or VGAT+ presynaptic terminals. Scale bar, 10 µm. PI(3)P intensity in VGlut1+ and VGAT+ presynaptic terminals. PI(3)P is equally abundant in excitatory and inhibitory presynaptic terminals. N = 3 independent experiments (≥ 15 images per condition); Student's t-test. Relative PI(3)P levels in the MAP2+ (Soma) and Syp+ (Synapse) areas in DMSO and VPS34IN1-treated neurons. N = 3 independent experiments (≥ 35 images per condition); Student's t-test. Cultured hippocampal neurons were field-stimulated with four trains of 200 APs (40 Hz, 5 s; 90 s gap between each train) (Stim) or left unstimulated (−), fixed and stained for MAP2, Synaptophysin (Syp), and PI(3)P. Arrows indicate PI(3)P puncta in synapses. Scale bar, 10 µm. PI(3)P puncta that do not overlap with neuronal soma and synapses belong to astrocytes in culture and are disregarded from the quantification. Relative intensity of PI(3)P in neuronal somata (MAP2+) or synapses (Syp+) from the images in F. N = 5 independent experiments; (≥ 40 images per condition); Student's t-test. Relative intensity of PI(3)P in neuronal somata (MAP2+) or synapses (Syp+) of cultured hippocampal neurons treated with DMSO or Gabazine (10 µM) overnight. N = 3 independent experiments (≥ 65 images per condition); Student's t-test. Cultured hippocampal neurons treated with AP5 (20 µM), CNQX (10 µM) or TTX (0.2 µM) overnight, fixed and stained for MAP2, Syp and PI(3)P. Arrows indicate PI(3)P puncta in synapses. Scale bar, 10 µm. Relative intensity of PI(3)P in neuronal somata (MAP2+) or synapses (Syp+) from the images in I. N ≥ 4 independent experiments (≥ 60 images per condition); Student's t-test. Relative intensity of PI(3)P in neuronal somata (MAP2+) or synapses (Syp+) of cultured hippocampal neurons were treated with Roscovitine (10 µM) for 1 h and field-stimulated with four trains of 200 APs (40 Hz, 5 s; 90 s gap between each train) (Stim) or left unstimulated (−). N = 3 independent experiments (≥ 40 images per condition); Two-way ANOVA; Fisher's Least Significant Difference Test. Data information: Data presented as mean ± SEM in all panels; n.s. not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; p, t, q, df values are provided in the Source Data Statistics file. Source data are available online for this figure. Source Data for Figure 1 [embj2021109352-sup-0003-SDataFig1.zip] Download figure Download PowerPoint To obtain further insights into the dynamics and function of PI(3)P-containing endosomes at synapses, we analyzed the mechanism of neuronal PI(3)P synthesis. Synthesis of PI(3)P in most mammalian cells and tissues is mediated predominantly by the endosomally localized class III PI 3-kinase VPS34, an essential factor for neuronal survival to prevent neurodegeneration (Zhou et al, 2010; Morel et al, 2013), and, to a minor extent, by the class II PI 3-kinases PI3K-C2α and PI3K-C2β (Di Paolo & De Camilli, 2006; Balla, 2013). We confirmed the expression of VPS34, PI3K-C2α, and PI3K-C2β in brain and found these enzymes to be enriched in crude SV fractions prepared from lysed presynaptic nerve terminals (Fig EV1A). Selective inhibition of VPS34 by the well-established specific small molecule inhibitor VPS34IN1 (Bago et al, 2014; Ketel et al, 2016) effectively reduced somatic and synaptic PI(3)P to nearly undetectable levels (Figs 1E and EV1B). A similar efficacy of VPS34IN1-mediated PI(3)P depletion was observed at excitatory and inhibitory presynaptic sites (Fig EV1C and D). Hence, the endosomal class III PI 3-kinase VPS34 is the major enzyme that produces PI(3)P at excitatory and inhibitory synapses in hippocampal neurons. Click here to expand this figure. Figure EV1. PI(3)P is localized at neuronal