Fast release regulated by the endocytic scaffold intersectin

Takeshi Sakabaa,b,1, Natalia L. Kononenkoc,2, Jelena Baceticc,2, Arndt Pechsteinc,d,2, Jan Schmoranzerc,2, Lijun Yaoa, Holger Barthe, Oleg Shupliakovd, Oliver Koblerf, Klaus Aktoriesg, and Volker Hauckec,1

aGraduate School of Brain Science, Doshisha University, Kyoto 6190225, Japan; bIndependent Junior Group of Biophysics of Synaptic Transmission, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany; cLeibniz Institut für Molekulare Pharmakologie, Department of Molecular Pharmacology and Cell Biology, 13125 Berlin, Germany; dDepartment of Neuroscience, Center of Excellence in Developmental Biology and Regenerative Medicine, Karolinska Institute, 17177 Stockholm, Sweden; eInstitute of Pharmacology and Toxicology, University of Ulm Medical Center, 89081 Ulm, Germany; fLeibniz Institute for Neurobiology, 39118 Magdeburg, Germany; and gInstitute of Experimental and Clinical Pharmacology and Toxicology, and BIOSS Centre for Biological Signalling Studies, Universität of Freiburg, 79104 Freiburg, Germany

Edited by Yukiko Goda, RIKEN, Wako, Japan, and accepted by the Editorial Board April 1, 2013 (received for review November 5, 2012) Sustained fast requires the rapid replenishment of intersectin might not be mandatory for SV membrane retrieval or release-ready synaptic vesicles (SVs) at presynaptic active zones. might regulate other steps in endocytosis that are not rate-limiting Although the machineries for exocytic fusion and for subsequent for the process per se (21). endocytic membrane retrieval have been well characterized, little is Using simultaneous recordings from presynaptic and post- known about the mechanisms underlying the rapid recruitment of synaptic compartments at the calyx of Held (22, 23), we have SVs to release sites. Here we show that the Down syndrome- shown that intersectin 1 is a crucial factor in the replenishment of associated endocytic scaffold intersectin 1 is a crucial factor release-ready SVs within the RRP. We found that acute pertur- for the recruitment of release-ready SVs. Genetic deletion of inter- bation or genetic deletion of intersectin 1 inhibited the recruitment sectin 1 expression or acute interference with intersectin function of release-ready SVs without affecting the rate of endocytic inhibited the replenishment of release-ready vesicles, resulting in membrane retrieval under the same conditions. These data in- short-term depression, without significantly affecting the rate of dicate an important role of the endocytic scaffold intersectin in fast endocytic membrane retrieval. Acute perturbation experiments neurotransmitter release. suggest that intersectin-mediated vesicle replenishment involves the association of intersectin with the fissioning enzyme dynamin Results and with the actin regulatory GTPase CDC42. Our data indicate a role To test whether early-acting endocytic such as intersectin for the endocytic scaffold intersectin in fast neurotransmitter release, may regulate fast neurotransmission, we first explored the sub- which may be of prime importance for information processing in synaptic distribution of intersectin by confocal and superresolution the brain. stimulated emission depletion (STED) microscopy in situ by pre- paring thin sections from the mature mouse calyx of Held, a giant endocytosis | synaptic transmission | synaptic vesicle recruitment terminal in the auditory brainstem (22, 23) (Fig. 1). Immuno- staining for the AZ marker bassoon (24) revealed punctuate sig- eurotransmission depends on the exocytosis of synaptic vesi- nals representing sites for rapid neurotransmitter release along the Ncles (SVs) at active zones (AZs) and the subsequent retrieval cup-like calyx terminal and at small conventional boutons (Fig. of SV membranes by endocytosis. Sustained fast neurotransmission 1A). Intersectin was found at the calyx terminal and within post- requires rapid replenishment of release-ready SVs composing the synaptic neurons (red fluorescence in Fig. 1A). High-magnification readily releasable pool (RRP) (1–3). Whereas the machinery for STED imaging revealed a subset of intersectin puncta that over- SV fusion is well understood, the mechanisms controlling the lapped with bassoon-containing AZs (Fig. 1B), as evident on in- number of SVs within the RRP and the rate of replenishment re- tensity line profiles along the terminal, whereas a large fraction of main enigmatic (4, 5). It has been postulated that the rate of re- intersectin puncta was also located peripheral to the bassoon hot plenishment of release-ready SVs in addition to molecular priming spots (Fig. 1 C–E). The partial but clearly nonrandom colocali- reactions is regulated by the availability of SV release sites (6–8). zation of bassoon and intersectin clusters was corroborated by The reuse of release sites appears to depend on components of the Pearson correlation analysis (Fig. S1). endocytic machinery, including dynamin (9–12). However, consid- To study the role of intersectin 1 in fast neurotransmission, we ering that the rate of endocytosis is ∼10–100 times slower than that examined synaptic responses in intersectin 1 KO mice (20), in of replenishment of release-ready SVs, the question arises as to which expression of all isoforms of intersectin 1 is abrogated. how endocytic proteins affect rapid neurotransmitter release. Loss of intersectin 1 did not cause any overt differences in Data from dynamin mutants can be interpreted as a deficit in the morphological architecture or composition of the calyx or of SV recycling (e.g., loss of the SV reserve pool; ref. 13). Alter- the medial nucleus of the trapezoid body (MNTB) area in the natively, endocytic proteins may regulate exocytosis more directly, brainstem. Specifically, calyces from KO mice displayed a WT-like independent of membrane retrieval. If so, interference with select endocytic factors might impair replenishment of release-ready SVs without affecting the rate of SV membrane retrieval. Author contributions: T.S., N.L.K., J.B., A.P., and V.H. designed research; T.S., N.L.K., J.B., A possible candidate for such regulation is the early-acting A.P., J.S., L.Y., O.K., and V.H. performed research; H.B., O.S., and K.A. contributed new – reagents/analytic tools; T.S., N.L.K., J.B., A.P., J.S., L.Y., O.K., and V.H. analyzed data; and multidomain endocytic protein intersectin (14 18), a protein T.S. and V.H. wrote the paper. overexpressed in Down syndrome, which also associates with com- The authors declare no conflict of interest. ponents of the exocytic machinery [i.e., synaptosomal-associated This article is a PNAS Direct Submission. Y.G. is a guest editor invited by the Editorial protein 25 (SNAP-25)] (19). Although a role for intersectin in Board. endocytosis has been established in Drosophila (16, 17), the pre- 1To whom correspondence may be addressed. E-mail: [email protected] or cise function of intersectin 1 in mammalian central synapses has [email protected]. not yet been analyzed extensively. Optical measurements of en- 2N.L.K., J.B., A.P., and J.S. contributed equally to this work. docytosis in cultured neurons suggest mild effects on endocytosis as This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. a consequence of intersectin 1 KO (20). These results indicate that 1073/pnas.1219234110/-/DCSupplemental.

