Molecular Interface of Neuronal Innate Immunity, Synaptic Vesicle Stabilization, and Presynaptic Homeostatic Plasticity

Nathan Harris, Richard D Fetter, Daniel J Brasier, Amy Tong, Graeme W Davis, Nathan Harris, Richard D Fetter, Daniel J Brasier, Amy Tong, Graeme W Davis

Abstract

We define a homeostatic function for innate immune signaling within neurons. A genetic analysis of the innate immune signaling genes IMD, IKKβ, Tak1, and Relish demonstrates that each is essential for presynaptic homeostatic plasticity (PHP). Subsequent analyses define how the rapid induction of PHP (occurring in seconds) can be coordinated with the life-long maintenance of PHP, a time course that is conserved from invertebrates to mammals. We define a novel bifurcation of presynaptic innate immune signaling. Tak1 (Map3K) acts locally and is selective for rapid PHP induction. IMD, IKKβ, and Relish are essential for long-term PHP maintenance. We then define how Tak1 controls vesicle release. Tak1 stabilizes the docked vesicle state, which is essential for the homeostatic expansion of the readily releasable vesicle pool. This represents a mechanism for the control of vesicle release, and an interface of innate immune signaling with the vesicle fusion apparatus and homeostatic plasticity.

Keywords: Drosophila; IKK; NMJ; Rel; Tak1; docking; homeostatic plasticity; innate immunity; motoneuron; motor neuron; presynaptic release; priming; readily releasable pool; synapse; synaptic vesicle; vesicle.

Conflict of interest statement

Declaration of interests: None

Copyright © 2018 Elsevier Inc. All rights reserved.

