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• W2148447088 abstract "Article27 July 2012Open Access Modulation of synaptic function by VAC14, a protein that regulates the phosphoinositides PI(3,5)P2 and PI(5)P Yanling Zhang Yanling Zhang Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, USA Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Amber J McCartney Amber J McCartney Neuroscience Graduate Program, University of Michigan, Ann Arbor, MI, USA Molecular and Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Sergey N Zolov Sergey N Zolov Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, USA Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Cole J Ferguson Cole J Ferguson Neuroscience Graduate Program, University of Michigan, Ann Arbor, MI, USA Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Miriam H Meisler Miriam H Meisler Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Michael A Sutton Corresponding Author Michael A Sutton Molecular and Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, MI, USA Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Lois S Weisman Corresponding Author Lois S Weisman Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, USA Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Yanling Zhang Yanling Zhang Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, USA Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Amber J McCartney Amber J McCartney Neuroscience Graduate Program, University of Michigan, Ann Arbor, MI, USA Molecular and Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Sergey N Zolov Sergey N Zolov Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, USA Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Cole J Ferguson Cole J Ferguson Neuroscience Graduate Program, University of Michigan, Ann Arbor, MI, USA Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Miriam H Meisler Miriam H Meisler Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Michael A Sutton Corresponding Author Michael A Sutton Molecular and Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, MI, USA Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Lois S Weisman Corresponding Author Lois S Weisman Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, USA Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Author Information Yanling Zhang1,2,‡, Amber J McCartney3,4,‡, Sergey N Zolov1,2, Cole J Ferguson3,5, Miriam H Meisler5, Michael A Sutton 4,6 and Lois S Weisman 1,2 1Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, USA 2Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA 3Neuroscience Graduate Program, University of Michigan, Ann Arbor, MI, USA 4Molecular and Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, MI, USA 5Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA 6Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA ‡These authors contributed equally to this work *Corresponding authors: Molecular and Behavioral Neuroscience Institute, University of Michigan, 5067 BSRB, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200, USA. Tel.: +1 734 615 2445; Fax: +1 734 936 3690; E-mail: [email protected] Sciences Institute, The University of Michigan, 210 Washtenaw Avenue, Room 6437, Ann Arbor, MI 48109-2216, USA. Tel.: +1 734 647 2537; Fax: +1 734 615 5493; E-mail: [email protected] The EMBO Journal (2012)31:3442-3456https://doi.org/10.1038/emboj.2012.200 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 Normal steady-state levels of the signalling lipids PI(3,5)P2 and PI(5)P require the lipid kinase FAB1/PIKfyve and its regulators, VAC14 and FIG4. Mutations in the PIKfyve/VAC14/FIG4 pathway are associated with Charcot-Marie-Tooth syndrome and amyotrophic lateral sclerosis in humans, and profound neurodegeneration in mice. Hence, tight regulation of this pathway is critical for neural function. Here, we examine the localization and physiological role of VAC14 in neurons. We report that endogenous VAC14 localizes to endocytic organelles in fibroblasts and neurons. Unexpectedly, VAC14 exhibits a pronounced synaptic localization in hippocampal neurons, suggesting a