8266–8271 | PNAS | May 14, 2013 | vol. 110 | no. 20 www.pnas.org/cgi/doi/10.1073/pnas.1219234110 Downloaded by guest on September 29, 2021 Fig. 1. Intersectin 1 is localized at and around AZs. (A) STED microscopy image of a mature mouse calyx of Held synapse (age P50–P70) immunostained for NEUROSCIENCE endogenous bassoon (BSN; green) and intersectin 1 (ITSN1; red). The postsynaptic neuron is surrounded by the calyx terminal immunopositive for the AZ marker bassoon. Note that bassoon staining is also detectable at noncalycial terminals. (Inset) Confocal image of bassoon immunopositive puncta. For the scale bar, see the line scan profiles in C–E.(B) Magnified images of the data shown in A (from line C). (B, i) ITSN1; (B, ii)BSN;(B, iii) merged image. (Insets) Intersectin-immunopositive puncta confined to the active zones. (Scale bars: 750 nm; 350 nm in Insets.) (C–E) Spatial intensity profiles of bassoon (green) and intersectin 1 (red). The regions of interest (lines C, D, and E in A) were selected where the presynaptic compartment showed a finger-like structure.

intensity and distribution of the abundant SV protein vesicular similar release kinetics were seen in control and KO, indicating glutamate transporter 1 (VGLUT1), suggesting a normal number that loss of intersectin 1 does not impair SV fusion per se. This of SVs, at the level of light microscopy. Calyces from KO also finding is also in agreement with our biochemical and morpho- showed no alterations in the localization or the amount of major logical analysis noted earlier (Fig. S2). endocytic proteins, such as clathrin and adaptor protein complex- When the same stimulation was repeated after a resting inter- 2 (AP-2) (Fig. S2), arguing against major alterations or compen- val of 500 ms, the presynaptic Ca current amplitude was almost satory changes with respect to the endocytic machinery (20). identical. The EPSC recovered to ∼60% of its initial value in WT Consistent with the fact that heterozygous animals exhibit no (Fig. 2A, solid traces), whereas the recovery of the synaptic re- phenotypic changes (20), we detected no differences in synaptic sponse during the second pulse was significantly reduced in KO responses between calyces from WT or heterozygous mice; thus, (Fig. 2A, Lower). We then changed the interval of the two con- these data were pooled to serve as a control. Simultaneous pre- secutive pulses to monitor SV replenishment following depletion of synaptic and postsynaptic recordings were conducted in WT the RRP. When the recovered amount of the FRP was plotted (n =4), heterozygous (n =4), and KO (n =8) calyces of Held at against the interstimulus interval (ISI) between the two pulses, the postnatal days 8–11 (P8–11) using a pulse protocol that allows for control data were fitted with a double-exponential function with separation of two components of transmitter release within the time constants of a few hundreds of ms and several seconds (Fig. RRP, a fast-releasing pool (FRP) and a slowly releasing pool (SRP) 2B) (25). Recovery of the FRP was markedly slowed in the inter- (Fig. 2A, dotted traces) (25), as described in Materials and Methods. sectin 1 KO and could be fitted by a single exponential function Presynaptic terminals were depolarized from −80 mV to +70 mV with a time constant of several seconds (Fig. 2B). Importantly, the for 2 ms, followed by repolarization to 0 mV for 50 ms. During the FRP recovered completely when given a sufficient time period for 0-mV period, a constant calcium (Ca) influx at the presynaptic side recovery, unlike manipulations