Figures

Figure 1.. Innate immune signaling during the…
Figure 1.. Innate immune signaling during the rapid induction of PHP
(A) Diagram of the Drosophila melanogaster Immune Deficient (IMD) pathway and homologous mammalian innate immune signaling. (B-E) Gene loci for Imd, Tak1, IKKβ, and Rel, with genetic reagents indicated. (F) Representative traces for EPSP (scale, 5 mV, 50 ms) and mEPSP (scale, 2 mV, 1 s) at baseline, and in the presence of philanthotoxin (PhTx) for the indicated genotypes. (G) Average mEPSP amplitude for each genotype in the absence (light bars) or presence (dark bars) of PhTx. (H) Average EPSP amplitude in the absence (light bars) or presence (dark bars) of PhTx. (I) Average quantal content in the absence (light bars) or presence (dark bars) of PhTx. (J) mEPSP amplitudes (filled bars) and quantal content (open bars) for each genotype in the presence of PhTx, normalized to baseline values in the absence of PhTx. Data are presented as average (+/− SEM) and statistical significance determined by Student’s t-test (unpaired, two-tailed). See also Supplemental Figures 1 and 3.
Figure 2.. Innate immune signaling during the…
Figure 2.. Innate immune signaling during the sustained expression of PHP
(A) Representative traces for EPSP (scale, 5 mV, 50 ms) and mEPSP (scale, 2 mV, 1 s) at baseline, and in the background of the GluRIIA mutation, for the indicated genotypes. (B) Average quantal content for each genotype in the absence (open bars) or presence (filled bars) of the GluRIIA mutation. (C) mEPSP amplitudes (filled bars) and quantal content (open bars) for each genotype in the GluRIIA background, normalized to the control genotype in the absence of GluRIIA. (D) Average mEPSP amplitude for each genotype in the absence (open bars) or presence (filled bars) of the GluRIIA mutation. (E) Average EPSP amplitude for each genotype in the absence (open bars) or presence (filled bars) of the GluRIIA mutation. (F) Average quantal content in the absence (open bars) or presence (filled bars) of the GluRIIA mutation. (G) Average mEPSP and quantal content normalized to genotypic controls as in C. Data are presented as average (+/−SEM) and statistical significance determined by Student’s t-test (unpaired, two-tailed). See also Supplemental Figure 2.
Figure 3.. Tak1 Interacts genetically with PGRP-LC
Figure 3.. Tak1 Interacts genetically with PGRP-LC
(A) Representative traces for EPSP (scale, 5 mV, 50 ms) and mEPSP (scale, 2 mV, 1 s) at baseline, and in the presence of PhTx, for the indicated genotypes. (B) Average mEPSP amplitude for each genotype in the absence (light bars) or presence (dark bars) of PhTx. (C) Average EPSP amplitude in the absence (light bars) or presence (dark bars) of PhTx. (D) Average quantal content in the absence (light bars) or presence (dark bars) of PhTx. (E) mEPSP amplitudes (filled bars) and quantal content (open bars) for each genotype in the presence of PhTx, normalized to baseline values in the absence of PhTx. (F) Average mEPSP amplitude for each genotype in the absence (light bars) or presence (dark bars) of PhTx. (G) Average EPSP amplitude in the absence (light bars) or presence (dark bars) of PhTx. (H) Average quantal content in the absence (light bars) or presence (dark bars) of PhTx. (I) mEPSP amplitudes (filled bars) and quantal content (open bars) for each genotype in the presence of PhTx, normalized to baseline values in the absence of PhTx. Data are presented as average (+/− SEM) and statistical significance determined by Student’s t-test (unpaired, two-tailed). See also Supplemental Figure 4.
Figure 4.. Tak1 is necessary for baseline…
Figure 4.. Tak1 is necessary for baseline vesicle fusion and PHP under physiological conditions.
(A) Representative traces for EPSC (scale, 50 nA, 5 ms) at baseline, and in the presence of PhTx, for the indicated genotypes. (B) Average mEPSP amplitude for each genotype in the absence (light bars) or presence (dark bars) of PhTx. (C) Average EPSC amplitude in the absence (light bars) or presence (dark bars) of PhTx. (D) mEPSP amplitudes (filled bars) and quantal content (open bars) for each genotype in the presence of PhTx, normalized to baseline values in the absence of PhTx. (E) Calcium cooperativity curves for the indicated genotypes. Neurotransmitter release was measured at 0.4, 0.7, 1.5, and 3mM extracellular calcium concentration. Quantal content was calculated by dividing EPSC amplitudes by mEPSC amplitudes. (F) Representative traces for mEPSP (scale, 1 mV, 0.5 s) for the indicated genotypes. (G) Average mEPSP frequency for the indicated genotypes. Data are presented as average (+/− SEM) and statistical significance determined by Student’s t-test (unpaired, two-tailed) (C) or by one-way ANOVA with Tukey’s multiple comparisons test (G).
Figure 5.. Tak1 functions downstream of action-potential…
Figure 5.. Tak1 functions downstream of action-potential induced presynaptic calcium influx.