role in regulating synaptic function. Indeed, the amplitude of miniature excitatory postsynaptic currents is enhanced in both Vac14−/− and Fig4−/− neurons. Re-introduction of VAC14 in postsynaptic Vac14−/− cells reverses this effect. These changes in synaptic strength in Vac14−/− neurons are associated with enhanced surface levels of the AMPA-type glutamate receptor subunit GluA2, an effect that is due to diminished regulated endocytosis of AMPA receptors. Thus, VAC14, PI(3,5)P2 and/or PI(5)P play a role in controlling postsynaptic function via regulation of endocytic cycling of AMPA receptors. Introduction Phosphorylated phosphoinositide (PI) lipids reside on the cytoplasmic side of eukaryotic membranes and regulate a diverse array of cellular functions. The sn-3, 4 and 5 positions of the inositol head group have the potential to be phosphorylated in all seven combinations and each type of PI plays unique roles in mammals. Each PI recruits a distinct set of protein effectors and regulates multiple cellular events including membrane traffic (Corvera et al, 1999; Roth, 2004), protein sorting (Saksena et al, 2007), growth factor signalling (Cantley, 2002), ion homeostasis (Balla, 2006; Dong et al, 2010), cell survival (Brunet et al, 2001) and cell motility (Yin and Janmey, 2003). PI lipids are tightly regulated and inter-converted by an array of lipid kinases and phosphatases. A conserved protein complex including PIKfyve/FAB1/PIP5K3 (GenBank accession # NP_035216), VAC14 (NP_666328) and FIG4/SAC3 (NP_598760) is responsible for the biosynthesis and turnover of PI(3,5)P2 (Jin et al, 2008; Ikonomov et al, 2009). PIKfyve is the PI(3)P 5-kinase that phosphorylates PI(3)P to form PI(3,5)P2 (Gary et al, 1998), whereas FIG4 dephosphorylates PI(3,5)P2 back to PI(3)P (Rudge et al, 2004; Duex et al, 2006a). The presence of both a kinase and a phosphatase in the same complex allows for tight regulation of PI(3,5)P2 levels. VAC14, a HEAT repeat protein, forms a scaffold for formation of the complex and also brings in other regulatory factors (Jin et al, 2008). Loss of PIKfyve or VAC14 causes a loss or decrease in PI(3,5)P2 levels, respectively (Duex et al, 2006b; Zhang et al, 2007; Ikonomov et al, 2011). While knockout of FIG4 might be predicted to increase PI(3,5)P2, FIG4 also activates PIKfyve and thus, PI(3,5)P2 is decreased in Fig4−/− fibroblasts (Chow et al, 2007) and yeast (Duex et al, 2006a). Interestingly, impairment of PIKfyve activity by either loss of VAC14 (Zhang et al, 2007) or pharmacological inhibition with YM201636 (Sbrissa et al, 2012) also causes a decrease in PI(5)P levels in mammalian cells. Note that the yeast S. cerevisiae does not produce PI(5)P. In mammals, the PI(5)P pool could come either from direct phosphorylation of phosphatidylinositol by PIKfyve (Sbrissa et al, 1999), or via dephosphorylation of PI(3,5)P2 by myotubularin family phosphatases (Tronchère et al, 2004); both pathways are dependent on PIKfyve activity. The PIKfyve/VAC14/FIG4 complex is critical for endomembrane homeostasis and has been implicated in an ever-growing list of processes. In yeast, PI(3,5)P2 regulates vacuole fission, vacuole acidification (Bonangelino et al, 1997, 2002; Gary et al, 1998), retrograde traffic from the vacuole (Bryant et al, 1998; Dove et al, 2004) and is involved in the assembly of transcriptional regulators (Han and Emr, 2011). In metazoans, the PIKfyve/VAC14/FIG4 pathway regulates endosome-to-trans-Golgi network retrograde transport (Rutherford et al, 2006), autophagy (Rusten et al, 2007; de Lartigue et al, 2009; Ferguson et al, 2009), exocytosis (Osborne et al, 2008), calcium channel activation (Shen et al, 2009; Dong et al, 2010) and degradation (Tsuruta et al, 2009). In plants, the PIKfyve/VAC14/FIG4 pathway is involved in endocytosis, vacuole formation, auxin transporter recycling, and pollen development (Hirano and Sato, 2011; Hirano et al, 2011). It remains to be determined which of the mammalian pathways are regulated by PI(3,5)P2 and/or PI(5)P. Analysis of several mouse mutants point to critical roles for the PIKfyve/VAC14/FIG4 