causing defective SV recycling (13, was observed. Similar Ca currents were elicited between control 27). In contrast, recovery of the SRP remained unchanged (Fig. (1,301 ± 105 pA) and KO (1,150 ± 66 pA). The excitatory post- 2C). These data show that intersectin 1 specifically regulates the synaptic current (EPSC) decayed during the pulse protocol, in- replenishment of release-ready SVs. dicating depletion of the RRP of SVs. To further analyze whether intersectin 1-mediated SV replen- Two components of transmitter release were observed during ishment reflects a role for intersectin 1 in endocytosis, we con- the pulse. The FRP had a time constant of a few ms [control: 1.8 ± ducted presynaptic capacitance measurements (27–29) after 0.4 ms (50% ± 3.4% of total release); KO: 3.1 ± 0.4 ms (49% ± pulses of 50 ms (Fig. 2D) or 500 ms (to elicit rapid endocytosis; Fig. 3.3% of total release)], reflecting fusion of SVs, which can be re- 2E) in WT and KO calyces. Capacitance jumps in response to the leased synchronously in response to action potentials (26). The SRP 50-ms pulse were similar in control [280 ± 40 femtofarads (fF)] and had a time constant of 20–30 ms (control: 21.7 ± 2.4 ms vs. KO: KO (224 ± 28 fF). In response to the 500-ms pulse, the jumps were 18.6 ± 1.1 ms) and requires more accumulation of residual Ca. larger in WT (969 ± 86 fF) than in KO (689 ± 80 fF), presumably During a 50-ms pulse, cumulative release reached a plateau level, reflecting slow SV replenishment after vesicle pool depletion in indicating fusion of all readily releasable SVs. Importantly, KO. Strikingly, in both stimulation protocols, the time course of

Sakaba et al. PNAS | May 14, 2013 | vol. 110 | no. 20 | 8267 Downloaded by guest on September 29, 2021 conditions, revealing a hitherto unknown function for intersectin 1 in regulating fast neurotransmitter release. To unravel the molecular mechanism of the exocytic function of intersectin 1, we turned to acute perturbation experiments to se- lectively interfere with intersectin-based protein–protein inter- actions. The major neuronal isoform of intersectin 1 (intersectin 1L) contains five SH3 domains that associate among other pro- teins with the membrane fissioning GTPase dynamin. Intersectin 1L also harbors pleckstrin homology (PH) and disabled (Dbl) ho- mology (DH) domains, which facilitate guanine nucleotide ex- change on CDC42 (14–18). Given the postulated role of dynamin in regulating the reuse of release sites (9–12), we first focused on the dynamin-binding SH3A domain of intersectin 1. To this aim, we analyzed whether acute perturbation of the intersectin 1 SH3A domain function (5 μM) affected synaptic depression and recovery at the rat calyx of Held. A train of AP-like stimuli was applied 20 times at 10 Hz. Subsequently, the stimulation frequency was switched to 50 Hz, to 100 Hz, and then back to 10 Hz. As shown in Fig. 3 A and C, the 10-Hz stimulation caused a depression of postsynaptic responses. In the presence of the SH3A domain (Fig. 3B), the depression deepened and led to smaller steady-state EPSC amplitudes (P < 0.05). At higher frequency, such differences became less overt, presumably because of a more pronounced overall synaptic depression. When the stimulation frequency was switched back from 100 Hz to 10 Hz, recovery from synaptic de- pression was profoundly slowed by the presence of SH3A (P < 0.01). These data suggest that perturbation of intersectin 1 SH3A domain function affects synaptic depression and recovery. Con- sistent with this interpretation, ATTO647N-labeled SH3A domain