(A) Representative traces for EPSC trains (scale, 50 nA, 40 ms). Stimulation frequency was 50 Hz, and extracellular calcium concentration was 1.5 mM. UAS-Tak1DN was expressed presynaptically with OK371-GAL4. (B) Average ratio of EPSC #4 in the train divided by EPSC #1 for the indicated genotypes. (C) Representative line scans and calcium transients (scale, 0.5 ΔF/F, 200 ms) for WT (grey bar) and Tak1179 (green bar). (D) Average ΔF/F for the indicated genotypes. (E) Representative calcium transients (scale, 1 ΔF/F, 400 ms) for the indicated genotypes. Stimulation was identical to that used in F. (F) Average ΔF/F for the indicated genotypes. (G) Cumulative frequency distributions of ΔF/F for WT (grey line) and Tak1179 (green line). (H) Average baseline fluorescence as a measure of dye-loading for the indicated genotypes. (I) Average decay constants of the transients in H and I for the indicated genotypes. Data are presented as average (+/− SEM) and statistical significance determined by Student’s t-test (unpaired, two-tailed) (D-I) or by one-way ANOVA with Tukey’s multiple comparisons test (B).
Figure 6.. Tak1 controls the homeostatic modulation…
Figure 6.. Tak1 controls the homeostatic modulation and dynamics of the readily releasable pool.
(A, B) Representative EPSC trains (scale, 100 nA, 100 ms) at baseline, and in the presence of PhTx, for the indicated genotypes. Trains are 30 stimuli delivered at 60 Hz. Extracellular calcium concentration is 3 mM. (C) Average data for mEPSP amplitudes (filled bars) and readily releasable pool size (open bars) in the presence of PhTx, normalized to baseline, for the indicated genotypes. (D) Average data for probability of release calculated as PTrain = amplitude of the first EPSC divided by the cumulative EPSC in the indicated genotypes. (E) Representative EPSC traces (scale, 100 nA, 40 ms) at baseline (light traces), and after incubation with EGTA-AM (50 µM) (dark offset traces) for the indicated genotypes. (F) Average data for EPSC amplitude at baseline (light bars), and after incubation with EGTA-AM (dark bars) for the indicated genotypes. (G) Average data for the amplitude of the 4th EPSC in a train divided by the 1st EPSC in a train. Data are presented as average (+/− SEM) and statistical significance determined by Student’s t-test (unpaired, two-tailed).
Figure 7.. Tak1 stabilizes a high release…
Figure 7.. Tak1 stabilizes a high release probability synaptic vesicle pool.
(A) Representative traces for trains used to deplete the vesicle pool (scale, 100 nA, 100 ms), and paired EPSC pulses at recovery intervals of 200 ms and 5 s after the train (scale, 100 nA, 10 ms), for the indicated genotypes. (B) Summary data showing average recovery time courses in the indicated genotypes. (C) Average paired-pulse ratio, calculated as the amplitude of EPSC #2 divided by that of EPSC #1, at each recovery interval, for the indicated genotypes. Inter stimulus interval for paired pulse is 20 ms. (D) Average data for recovery time courses of raw EPSC amplitudes in the indicated genotypes. The data labeled “subtract” (blue) are calculated by subtraction of the average amplitude in Tak1179 from that of WT, at each tested recovery interval. (E) View of the first 6 seconds of the data presented in D, now plotted on a linear x axis. WT data are fit with a double-exponential function (τfast=0.053 s, τslow=6.25 s). Subtraction data could not be fit with a double-exponential function and are fit with a single exponential function (τ=1.23 s). Fits were calculated using all the data out to 90 s, but the x axis is limited to 6 s for ease of visualization. (F) Average data for recovery time courses of raw EPSC amplitudes in the indicated genotypes. WT data are repeated from D. “Subtract” was generated as in D, subtracting Tak12 amplitudes from WT. (G) View of the first 6 seconds of the data in F. Subtraction data are again fit with a single exponential function (τ=1.04 s). WT data are repeated from E. Data are presented as average (+/− SEM).
Figure 8.. Impaired synaptic vesicle distribution and…
Figure 8.. Impaired synaptic vesicle distribution and docking at the active zones of Tak1 mutants.
(A) Representative electron micrographs of presynaptic active zones in the indicated genotypes. Scale bar represents 100 nm. (B) Average number of vesicles within 150 nm of the base of the T-bar for the indicated genotypes. (C) Average number of vesicles within 400 nm of the base of the T-bar for the indicated genotypes. (D) Cumulative frequency distribution of the distance between each vesicle and its nearest neighboring vesicle for the indicated genotypes. (E) Average number of docked vesicles per active zone in the indicated genotypes. Only Tak12 is statistically different from wild type. Data are presented as average (+/− SEM) and statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. (F) Individual data points for number of docked vesicles in the indicated genotypes, re-plotted from (E) where statistical significance is indicated. Each dot represents one active zone. Line indicates the mean. (G) Model. At right: the IMD signaling cascade couples the mechanisms of rapid induction with the long-term expression of PHP. Center: diagram of the IMD signaling cascade initiated by the PGRP receptor, which catalyzes the assembly of the IMD complex inclusive of the proteins IMD, IKK and Tak1. Left: Tak1 inhibits the rate of vesicle un-docking (orange arrow) at an individual presynaptic release site. The opposing rate of vesicle docking is potentiated by intracellular calcium, among other factors. The readily releasable vesicle pool (RRP) is indicated.