pathway in the central and peripheral nervous systems. Vac14 gene trap (Vac14−/−) mice have half of the normal levels of PI(3,5)P2 and PI(5)P. They develop normally, yet die perinatally with numerous neural defects including spongiform-like lesions and increased apoptosis in the brain, and intracellular vacuolation in peripheral neurons (Zhang et al, 2007). Fig4−/− (Chow et al, 2007; Ferguson et al, 2009; Lenk et al, 2011) and Vac14L156R/L156R (ingls) (Jin et al, 2008), mouse mutants defective in PI(3,5)P2 and PI(5)P regulation, have similar patterns of neurodegeneration to that observed in the Vac14−/− mouse. Importantly, human patients with minor defects in the PIKfyve/VAC14/FIG4 pathway also display severe neurological problems. Mutations in FIG4 are responsible for human Charcot-Marie-Tooth disease type 4J (CMT4J), a recessive disorder affecting the peripheral nervous system (Chow et al, 2007; Nicholson et al, 2011). The most common CMT4J allele, I41T, destabilizes the mutant FIG4 protein and impairs its binding to VAC14 (Ikonomov et al, 2010; Lenk et al, 2011). Heterozygous FIG4 mutations have also been identified in patients with amyotrophic lateral sclerosis (ALS) and primary lateral sclerosis (PLS), two forms of motor neuron disease (Chow et al, 2009). These observations suggest that the PIKfyve/VAC14/FIG4 pathway has specialized functions in the nervous system. Defects in multiple neural cell types likely contribute to the pathologies observed in VAC14/FIG4-deficient mouse models. Both neurons (Chow et al, 2007; Zhang et al, 2007, 2008; Katona et al, 2011) and astrocytes (Jin et al, 2008; Ferguson et al, 2009) are affected by mutations in VAC14 or FIG4. However, expression of FIG4 in neurons, but not astrocytes, rescues the spongiform-like lesions, gliosis and early lethality in Fig4−/− mice (Ferguson et al, 2012), emphasizing the importance of the PIKfyve/VAC14/FIG4 pathway in neuronal function. Neurons are highly polarized cells that process electrochemical signals by extending long specialized processes—axons and dendrites—to facilitate information transfer through neural circuits. Accordingly, the endosomal system in neurons has both general and specialized pathways, such as long-range trafficking along neurites (Ibáñez, 2007), as well as specialized recycling in both the presynaptic and postsynaptic terminals (Kennedy and Ehlers, 2006; Dittman and Ryan, 2009). These membrane events are critical for multiple aspects of neuronal function such as neurite outgrowth, neurotrophic factor signalling and synaptic plasticity (Lasiecka and Winckler, 2011). However, the role of PIKfyve/VAC14/FIG4 in neuronal function remains unknown. Here, we address the functional significance of the PIKfyve/VAC14/FIG4 pathway in cultured neurons from the hippocampus of wild-type and Vac14−/− mice. To gain insight into the cellular distribution of PIKfyve/VAC14/FIG4 pathway, we developed an antibody to VAC14, suitable for immunofluorescence microscopy, and found that endogenous VAC14 localizes to multiple organelles, consistent with multiple roles for PI(3,5)P2 in the endomembrane system. VAC14 partially colocalizes with early endosomes, late endosomes, lysosomes and autophagosomes. In neurons, VAC14 is found in both somatodendritic regions and axons. Notably, a substantial amount of endogenous VAC14 is present at synaptic sites, suggesting a role for VAC14 in the regulation of synaptic efficacy. Indeed, we find that synaptic function is altered in neurons cultured from Vac14−/− and Fig4−/− mice. Both postsynaptic function and surface expression of AMPA-type glutamate receptors are enhanced in Vac14−/− hippocampal neurons. Expression of VAC14 in Vac14−/− neurons reverses the synaptic phenotype, indicating a cell-autonomous and post-developmental role for VAC14 in regulating excitatory synaptic strength. We further show that the elevated surface AMPA receptor levels in Vac14−/− neurons are due to decreased endocytosis at postsynaptic sites. Together, our results identify control of PI(3,5)P2 and/or PI(5)P synthesis as a novel regulatory pathway at synapses