Fig. 2. SV replenishment in intersectin 1 KO mice. (A) Simultaneous re- cordings of the presynaptic and postsynaptic compartments at the calyx of Held synapse. A pair of depolarizing pulses (0 mV for 50 ms, after a prepulse to +70 mV for 2 ms) was applied to the presynaptic terminal with an interval of 500 ms. During a 0-mV period, presynaptic Ca current was elicited. Evoked postsynaptic EPSCs (dotted line, first stimulation; solid line, second stimula- tion with an interval of 500 ms) and the cumulative release are shown. Cu- mulative release in response to the first pulse showed a double-exponential time course. (Upper) WT. (Lower) Intersectin 1 KO littermates. (B and C) Recovery of the fast (FRP; B) and the slow (SRP; C) components of release plotted against the stimulation interval. Dotted and solid circles represent data from control (four WT cells and four +/− cells) and KO (eight cells) mice, respectively. (D) The capacitance trace in response to a 50-ms pulse was normalized to the peak value and averaged among cells. Open and filled circles represent data from control and KO mice, respectively (n = 6 cells each). (E) The pulse duration increased to 500 ms. Note that for the open circle data, only downward error bars are displayed. Upward error bars are shown for the closed circle data for clarity.

membrane retrieval was similar in control and KO terminals (Fig. Fig. 3. Acute perturbation of intersectin 1 SH3A domain function causes short-term synaptic depression. Simultaneous recordings of the presynaptic S3 shows fast measurements). Of importance, given that mem- and postsynaptic compartments at the calyx of Held synapse (rats, P8–P11). A brane retrieval in KO terminals was affected only mildly, our train of AP-like stimuli (depolarization to +40 mV for 1.5 ms) was applied (20 observations here differ from kiss-and-run type exocytosis (30). In pulses), at 10, 50, and 100 Hz, and then at 10 Hz. Presynaptic Ca currents addition, WT and KO terminals responded similarly to perturba- (Top), EPSCs (Middle), and magnified EPSCs (with initial EPSC peaks trun- tion of AP2 by dialysis of a Synaptotagmin 2 (Syt2) 2-derived AP2- cated; Bottom) are shown. (A and B) Data under control conditions (A)and in the presence of SH3A domain (5 μM) (B). (C) Normalized time course of binding peptide (9), resulting in nearly complete elimination of the EPSC amplitudes plotted over time. Red and blue symbols indicate the membrane retrieval and of SV replenishment in both genotypes data under control and in the presence of the SH3A domain, respectively. (D (Fig. S4). This phenotype is clearly distinct from that induced by and E) Confocal images of a calyx terminal (rats, P8–P11) preloaded with loss of intersectin 1 and argues against major changes in the mode Alexa Fluor 488 (200 μM; D) and the Atto647N-conjugated SH3A domain of intersectin (10 μM; E). (F) STED images of magnified data outlined by the box of endocytosis in intersectin 1 KO mice. We conclude that deletion in E, illustrating the localization of the AZ markers bassoon (F, i; green), and of intersectin 1 selectively perturbs SV replenishment without Atto-SH3A (F, ii; red). (F, iii) Composite image, with colocalizing puncta in significantly affecting endocytic membrane retrieval under the same yellow. (Scale bar: 500 nm.)