soma and synapses and endosomal PI(3)P levels are regulated by neuronal activity A. Subcellular fractionation of mouse brains followed by western blot reveals the presence of Class II and Class III PI3Ks in the synaptic vesicle enriched LP2 fraction. B. Representative 2-color confocal images of cultured hippocampal neurons treated with DMSO (0.1%) or VPS34IN1 (10 µM) for 1 h, fixed and stained for MAP2, synaptophysin (Syp), and PI(3)P. Arrows indicate presynaptic terminals that contain (DMSO) or lack (VPS34IN1) PI(3)P. Scale bar, 10 µm. C, D. Representative 3-color STED images of synapses of cultured hippocampal neurons treated with DMSO (0.1%) or VPS34IN1 (10 µM) for 1 h, fixed and stained for PI(3)P and VGlut1-PSD95 pair (markers for excitatory synapses) in C and VGAT-Gephyrin pair (markers for inhibitory synapses) in D. Scale bar, 0.5 µm. Both excitatory and inhibitory synaptic terminals contain PI(3)P-positive structures, which disappear upon VPS34 inhibition. E. Cultured hippocampal neurons treated with Gabazine (10 µM) overnight, fixed and stained for MAP2, Syp and PI(3)P. Scale bar, 10 µm. F. Cultured hippocampal neurons were treated with Roscovitine (10 µM) for 1 h and field-stimulated with four trains of 200 APs (40 Hz, 5 s; 90 s gap between each train) (Stim) or left unstimulated (−), fixed and stained for MAP2, Syp, and PI(3)P. Scale bar, 10 µm. G. Cultured hippocampal neurons were treated with Dinaciclib (10 µM) for 1 h and field-stimulated with four trains of 200 APs (40 Hz, 5 s; 90 s gap between each train) (Stim) or left unstimulated (−), fixed and stained for MAP2, Syp, and PI(3)P. Scale bar, 10 µm. H. Relative intensity of PI(3)P in neuronal somata (MAP2+) or synapses (Syp+) of cultured hippocampal neurons were treated with Dinaciclib (10 µM) for 1 h and field-stimulated with four trains of 200 APs (40 Hz, 5 s; 90 s gap between each train) (Stim) or left unstimulated (−). N = 3 independent experiments (≥ 15 images per condition); Mean ± SEM; *P < 0.05; Two-way ANOVA. I. Cultured hippocampal neurons transfected with VPS34-SNAP or VPS34T159A-SNAP at DIV7, treated with Gabazine (10 µM) overnight at DIV13, treated 1 h with SNAP-tag ligand JF646 to label VPS34-SNAP at DIV14 and subsequently fixed and stained for PI(3)P. Scale bar, 10 µm. J. Relative intensity of PI(3)P in neuronal somata of cultured hippocampal neurons expressing VPS34-SNAP or VPS34T159A-SNAP, treated with DMSO or Gabazine (10 µM) overnight. N = 6 independent experiments (≥ 30 images per condition); Mean ± SEM; *P < 0.05; Two-way ANOVA. Source data are available online for this figure. Download figure Download PowerPoint Given these data and the alleged functions of neuronal or synaptic endosomes (Park et al, 2004; Uytterhoeven et al, 2011; van der Sluijs & Hoogenraad, 2011; Saheki & De Camilli, 2012; Rizzoli, 2014; Watanabe et al, 2014; Wu et al, 2014; Chanaday et al, 2019), we hypothesized that endosomal PI(3)P may impinge on or be controlled by neuronal network activity. Sustained stimulation of hippocampal neurons with repetitive trains of APs (200 APs, 40 Hz, 5 s) resulted in the depletion of PI(3)P from endosomal puncta present in neuronal somata and at presynaptic nerve terminals marked by the SV protein Synaptophysin (Fig 1F and G). In striking contrast, we confirmed the previously observed stimulation-induced elevation of presynaptic phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] levels at active synapses (not shown; (Micheva et al, 2001)). Elevation of neuronal network activity by disinhibition in the presence of the γ-amino-butyric acid (GABA) type A (GABAA) receptor antagonist Gabazine (Zhao et al, 2011; Jeans et al, 2017), led to a similar