that influences surface levels of glutamate receptors and synaptic function. Results Vac14−/− hippocampal neurons exhibit vacuolation, but otherwise develop normally in culture Consistent with its importance in the nervous system, expression of VAC14 is abundant in the brain relative to other tissues (Supplementary Figure S1A and B). In this study, we sought insights into the neuronal-specific functions of the PIKfyve/VAC14/FIG4 complex. We focused on hippocampal neurons because VAC14 expression in the hippocampus is similar to other brain regions (Supplementary Figure S1C) and the hippocampus is largely spared from neurodegeneration, even at the time of death in Vac14−/− and Fig4−/− animals (Chow et al, 2007; Zhang et al, 2007). Hippocampal neurons from Vac14−/− embryos remain viable for several weeks, which enabled us to examine the impact of Vac14 deletion in these cells. Although no spongiform lesions were observed in hippocampal regions in vivo, cultured Vac14−/− hippocampal neurons developed small vacuoles in the soma as early as 1 day in vitro (DIV) (Supplementary Figure S2A). Similarly to fibroblasts, the neuronal vacuoles are positive for the late endosome/lysosome marker LAMP1 and negative for the early endosome marker EEA1 (Supplementary Figure S2B). Notably, at the neuron density used (2 × 104 per 1.91 cm2), vacuoles are also observed in neurites by 12 DIV, suggesting that VAC14 functions in both the soma and neurites. Vacuole formation in Vac14−/− neurons appears to be activity independent; the degree of vacuolation in cells subjected to activity blockade (1 μM TTX, 40 μM CNQX, 20 μM APV) from DIV3 to DIV18 was similar to that in untreated neurons (Supplementary Figure S2C). The presence of vacuoles in neurons lacking VAC14 did not appear to affect axon and dendrite development in culture, as assessed using the five stage model (Dotti et al, 1988; Supplementary Figure S3A). Vac14−/− neurons progressed from stage I (lamellipodia) to stage IV/V (complicated networks) at a rate similar to wild-type neurons (Supplementary Figure S3B), implying normal neurite outgrowth and differentiation. Subcellular localization of VAC14 in cultured fibroblasts To determine the sites of action of the PIKfyve/VAC14/FIG4 complex in neurons, we determined the localization of endogenous VAC14. Previous attempts to localize components of the PIKfyve complex in non-neuronal cells relied on overexpression of tagged proteins, and have produced divergent results. An earlier study indicated that overexpressed, tagged PIKfyve is confined to late endosome/lysosome compartments (Ikonomov et al, 2001), whereas other analyses found tagged FAB1/PIKfyve primarily localized to early endosomes (Cabezas et al, 2006; Rutherford et al, 2006). To better understand the endogenous cellular distribution of the PIKfyve/VAC14/FIG4 complex, we raised a rabbit polyclonal antibody against full-length human VAC14 protein. After extensive affinity purification, we obtained a reagent that, in western blot analysis, revealed a major band at the expected molecular weight (88 kD) in wild-type but not in Vac14−/− brain (Supplementary Figure S1A). In wild-type fibroblasts, permeabilized with saponin prior to fixation, VAC14 was present on punctate organelles distributed throughout the cytoplasm; these structures were absent from Vac14−/− fibroblast controls (Figure 1A). Nuclear staining was frequently observed in both wild-type and Vac14−/− cells (Supplementary Figure S4A); thus, the antibody is not suitable to test whether VAC14 is also localized in the nucleus. Figure 1.Endogenous VAC14 partially colocalizes with multiple endocytic organelles. (A) Polyclonal VAC14 antibody recognizes punctate structures in wild-type cells. Fibroblasts were permeabilized with saponin followed by fixation, then labelled with anti-VAC14 antibody. Bottom panels, DIC images. (B) In fibroblasts, endogenous VAC14 colocalizes with both EEA1 and LAMP1. Wild-type fibroblasts were triple labelled with rabbit anti-VAC14, chicken anti-EEA1 and rat anti-LAMP1. The majority of VAC14 colocalized with either EEA1 (yellow