8268 | www.pnas.org/cgi/doi/10.1073/pnas.1219234110 Sakaba et al. Downloaded by guest on September 29, 2021 (Fig. 3E) coinjected together with Alexa Fluor 488 (to identify (compare Fig. 2). A mutant SH3A domain unable to associate with injected calyces; Fig. 3D) was detectable as small puncta in close proline-rich ligands, including dynamin (Fig. S6 C and D), did not apposition to and partly colocalizing with bassoon-containing AZs block recovery (Fig. 4D), nor did the related SH3 domain of in dual-color STED images (Fig. 3F). These results are consistent endophilin A1 (recovery of 60% ± 6%, n = 3 cells; Table S1), with the localization of endogenous intersectin 1 (Fig. 1 A–E and indicating that the effect is specific to the SH3A domain of Fig. S1) and dynamin 1 (Fig. S5) detected by confocal and two- intersectin 1. color STED microscopy. Given that dominant-negative interference with protein func- To further corroborate these findings, we measured the rates of tion may cause side effects through sequestration of endogenous SV recruitment to the FRP and SRP. We introduced the SH3A components, we sought to corroborate our results using other fi domain of intersectin 1 via the presynaptic patch pipette, thereby reagents. To this aim, we dialyzed an antibody that speci cally interfering with the recruitment of SH3A domain binding part- recognizes the SH3A domain of intersectin 1 (31) into the termi- ners, such as dynamin 1 (Fig. S6 C and D), to endogenous inter- nal, thereby occluding the binding site on SH3A for proline-rich ligands including dynamin. Slowed recovery from vesicle pool sectin. Presynaptic Ca currents were comparable with control depletion similar to that seen for the injected SH3A domain was conditions, as was the synaptic response during the first pulse seen after anti-SH3A antibody injection (20–30 μg/mL; Fig. 4C). (Table S1), whereas recovery of the synaptic response (FRP) fi Analysis of SV replenishment after depletion of the RRP by two during the second pulse was reduced signi cantly (Fig. 4 A and B), consecutive pulses applied at changing ISIs in calyces injected with similar to the phenotype observed in intersectin 1 KO calyces the SH3A domain or anti-SH3A antibodies confirmed a selective role of intersectin 1 in the recovery of the FRP (Fig. 4D, Left) without perturbing the SRP (Fig. 4D, Right). If the effect of intersectin 1 SH3A on replenishment of the FRP were mediated by control SH3A domain C SH3A domain ab A B its association with dynamin, then dialysis of a dynamin-derived 0.5 I 0.5 0.5 pre nA proline-rich SH3A should result in a phenocopy. Indeed, nA nA – 5 nA application of the dynamin proline-rich domain (Dyn1 509 864; EPSC 5 3 μM) [but not the proline-rich domain from neuronal Wiskott 3 nA nA 1st Aldrich syndrome protein (N-WASP)] or of a SH3 domain-binding 2nd 400 peptide (Dyn1 773–794; 1 mg/mL) that competes with endogenous cum ves dynamin for the same site on intersectin 1 SH3A (Fig. S6 A and B) NEUROSCIENCE rel slow 1000 500 fast ves ves slowed recovery of the FRP (Fig. 4E). Inhibition of FRP refilling 10 ms was also seen in the dynamin-binding SH3E domain of intersectin 1 (Fig. 4F), whereas SRP refilling was unaffected (Fig. S7). These D fast component (FRP) slow component (SRP) findings suggest that the proline-rich domain of dynamin 1 is in- 1.0 1.4 volved in the recruitment of SVs to the FRP (9). 0.8 1.2 1.0 Consistent with the KO data, endocytic membrane retrieval 0.6 0.8 assayed by capacitance measurements proceeded relatively un- 0.4 control 0.6 perturbed in the presence of either the SH3A domain or anti- SH3A 0.4 0.2 SH3A mutant intersectin 1 SH3A-directed antibodies under different stimulation

recovered fraction 0.2 SH3A ab recovered fraction 0.0 0.0 conditions (Fig. S8). Moreover, no differences between micro-

0 2 4 6 8 0 2 4 6 8 injected calyces and control conditions were observed when ca- ISI (s) ISI (s) pacitance traces were monitored at fast time resolution or after a strong depolarizing pulse of 500 ms (Fig. S8). The findings from E F these acute perturbation experiments are consistent with our results 1.0 fast component (FRP) 1.0 fast component (FRP) for intersectin 1 KO mice and establish a function of intersectin 1 0.8 0.8 and its SH3 domains in fast neurotransmission via regulation of 0.6 0.6 recovery from SV pool depletion independent of alterations in