References

    1. Ahn HJ, Hernandez CM, Levenson JM, Lubin FD, Liou H-C, and Sweatt JD (2008). c-Rel, an NF-kappaB family transcription factor, is required for hippocampal long-term synaptic plasticity and memory formation. Learn. Mem 15, 539–549.
    1. Boutros M, Agaisse H, and Perrimon N (2002). Sequential Activation of Signaling Pathways during Innate Immune Responses in Drosophila. Developmental Cell 3, 711– 722.
    1. Chen X, Rahman R, Guo F, and Rosbash M (2016). Genome-wide identification of neuronal activity-regulated genes in Drosophila. eLife 5, e19942.
    1. Choe K-M, Lee H, and Anderson KV (2005). Drosophila peptidoglycan recognition protein LC (PGRP-LC) acts as a signal-transducing innate immune receptor. Proc. Natl. Acad. Sci. U.S.A 102, 1122–1126.
    1. Choe K-M, Werner T, Stöven S, Hultmark D, and Anderson KV (2002). Requirement for a Peptidoglycan Recognition Protein (PGRP) in Relish Activation and Antibacterial Immune Responses in Drosophila. Science 296, 359–362.
    1. Corriveau RA, Huh GS, and Shatz CJ (1998). Regulation of class I MHC gene expression in the developing and mature CNS by neural activity. Neuron 21, 505–520.
    1. Cull-Candy SG, Miledi R, Trautmann A, and Uchitel OD (1980). On the release of transmitter at normal, myasthenia gravis and myasthenic syndrome affected human end-plates. The Journal of Physiology 299, 621–638.
    1. Davis GW (2013). Homeostatic signaling and the stabilization of neural function. Neuron 80, 718–728.
    1. Davis GW, and Goodman CS (1998). Synapse-specific control of synaptic efficacy at the terminals of a single neuron. Nature 392, 82–86.
    1. Delaney JR, Stöven S, Uvell H, Anderson KV, Engström Y, and Mlodzik M (2006). Cooperative control of Drosophila immune responses by the JNK and NF-kappaB signaling pathways. EMBO J 25, 3068–3077.
    1. Frank CA, Kennedy MJ, Goold CP, Marek KW, and Davis GW (2006). Mechanisms underlying the rapid induction and sustained expression of synaptic homeostasis. Neuron 52, 663–677.
    1. Gaviño MA, Ford KJ, Archila S, and Davis GW (2015). Homeostatic synaptic depression is achieved through a regulated decrease in presynaptic calcium channel abundance. Elife 4.
    1. Gottar M, Gobert V, Michel T, Belvin M, Duyk G, Hoffmann JA, Ferrandon D, and Royet J (2002). The Drosophila immune response against Gram-negative bacteria is mediated by a peptidoglycan recognition protein. Nature 416, 640–644.
    1. Häcker H, and Karin M (2006). Regulation and Function of IKK and IKK-Related Kinases. Sci. STKE 2006, re13–re13.
    1. Hallermann S, Heckmann M, and Kittel RJ (2010). Mechanisms of short-term plasticity at neuromuscular active zones of Drosophila. HFSP J 4, 72–84.
    1. Harris N, Braiser DJ, Dickman DK, Fetter RD, Tong A, and Davis GW (2015). The Innate Immune Receptor PGRP-LC Controls Presynaptic Homeostatic Plasticity. Neuron 88, 1157–1164.
    1. He E, Wierda K, van Westen, R., Broeke JH, Toonen RF, Cornelisse LN, and Verhage M (2017). Munc13–1 and Munc18–1 together prevent NSF-dependent de-priming of synaptic vesicles. Nature Communications 8, 15915.
    1. Hedengren M, Asling B, Dushay MS, Ando I, Ekengren S, Wihlborg M, and Hultmark D (1999). Relish, a central factor in the control of humoral but not cellular immunity in Drosophila. Mol. Cell 4, 827–837.