arrows) or LAMP1 (turquoise arrows). Some VAC14 colocalized with both (white arrows) or neither (green arrow) markers. (C) VAC14 partially colocalizes with the late endosome marker LBPA (arrow). Fibroblasts were double labelled with rabbit anti-VAC14 and mouse anti-LBPA. (D) VAC14 partially colocalized with lysosomes (arrow). To label lysosomes, prior to fixation, fibroblasts were pulsed with Texas Red-Dextran (Mw 70 kD) for 1 h and chased in the absence of dextran for 24 h. (E) The limiting membrane of vacuoles in Vac14−/− cells is positive for LAMP1 while negative for LBPA, suggesting a lysosomal origin. Vac14−/− fibroblasts were double labelled with rat anti-LAMP1 and mouse anti-LBPA. (D, E) DAPI (blue) used to label nuclei. (F) VAC14 partially colocalizes with LC3-RFP puncta (arrows). Fibroblasts transfected with LC3-RFP were fixed and labelled with anti-VAC14. (A–F) Bar=10 μm. Download figure Download PowerPoint To determine the relative distribution of VAC14 on endosomal and lysosomal membranes, we performed triple labelling experiments in primary fibroblasts and determined the distribution of VAC14, EEA1 and LAMP1 puncta (Figure 1B; Supplementary Figure S4B and F). Consistent with earlier studies, EEA1 and LAMP1 labelled distinct compartments. The majority of VAC14 puncta colocalized with EEA1 (20±5%), LAMP1 (30±5%) or both markers (19±7%). These triple-labelled puncta likely represent intermediate endosomes. Thus, VAC14, PI(3,5)P2 and potentially PI(5)P, are present in multiple locations within the endomembrane system, including early endosomes, late endosomes and lysosomes (Supplementary Figure S5). Some VAC14 puncta (31±8%) did not colocalize with either EEA1 or LAMP1, suggesting that VAC14 may also function on other compartments. LAMP1 is present on both late endosomes and lysosomes. To determine whether VAC14 is found on one or both of these compartments, we examined VAC14 localization relative to LBPA (late endosomes) or internalized dextran (lysosomes). Partial colocalization was observed between VAC14 (15±6%) and LBPA (Figure 1C; Supplementary Figure S4C and G), which indicates that some VAC14 resides on late endosomes. To determine whether lysosomes also contain VAC14, cells were incubated with a fluid phase marker, 70 kD Texas Red-dextran, and then chased in the absence of dextran for 24 h to allow it to reach lysosomes. Partial colocalization was observed between VAC14 (23±9%) and lysosomes loaded with dextran (Figure 1D; Supplementary Figure S4D and G), suggesting that some VAC14 is also localized on lysosomes. Interestingly, the limiting membrane of vacuoles in Vac14−/− fibroblasts is positive for LAMP1, but negative for LBPA (Figure 1E), implying that the large vacuoles derive solely from lysosomes. In metazoans, the PIKfyve/VAC14/FIG4 pathway is thought to play a role in autophagy, either during fusion of autophagosomes with endosomes/lysosomes, or recycling of lysosomes from autolysosomes (Rusten et al, 2007; de Lartigue et al, 2009; Ferguson et al, 2009). LC3 is a common marker of autophagosomes. We transfected wild-type or Vac14−/− fibroblasts with LC3-RFP and colabelled transfected cells with anti-VAC14. VAC14 (17±13%) partially colocalized with LC3 (Figure 1F; Supplementary Figure S4E and G), suggesting that autophagosomes may contain PI(3,5)P2 and/or PI(5)P. Alternatively, these PI(3,5)P2 and/or PI(5)P containing regions may represent the interface between autophagosomes and endosomes/lysosomes. Localization of VAC14 in neurons To determine the localization of VAC14 in neurons, we first examined its distribution in the soma. In this case, neurons were not permeabilized with saponin prior to fixation; thus, the images indicate both membrane bound and cytosolic pools of VAC14. A significant portion of the VAC14 localized to punctate structures (Supplementary Figure S6). As in fibroblasts, VAC14 puncta colocalized both with the early endosome marker, EEA1 (Supplementary Figure S6A), and with the late endosome/lysosome marker, LAMP2 (Supplementary Figure S6B). To test whether VAC14 is present in dendrites, hippocampal