0.4 control 0.4 endocytic membrane retrieval. control Dyn PRD peptide SH3E domain How can intersectin 1 regulate recruitment of release-ready 0.2 Dyn PRD 0.2 recovered fraction recovered fraction N-WASP PRD SVs? The long neuronal isoform (L) of intersectin 1 serves as 0.0 0.0 a molecular scaffold that, along with its ability to associate with 0 2 4 6 8 0 2 4 6 8 ISI (s) ISI (s) endocytic proteins such as dynamin, also contains a protein module that regulates actin polymerization. This module com- Fig. 4. Acute perturbation of the intersectin 1 SH3A domain slows SV re- prises DH and PH domains that together serve as a guanine nu- plenishment. A pair of depolarizing pulses (0 mV for 50 ms after a prepulse cleotide exchange factor (GEF) for the Rho family small GTPase + to 70 mV for 2 ms) was applied to the presynaptic terminal with an interval CDC42, a crucial regulator of actin reorganization of 500 ms. (A–C) EPSCs (dotted line, first stimulation; solid line, second – fi stimulation, with an interval of 500 ms) and cumulative release (cum rel) are (14 18). This structure, together with our nding that loss of shown. (A) Control conditions. (B and C) Terminals dialyzed with SH3A dynamin function is associated with a profound increase in cortical domain (5 μM) (B) or antibodies directed against intersectin 1 SH3A (2,030 actin polymerization, led us to speculate that CDC42 may serve as μg/mL) (C). (D) Similar experiments as in A–C, but with the stimulus interval another downstream target of intersectin 1 in FRP replenishment. of the two pulses varied. Recovery of the FRP (Left) and the SRP (Right) We explored this possibility by introducing the DH-PH domain after RRP depletion was plotted against the ISI. Data from control con- of intersectin (5 μM) into the presynaptic terminal to interfere ditions (open circles) and obtained in the presence of SH3A (filled circles), with intersectin 1–CDC42 complex formation. We found that re- mutant SH3A (filled squares), or antibodies against the intersectin 1 SH3A covery of the fast component of release was significantly slowed fi domain ( lled triangles) are shown. (E) Recovery of the fast-releasing com- in DH-PH domain-injected terminals (Fig. 5A). To corroborate ponent is plotted against ISI. The proline-rich domain of dynamin (filled triangles), a dynamin 1-derived proline-rich peptide (filled circles), or the these data, we probed the function of Rho family small G proteins, proline-rich domain of N-WASP (filled squares) were introduced into the including CDC42, directly. We examined the effect of introducing terminal (ISI = 0.5 and 1 s). Open circles, control condition. (F) Same as in E, the enzymatic domains of toxin B from Clostridum botulinum except that the SH3E domain of intersectin 1 was introduced into the ter- (1 μM), a well-established inhibitor of Rho family small G pro- minal (filled circles). Open circles, control condition. teins, including Rho, CDC42, and Rac (32). Application of toxin

Sakaba et al. PNAS | May 14, 2013 | vol. 110 | no. 20 | 8269 Downloaded by guest on September 29, 2021 A DHPH domain intersectin 1 may be involved in endocytosis under certain stimu- fast component (FRP) lation conditions distinct from those used in the present study or at I different synapses. In addition, our data were obtained in young pre 1 nA 0.8 2 nA animals and at room temperature, and endocytic mechanisms may 0.6 undergo developmental changes that could involve intersectin 1. EPSC 0.4 Indeed, a role for intersectin (including other isoforms) in endo- control cytic SV recycling has been reported at other synapses and under 0.2 DHPH domain different physiological conditions (14–18). Finally, intersectin 1 cum recovered fraction 0.0 may regulate discrete steps within the endocytic limb of the SV cycle rel 500 ves 0 2 4 6 8 10 (e.g., sorting of SV cargo) that do not result in altered membrane ISI (s) retrieval and thus would have escaped detection by capacitance measurements. Irrespective of these considerations, our data clearly B Toxin B show that loss of intersectin 1 at the calyx of Held (P8–P12) impairs replenishment of release-ready SVs with no apparent change Ipre in capacitative membrane retrieval assayed under the same con- 1 nA 0.8 2 nA ditions, revealing an unexpected function for intersectin 1 in fast 0.6 neurotransmission. EPSC 0.4 How might an endocytic protein affect replenishment of fast- control 0.2 ToxinB releasing SVs? One possibility is that release sites may be mutually occupied by exocytic or endocytic protein complexes. In this sce-