    1. Huh GS, Boulanger LM, Du H, Riquelme PA, Brotz TM, and Shatz CJ (2000). Functional requirement for class I MHC in CNS development and plasticity. Science 290, 2155–2159.
    1. Jorquera RA, Huntwork-Rodriguez S, Akbergenova Y, Cho RW, and Littleton JT (2012). Complexin Controls Spontaneous and Evoked Neurotransmitter Release by Regulating the Timing and Properties of Synaptotagmin Activity. J. Neurosci 32, 18234–18245.
    1. Kazama H, and Wilson RI (2008). Homeostatic matching and nonlinear amplification at identified central synapses. Neuron 58, 401–413.
    1. Kim SH, and Ryan TA (2010). CDK5 serves as a major control point in neurotransmitter release. Neuron 67, 797–809.
    1. Kittel RJ, Wichmann C, Rasse TM, Fouquet W, Schmidt M, Schmid A, Wagh DA, Pawlu C, Kellner RR, Willig KI, et al. (2006). Bruchpilot promotes active zone assembly, Ca2+ channel clustering, and vesicle release. Science 312, 1051–1054.
    1. Kondo S, and Ueda R (2013). Highly Improved Gene Targeting by Germline-Specific Cas9 Expression in Drosophila. Genetics 195, 715–721.
    1. Lee H, Brott BK, Kirkby LA, Adelson JD, Cheng S, Feller MB, Datwani A, and Shatz CJ (2014). Synapse elimination and learning rules co-regulated by MHC class I H2-Db. Nature 509, 195–200.
    1. Lemaitre B, Kromer-Metzger E, Michaut L, Nicolas E, Meister M, Georgel P, Reichhart JM, and Hoffmann JA (1995). A recessive mutation, immune deficiency (imd), defines two distinct control pathways in the Drosophila host defense. Proc. Natl. Acad. Sci. U.S.A 92, 9465–9469.
    1. Mahoney RE, Rawson JM, and Eaton BA (2014). An age-dependent change in the set point of synaptic homeostasis. J. Neurosci 34, 2111–2119.
    1. Mahr A, and Aberle H (2006). The expression pattern of the Drosophila vesicular glutamate transporter: a marker protein for motoneurons and glutamatergic centers in the brain. Gene Expr. Patterns 6, 299–309.
    1. Meffert MK, Chang JM, Wiltgen BJ, Fanselow MS, and Baltimore D (2003). NF-kappa B functions in synaptic signaling and behavior. Nat. Neurosci 6, 1072–1078.
    1. Mitra A, Mitra SS, and Tsien RW (2011). Heterogeneous reallocation of presynaptic efficacy in recurrent excitatory circuits adapting to inactivity. Nat. Neurosci 15, 250–257.
    1. Müller M, Liu KSY, Sigrist SJ, and Davis GW (2012). RIM controls homeostatic plasticity through modulation of the readily-releasable vesicle pool. J. Neurosci 32, 16574–16585.
    1. Müller M, Genç Ö, and Davis GW (2015). RIM-b inding protein links synaptic homeostasis to the stabilization and replenishment of high release probability vesicles. Neuron 85, 1056–1069.
    1. Murthy VN, and Stevens CF (1999). Reversal of synaptic vesicle docking at central synapses. Nature Neuroscience 2, 503–507.
    1. Myllymäki H, Valanne S, and Rämet M (2014).heT Drosophila imd signaling pathway. J. Immunol 192, 3455–3462.
    1. Park JM, Brady H, Ruocco MG, Sun H, Williams D, Lee SJ, Kato T, Richards N, Chan K, Mercurio F, et al. (2004). Targeting of TAK1 by the NF-κB protein Relish regulates the JNK-mediated immune response in Drosophila. Genes Dev 18, 584–594.
    1. Petersen SA, Fetter RD, Noordermeer JN, Goodman CS, and DiAntonio A (1997). Genetic analysis of glutamate receptors in Drosophila reveals a retrograde signal regulating presynaptic transmitter release. Neuron 19, 1237–1248.