neurons were labelled with antibodies against VAC14 and against MAP2, a microtubule-associated protein that is highly expressed in dendrites but not in axons. Notably, discrete VAC14 puncta were found in MAP2-positive dendrites (Figure 2A; Supplementary Figure S7A). Moreover, another pool of VAC14 puncta was evident in MAP2-negative neurites, implying an axonal localization. To test this further, we labelled neurons with anti-VAC14 and anti-TAU-1, which preferentially labels axons in younger cultures (Horton et al, 2005). Again, VAC14 puncta were present in TAU-1-labelled axons (Figure 2A; Supplementary Figure S7A), although this axonal VAC14 pool was less prominent than the dendritic pool. In neurites, VAC14 puncta partially colocalized with both EEA1 (23±12%) and LAMP1 (29±9%) (Figure 2B; Supplementary Figure S7B and C), suggesting that VAC14 in neuronal processes functions in pathways that involve early and late endosomes as well as lysosomes. Figure 2.VAC14 is found in both dendrites and axons, and colocalizes with endocytic and synaptic markers in hippocampal neurons. (A) Wild-type and Vac14−/− neurons were double labelled with rabbit anti-VAC14 and mouse anti-MAP2 (dendrites) or mouse anti-TAU-1 (axons). Arrows indicate the localization of VAC14 on dendrites (MAP2-positive and TAU-negative neurites). Arrowheads indicate the localization of VAC14 on axons (MAP2-negative and TAU-1-positive neurites). Bar=10 μm. (B) VAC14 partially colocalizes with EEA1 or LAMP1 in the neurites (arrows). Wild-type and Vac14−/− neurons were triple labelled with rabbit anti-VAC14, chicken anti-EEA1 and rat anti-LAMP1. Bar=5 μm. (C) VAC14 displays significant colocalization with several synaptic markers: synapsin, synaptotagmin and synaptobrevin. Wild-type and Vac14−/− neurons were double labelled with rabbit anti-VAC14 and guinea pig anti-synapsin, mouse anti-synaptotagmin or mouse anti-synaptobrevin. Bar=5 μm. (D) VAC14 partially localizes at excitatory synapses (labelled with both the synaptic vesicle glutamate transporter vGlut1 and postsynaptic marker PSD95). Wild-type and Vac14−/− neurons were labelled with rabbit anti-VAC14, mouse anti-PSD95 and guinea pig anti-vGlut1. Arrows indicate examples of colocalization. (C, D) Lower panels show straightened dendrites from corresponding top panels. Bar=5 μm. Download figure Download PowerPoint Endogenous VAC14 localizes to synapses Interestingly, the most striking colocalization was observed between VAC14 and synaptic markers. A substantial number of VAC14 puncta colocalized with the presynaptic terminal markers synapsin, synaptotagmin and VAMP/synaptobrevin (Figure 2C; Supplementary Figure S7D). To test whether VAC14 colocalizes with excitatory synapses, we performed triple labelling against VAC14, vGlut1 (the glutamate transporter on presynaptic vesicles), and the postsynaptic scaffolding protein PSD95. VAC14 puncta colocalized extensively with vGlut1/PSD95 double-positive puncta (Figure 2D; Supplementary Figure S7E), suggesting a role for VAC14 in excitatory synapse function. Altered synaptic function in cultured Vac14−/− neurons To examine a functional role for the PIKfyve/VAC14/FIG4 pathway at the synapse, we measured miniature excitatory postsynaptic currents (mEPSCs) in pyramidal-like neurons from Vac14−/− hippocampal cultures and corresponding wild-type controls. mEPSCs represent unitary synaptic currents mediated by the spontaneous fusion of single synaptic vesicles, and are often used to reveal functional changes in synaptic strength. Pyramidal-like neurons with little to no vacuolation were targeted for electrophysiology. Given the neurodegeneration observed in other regions of the brain at the time of birth, one might expect synaptic function to be diminished in Vac14−/− neurons. Surprisingly, mEPSCs from Vac14−/− neurons displayed a significant increase (24±6%) in amplitude relative to wild-type mEPSCs (Figure 3A and B), suggesting an inhibitory role for VAC14 in synaptic function. We found no difference in mEPSC frequency or decay time in Vac14−/− mEPSCs (Figure 3C–E). In a parallel experiment, we