500 recovered fraction cum 0.0 rel ves nario, removal of an endocytic protein complex by intersectin 0 2 4 6 8 would be necessary for priming of SVs. This idea is consistent with ISI (s) a possible interaction between the SNARE complex (syntaxin/ SNAP-25) and dynamin (12, 19), and also with our preliminary C secramine A observation that the distribution of dynamin at and around AZs is altered in intersectin 1 KO mice. A second possibility may relate to Ipre 1 nA 0.8 a potential function of intersectin 1 in SV priming, although to our 0.6 knowledge, no data exist to support this idea. A third possibility is EPSC that released SV proteins or dead-end cis-SNARE complexes may 2 nA 0.4 control Secramine A jam the release site at AZ membranes. Such jammed release sites 0.2 may be unavailable for the recruitment of new release-ready SVs

cum recovered fraction until the material clogging the fusion zone has been removed. This rel 500 removal could involve lateral translocation of membrane proteins, 10 ms 0 2 4 6 8 10 ves ISI (s) including cis-SNARE complexes, within the plane of the mem- brane, for example, by intersectin 1-regulated actin-driven move- Fig. 5. Blocking the activity of the intersectin binding partner CDC42 impairs ment out of the AZ. Intersectin 1 consistently binds to SNAP-25 recruitment of release-ready SVs. (A) Simultaneous recordings of the pre- and postsynaptic compartments at the calyx of Held synapse (as depicted in Figs. 2 (19), and SNAP-25 mutants in Drosophila melanogaster suffer from and 4). The DH-PH domain of intersectin 1 was introduced into the terminal. short-term depression (35). (Left) panel is similar to Fig 4 and the traces from one cell pair are shown Based on our data, the function of intersectin 1 in replenishment (dotted: first stimulation, solid: second stimulation with an interval of 500 ms). of release-ready vesicles also necessitates its association with dy- Right) Plot of recovery of the fast component (FRP) against the stimulus interval. namin (Fig. 4), perhaps reflecting a requirement for membrane Filled circles, Dbl homology-pleckstrin homology (DH-PH) domain; open circles, remodeling in release site clearance. Irrespective of the precise control condition. (B)SameasinA,exceptwith1μM toxin B introduced into the mechanisms involved, the results reported here support a crucial presynaptic terminal. Toxin B potently inhibits Rho family small G proteins. Open regulatory role for the endocytic scaffold intersectin 1 in fast neu- and filled circles represent the data from controls and in the presence of toxin B, rotransmission and short-term plasticity independent of mem- respectively. (C)SameasinA,exceptthat20μMsecramineA(filled circles), aspecific small-molecule inhibitor of CDC42, was applied to the terminal. brane retrieval, suggesting that the machineries for exocytosis and endocytosis may be coupled more tightly than previously thought (6, 9, 12). Such coupling is likely of crucial physiological importance for fast neurotransmission and for information pro- B inhibited recovery of the FRP of release (Fig. 5B). A similar cessing in the brain, particularly in sensory systems that fire at effect was observed for secramine A, a recently developed specific hundreds of Hz. blocker of CDC42 (33) (Fig. 5C). Refilling of the SRP was not affected by any of these reagents (Fig. S7). Taken together, Materials and Methods these results are consistent with a model in which intersectin 1L- Electrophysiology. Transverse brainstem slices (200 μm thick) were prepared mediated activation of CDC42 regulates replenishment of re- from 8- to 12- day-old Wistar rats (22, 23) or from intersectin 1 KO mice, along with corresponding WT or heterozygous littermates used as controls lease-ready SVs at the calyx of Held. (20). Experiments have been approved by the review board at Doshisha Discussion University, Max Planck institute for biophysical chemistry and Leibniz Institut für Molekulare Pharmakologie, Berlin. The extracellular solution contained

The data reported herein suggest an unexpected function for the 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 25 mM glucose, 25 mM endocytic scaffold intersectin 1 in fast neurotransmission at the NaHCO3, 1.25 mM NaH2PO4, 0.4 mM ascorbic acid, 3 mM myoinositol, and calyx of Held through regulation of replenishment of a pool of fast- 2 mM Na-pyruvate [pH 7.4; bubbled with 95% (vol/vol) O2 and 5% (vol/vol) releasing SVs at or near AZs that is independent of endocytic CO2]. Experiments were performed at room temperature. During the recordings, 1 μM TTX and 10 mM tetraethylammonium chloride (TEA-Cl) membrane retrieval. This phenotype is distinct from that of other + + were included in the external solution to block Na and K channels. In endocytic proteins; for example, dynamin 1 KO mice exhibit nor- paired recordings, 50 μM D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5), mal endocytosis in response to weak stimulation, but their endo- 100 μM cyclothiazide (CTZ), and 1 mM or 2 mM (for the experiments in mice) cytic capacity is readily saturated in response to strong stimulation kynurenic acid (Kyn) were added to isolate postsynaptic AMPA - (34). Nonetheless, our results do not exclude the possibility that mediated EPSCs and to block desensitization and possible saturation of