    1. Polley S, Huang D-B, Hauenstein AV, Fusco AJ, Zhong X, Vu D, Schröfelbauer B, Kim Y, Hoffmann A, Verma IM, et al. (2013). A Structural Basis for IκB Kinase 2 Activation Via Oligomerization-Dependent Trans Auto-Phosphorylation. PLOS Biology 11, e1001581.
    1. Rothwarf DM, and Karin M (1999). The NF-kappa B activation pathway: a paradigm in information transfer from membrane to nucleus. Sci. STKE 1999, RE1.
    1. Sakaba T, and Neher E (2001). Calmodulin mediates rapid recruitment of fast-releasing synaptic vesicles at a calyx-type synapse. Neuron 32, 1119–1131.
    1. Sato S, Sanjo H, Takeda K, Ninomiya-Tsuji J, Yamamoto M, Kawai T, Matsumoto K, Takeuchi O, and Akira S (2005). Essential function for the kinase TAK1 in innate and adaptive immune responses. Nat. Immunol 6, 1087–1095.
    1. Schafer DP, and Stevens B (2013). Phagocytic glial cells: sculpting synaptic circuits in the developing nervous system. Current Opinion in Neurobiology 23, 1034–1040.
    1. Schneggenburger R, and Neher E (2005). Presynaptic calcium and control of vesicle fusion. Curr. Opin. Neurobiol 15, 266–274.
    1. Shim J-H, Xiao C, Paschal AE, Bailey ST, Rao P, Hayden MS, Lee K-Y, Bussey C, Steckel M, Tanaka N, et al. (2005). TAK1, but not TAB1 or TAB2, plays an essential role in multiple signaling pathways in vivo. Genes Dev 19, 2668–2681.
    1. Silverman N, Zhou R, Stöven S, Pandey N, Hultmark D, and Maniatis T (2000). A Drosophila IkappaB kinase complex required for Relish cleavage and antibacterial immunity. Genes Dev 14, 2461–2471.
    1. Smith PD, Liesegang GW, Berger RL, Czerlinski G, and Podolsky RJ (1984). A stopped-flow investigation of calcium ion binding by ethylene glycol bis(beta-aminoethyl ether)-N,N’-tetraacetic acid. Anal. Biochem 143, 188–195.
    1. Stellwagen D, and Malenka RC (2006). Synaptic scaling mediated by glial TNF-alpha. Nature 440, 1054–1059.
    1. Stevens B, Allen NJ, Vazquez LE, Howell GR, Christopherson KS, Nouri N, Micheva KD, Mehalow AK, Huberman AD, Stafford B, et al. (2007). The classical complement cascade mediates CNS synapse elimination. Cell 131, 1164–1178.
    1. Takatsu Y, Nakamura M, Stapleton M, Danos MC, Matsumoto K, O’Connor MB, Shibuya H, and Ueno N (2000). TAK1 participates in c-Jun N-terminal kinase signaling during Drosophila development. Mol. Cell. Biol 20, 3015–3026.
    1. Vidal S, Khush RS, Leulier F, Tzou P, Nakamura M, and Lemaitre B (2001). Mutations in the Drosophila dTAK1 gene reveal a conserved function for MAPKKKs in the control of rel/NF-kappaB-dependent innate immune responses. Genes Dev 15, 1900–1912.
    1. Wang T, Jones RT, Whippen JM, and Davis GW (2016). α2δ−3 Is Required for Rapid Transsynaptic Homeostatic Signaling. Cell Rep 16, 2875–2888.
    1. Weimer RM, Richmond JE, Davis WS, Hadwiger G, Nonet ML, and Jorgensen EM (2003). Defects in synaptic vesicle docking in unc-18 mutants. Nat Neurosci 6, 1023–1030.
    1. Wu MN, Littleton JT, Bhat MA, Prokop A, and Bellen HJ (1998). ROP, the Drosophila Sec1 homolog, interacts with syntaxin and regulates neurotransmitter release in a dosage‐dependent manner. The EMBO Journal 17, 127–139.
    1. Zhao C, Dreosti E, and Lagnado L (2011). Homeostatic synaptic plasticity through changes in presynaptic calcium influx. J. Neurosci 31, 7492–7496.
    1. Zhou R, Silverman N, Hong M, Liao DS, Chung Y, Chen ZJ, and Maniatis T (2005). The role of ubiquitination in Drosophila innate immunity. J. Biol. Chem 280, 34048–34055.

Source: PubMed

赞助者和合作者

医疗条件

药物干预

3
订阅