reduction in somatic and synaptic PI(3)P levels (Figs 1H and EV1E). Conversely, blockade of endogenous network activity by the N-methyl-D-aspartate (NMDA) receptor antagonist AP5, the sodium channel blocker tetrodotoxin (TTX), or the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor inhibitor CNQX caused a profound elevation of somatic and synaptic PI(3)P levels (Fig 1I and J). Endosomal PI(3)P levels are, thus, inversely correlated with and, possibly, controlled by neuronal activity. In non-neuronal cells, cyclin-dependent kinases have been shown to repress VPS34-mediated PI(3)P synthesis by phosphorylating VPS34 at Thr159 (Furuya et al, 2010). We therefore reasoned that the observed repression of PI(3)P synthesis by neuronal activity might be mediated by Cdk5, a major negative regulator of neurotransmission (Kim & Ryan, 2010, 2013) and SV endocytosis (Tan et al, 2003; Ferguson et al, 2007; Armbruster et al, 2013). Consistently, we found that pharmacological inhibition of Cdk5 activity by Roscovitine or Dinaciclib occludes the activity-induced repression of VPS34-mediated PI(3)P synthesis in neuronal somata and at synapses (Figs 1K and EV1F–H). Furthermore, overexpression of a nonphosphorylatable mutant of VPS34 (T159A) led to elevated PI(3)P levels in neuronal somata and occluded Gabazine-induced downregulation of PI(3)P synthesis (Fig EV1I and J). These results indicate that endosomal PI(3)P synthesis is repressed by neuronal activity via Cdk5-mediated feedback regulation of VPS34. PI(3)P depletion reduces neurotransmission and perturbs SV endocytosis To analyze the functional consequences of reduced PI(3)P synthesis at synapses, we probed the effects of pharmacological inhibition of VPS34-mediated PI(3)P synthesis on neurotransmission by electrophysiological recordings in acute hippocampal slice preparations from genetically unperturbed wild-type mice. Depletion of PI(3)P in the presence of VPS34IN1 resulted in a rapid and progressive decline of field excitatory postsynaptic potentials (fEPSP) (Figs 2A, Appendix Fig S1A and B). This was paralleled by an increased paired pulse ratio (Figs 2B and Appendix Fig S1C), a surrogate measure of presynaptic release probability. A similar depression of basal excitatory neurotransmission (Fig 2A) and elevated paired-pulse response (Appendix Fig S1D) was observed upon application of another VPS34 inhibitor, SAR405 (Ronan et al, 2014). These data indicate that repression of VPS34-mediated PI(3)P synthesis reduces basal excitatory neurotransmission in response to depolarizing stimuli, likely via alterations in presynaptic release probability. Figure 2. Loss of PI(3)P leads to reduced basal synaptic transmission and perturbs SV endocytosis Basal excitatory synaptic transmission of CA3-CA1 connections in acute hippocampal slices treated with DMSO (0.03%) or the VPS34 inhibitors, VPS34IN1 (3 µM) or SAR405 (20 µM). Representative fEPSP traces (above) and the relative slope of fEPSPs (below) are shown. VPS34 inhibition causes reduced excitatory synaptic transmission down to 46.0 ± 4.1% and 55.0 ± 4.4% for VPS34IN1 and SAR405 at the end of 60-min treatment. N = 6 slices per condition from ≥ 4 mice; Student's t-test. Increased paired paired-pulse facilitation (PPF) at hippocampal CA3-CA1 synapses after VPS34 inhibition. Representative traces of fEPSPs with a 20 ms interpulse interval is shown above. The ratio of the slope of the second to the first response over a range of interstimulus intervals (10–500 ms) is plotted below, indicating an increased facilitation of the second response in VPS34IN1-treated acute hippocampal slices. PI(3)P depletion causes a significant facilitation of the second fEPSP, indicative