8270 | www.pnas.org/cgi/doi/10.1073/pnas.1219234110 Sakaba et al. Downloaded by guest on September 29, 2021 AMPA receptors (36). A calyx of Held and its postsynaptic MNTB principal missed by using a single exponential with free parameters, which fit the data neuron were whole-cell voltage clamped at –70 mV (for the experiments in equally well in some cases. Data are shown as mean ± SEM. mice) or at −80 mV using an EPC 10/2 amplifier (HEKA). The presynaptic patch pipette (3–5MΩ) solution contained 135 mM Cs-gluconate, 20 mM Peptides and Protein Domains. The SH3 domain binding dynamin 1-derived 773 TEA-Cl, 10 mM Hepes, 5 mM Na2-phosphocreatine, 4 mM MgATP, 0.3 mM proline-rich peptide (amino acid sequence: WT peptide, RSPTSSPTPQRRA- GTP, and either 0.05 or 0.5 mM EGTA (pH 7.2). The presynaptic series re- PAVPPARPG794; mutant peptide, 773RSPTSSPTPQRRAAAVAPARPG794)wasob- sistance (5–20 MΩ) was compensated for by 30–70%. The postsynaptic pi- tained by chemical synthesis. The following hexahistidine-tagged recombinant pette (2–3.5 MΩ) contained the same solution as the presynaptic pipette, proteins were expressed and purified from overexpressing Escherichia coli except that the EGTA concentration was increased to 5 mM. The post- (BL21) using nickel nitrilo-triacetic acid (Ni-NTA) affinity chromatography synaptic series resistance (3–8MΩ) was compensated for by the amplifier, so according to the manufacturer’s (Sigma-Aldrich Inc., St. Louis) instructions: that the uncompensated resistance was below 3 MΩ. The remaining re- human intersectin 1 SH3A domain (residues 738–803), intersectin 1L-SH3A sistance was further compensated for offline. mutant (residues 738–803 with G794R/ P797L mutations), human intersectin 1 Presynaptic capacitance measurements were carried out using an EPC10/2 SH3E domain (residues 1152–1214), human intersectin 1 DH-PH domain (resi- amplifier in the sine + DC configuration. A sine wave (30 mV amplitude, 1,000 dues 1229–1581), and mouse endophilin A1 (residues 283–352). The affinity Hz) was superimposed on a holding potential of −80 mV. Measurements were purification experiments shown in Fig. S6 were carried out essentially as de- included in the dataset if the presynaptic series resistance was below 20 MΩ. scribed previously (31). Occasionally, baseline capacitance traces exhibited a slow shift that was cor- rected for analysis. ACKNOWLEDGMENTS. We thank the Leibniz Institute for Neurobiology, CTZ, D-AP5, and Kyn were obtained from Tocris. SH3 domains, antibodies. Magdeburg, Special Laboratory Electron- and Laserscanning Microscopy and peptides were dialyzed to the presynaptic solution. Center for instrument use and scientific and technical assistance. We also Quantal release rates were estimated by the deconvolution method, thank Dr. Melanie Pritchard (Monash University) for providing intersectin 1 KO adapted for the calyx of Held (36). Quantal release rates, determined by mice, Dr. Tanja Martizen for help with the experiments using KO mice, and Dr. T. Kirchhausen (Harvard Medical School) for kindly providing Secramine A. deconvolution, were integrated to obtain the cumulative release. Cumula- fi The study was supported by the Japan Society for the Promotion of Science tive release was tted by a double-exponential function after correction for (KAKENHI Grants 24300144 and 24650218 and Core-to-Core Program A, to SV replenishment. In the paired-pulse protocol, the time constants for the T.S.), the Toray Science Foundation (T.S.), the Uehara Foundation (T.S.), the second pulse were derived in the same manner as those for the first pulse, Swedish Research Council (A.P. and O.S.), and the German Research Founda- because a small fraction of the fast-releasing component might have been tion DFG (Grants EXC 257 and SFB958/A01 to V.H. and SFB958/Z02 to J.S.).

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