found mEPSC amplitude was similarly increased in Fig4−/− mice (Figure 3F and G), which also have reduced PIKfyve kinase activity. Together, these data suggest that the increase in mEPSC amplitude in both Vac14−/− and Fig4−/− neurons results from defects in PI(3,5)P2 and/or PI(5)P synthesis. Figure 3.Loss of VAC14 or FIG4 leads to an increase in excitatory synaptic function. (A) Representative mEPSC recordings of wild-type (N=32) and Vac14−/− neurons (N=32). (B) Mean mEPSC amplitude in Vac14−/− neurons is larger than in wild-type neurons, 20.86±1.03 pA versus 16.83±0.91 pA, respectively. *P=0.0045, t-test. (C) Mean mEPSC frequency is similar in wild-type (1.21±0.26 Hz) and Vac14−/− (1.16±0.24 Hz) neurons. (D, E) Summary of mEPSC kinetics in wild-type and Vac14−/− neurons. (D) Individual mEPSCs overlaid. Thick lines show the mean trace. Dashed line is aligned to the mean peak inward current of Vac14−/− mEPSC. Scaled overlay shows similar kinetics between wild-type and Vac14−/− mEPSCs. (E) Mean mEPSC decay is similar between wild type (3.80±0.15 ms) and Vac14−/− (3.69±0.14 ms). (F) Representative mEPSC traces of wild-type (Fig4+/+) (N=12) and Fig4−/− neurons (N=14). (G) Mean mEPSC amplitude in Fig4−/− neurons is larger than in Fig4+/+ neurons (14.64±0.39 pA versus 18.73±1.38 pA, respectively, *P=0.0164, t-test). (H) Mean mEPSC frequency is similar in Fig4+/+ and Fig4−/− (0.99±0.22 Hz versus 0.92±0.28 Hz, respectively, P=0.8542, t-test). Error bars are standard error of the mean (s.e.m.). Download figure Download PowerPoint Although we found no change in mEPSC frequency in either Vac14−/− (Figure 3C) or Fig4−/− neurons (Figure 3H), VAC14 is localized to axons (Figure 2A), and therefore, is well positioned to contribute to presynaptic function. The enlargement of endocytic compartments in Vac14−/− cells (Zhang et al, 2007) also suggested that the increase in mEPSC amplitude in Vac14−/− neurons could have resulted from increased glutamate release by enlarged presynaptic vesicles (increased quantal content). To examine this possibility, we performed transmission electron microscopy on thin sections from the hippocampus and hindbrain of wild-type and Vac14−/− mice at P0. The hindbrain was included in these studies because it is the most vacuolated brain region in the Vac14−/− animal at the time of death. We found similar synaptic vesicle diameter in wild-type and Vac14−/− presynaptic terminals of both brain regions (Figure 4A and B). Figure 4.Presynaptic probability of release is enhanced in Vac14−/− neurons. (A, B) Synaptic vesicles from Vac14−/− are not larger than synaptic vesicles observed in brains from wild type. (A) Electron microscopy of excitatory synapses, evident by the thickening of the postsynaptic membrane, in wild-type and Vac14−/− hippocampus and hindbrain. Bar=100 nm. (B) Quantitation of the diameter of synaptic vesicles. Cumulative probability distribution of synaptic vesicle diameter. No significance difference was found between wild type and Vac14−/− by a two-sample Kolmogorov-Smirnov test (kstest2, Matlab) (hippocampus, P=0.32; hindbrain, P=0.46). Three wild-type and three Vac14−/− animals were analysed. Hindbrain: N=567 vesicles from 33 terminals for wild type and 388 vesicles from 29 terminals for Vac14−/−. Hippocampus: N=433 vesicles from 33 terminals for wild type and 66 vesicles from 15 terminals for Vac14−/−. (C, D) The number of synapses is decreased in Vac14−/− neurons. (C) Wild-type and Vac14−/− hippocampal neurons were triple labelled with rabbit anti-MAP2 (blue), mouse anti-PSD95 (red) and guinea pig anti-vGlut (green). Examples of straightened dendrites are shown. Bar=5 μm. (D) Quantitation of the number of synapses on the fir" @default.
• W2148447088 created "2016-06-24" @default.
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• W2148447088 date "2012-07-27" @default.
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• W2148447088 title "Modulation of synaptic function by VAC14, a protein that regulates the phosphoinositides PI(3,5)P₂and PI(5)P" @default.
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