of reduced initial release probability. N = 6 slices per condition from 6 mice; Two-way ANOVA. Synaptophysin-pHluorin responses normalized to baseline from neurons stimulated with 12 consecutive trains of 20 APs (20 Hz, 1 s) with 30 s gap between each stimulus. Neurons are acutely treated with DMSO (0.1%) or VPS34 inhibitor1 (50 µM) starting from the onset of 2nd stimulation. The timing of the treatment and the stimulation trains are indicated. Quantification of data from C. Net difference of Synaptophysin-pHluorin fluorescence 2 s before and 28 s after each stimulation (2 s before the next stimulation) to estimate relative surface retention of SVs. SV endocytosis is blocked within 5 min of treatment with VPS34 Inhibitor1, leading to progressive accumulation of Synaptophysin-pHluorin signal. N = 4 or 5 independent measurements per condition; Student's t-test. Data information: Data presented as mean ± SEM in all panels; *P < 0.05; **P < 0.01; ***P < 0.001; p, t, q, df values are provided in the Source Data Statistics file. Source data are available online for this figure. Source Data for Figure 2 [embj2021109352-sup-0004-SDataFig2.zip] Download figure Download PowerPoint As VPS34 operates mainly on endosomes and PI(3)P is absent from the plasma membrane (Gaullier et al, 1998; Di Paolo & De Camilli, 2006; Balla, 2013; Morel et al, 2013; Raiborg et al, 2013; Ketel et al, 2016), we speculated that reduced basal neurotransmission in PI(3)P-depleted neurons might be accompanied or partially caused by impaired SV endocytosis and/ or recycling, which may involve endosome-like intermediates (Rizzoli, 2014; Watanabe et al, 2014; Jahne et al, 2015; Chanaday et al, 2019). We tested this by monitoring SV endocytosis using the pH-sensitive reporter Synaptophysin-pHluorin, a chimera between pH-sensitive super-ecliptic GFP and the SV protein Synaptophysin (Miesenbock et al, 1998; Kavalali & Jorgensen, 2014). Pharmacological inhibition of endosomal PI(3)P synthesis caused a progressive impairment of Synaptophysin-pHluorin endocytosis in hippocampal neurons stimulated with brief trains of APs (20APs, 20 Hz) (Fig 2C). Delayed kinetics of Synaptophysin-pHluorin retrieval were paralleled by a pronounced retention of nonretrieved Synaptophysin-pHluorin molecules on the neuronal surface (Fig 2D). Under these conditions of repeated AP train stimulation (see also Fig 4A–D) no overt defect in SV exocytosis was observed, suggesting that AP train-induced presynaptic calcium elevation can override a reduction in release probability caused by depletion of PI(3)P. To corroborate these results from optical imaging experiments, we analyzed the ultrastructure of synapses from VPS34IN1-treated hippocampal neurons stimulated with trains of 200 APs or kept at rest by electron microscopy (EM) and quantitative morphometry and by tomographic 3D reconstructions (Fig 3). Synapses from stimulated neurons depleted of PI(3)P by application of VPS34IN1 displayed a profound reduction in the SV pool size (Fig 3A and D) and an accumulation of endocytic plasma membrane invaginations (Fig 3B and E) and endosome-like vacuoles (Fig 3C and F), in addition to clathrin-coated endocytic pits and vesicles (Fig 3B, G and H) compared to DMSO-treated controls. No significant SV depletion or accumulation of unresolved intermediates of SV endocytosis were observed in nonstimulated hippocampal neurons (Fig 3D–H). Figure 3. Loss of PI(3)P leads to accumulation of SV recycling intermediates in stimulated synapses A. Electron micrographs of VPS34IN1- and DMSO-treated synapses following four consecutive trains of 200 AP (40 Hz, 5 s; 90 s gap betwe
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