Brain Struct Funct DOI 10.1007/s00429-014-0766-0

ORIGINAL ARTICLE

Tomosyn-2 is required for normal motor performance in mice and sustains neurotransmission at motor endplates Cornelia J. Geerts • Jaap J. Plomp • Bastijn Koopmans • Maarten Loos • Elizabeth M. van der Pijl • Martin A. van der Valk Matthijs Verhage • Alexander J. A. Groffen

•

Received: 28 October 2013 / Accepted: 26 March 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Tomosyn-1 (STXBP5) is a soluble NSF attachment protein receptor complex-binding protein that inhibits vesicle fusion, but the role of tomosyn-2 (STXBP5L) in the mammalian nervous system is still unclear. Here we generated tomosyn-2 null (Tom2KO/KO) mice, which showed impaired motor performance. This was accompanied by synaptic changes at the neuromuscular junction, including enhanced spontaneous acetylcholine release frequency and faster depression of muscle motor endplate potentials during repetitive stimulation. The postsynaptic geometric arrangement and function of

acetylcholine receptors were normal. We conclude that tomosyn-2 supports motor performance by regulation of transmitter release willingness to sustain synaptic strength during high-frequency transmission, which makes this gene a candidate for involvement in neuromuscular disorders. Keywords Neuromuscular junction
Release willingness
Short-term plasticity
STXBP5L
Synaptic transmission
Tomosyn-2

Introduction C. J. Geerts and J. J. Plomp contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s00429-014-0766-0) contains supplementary material, which is available to authorized users. C. J. Geerts
M. Verhage
A. J. A. Groffen (&) Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University, 1081 HV Amsterdam, The Netherlands e-mail: [email protected]; [email protected] J. J. Plomp
E. M. van der Pijl Department of Neurology, Leiden University Medical Centre, PO Box 9600, 2300 RC Leiden, The Netherlands B. Koopmans
M. Loos Sylics (Synaptologics BV), PO Box 71033, 1008 BA Amsterdam, The Netherlands M. A. van der Valk Department of Experimental Animal Pathology, Antoni van Leeuwenhoek-Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands M. Verhage
A. J. A. Groffen Department of Clinical Genetics, VU Medical Center, 1081 HZ Amsterdam, The Netherlands

Intracellular trafficking requires fusion of vesicular organelles, mediated by soluble NSF attachment protein receptor (SNARE) proteins. In neurons, vesicle fusion contributes to neurite outgrowth and is essential for neurotransmitter release. The neuronal SNARE complex is constituted by synaptobrevin (VAMP), anchored in the vesicular membrane, and syntaxin and SNAP25 in the target membrane. SNARE complex assembly and subsequent membrane fusion are tightly regulated in space and time by accessory proteins, such as Munc-18, Munc-13, Ca2? sensors, complexin and tomosyn (reviewed by Jahn and Fasshauer 2012). Tomosyn is a syntaxin-binding protein (Fujita et al. 1998) that inhibits secretion from PC12 cells (Fujita et al. 1998; Hatsuzawa et al. 2003), adrenal chromaffin cells (Gladycheva et al. 2007; Yizhar et al. 2004) and insulin secreting beta cells (Cheviet et al. 2006; Zhang et al. 2006). In neurons, synaptic transmission (Baba et al. 2005; Sakisaka et al. 2008; Yamamoto et al. 2010b) and neurite outgrowth (Kraut et al. 2001; Sakisaka et al. 2004) are suppressed by tomosyn. This is confirmed in vivo for transmission at the neuromuscular junction (NMJ) in C. elegans (Gracheva et al. 2006; McEwen et al. 2006) and Drosophila (Chen et al. 2011).

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Brain Struct Funct

The mammalian nervous system expresses two tomosyn genes, tomosyn-1 and tomosyn-2 (STXBP5 and STXBP5L respectively), with 77 % amino acid sequence similarity. While tomosyn-1 mRNA expression is stable during development, tomosyn-2 mRNA is upregulated between E18 and P12, concurrent with syntaxin and Munc-18. The spatial distribution also differs: tomosyn-1 is widely expressed in the brain, while tomosyn-2 expression seems more restricted to certain areas, including parts of the hippocampus and the granular layer of the cerebellum (Groffen et al. 2005). Both are expressed in the spinal cord (Lein et al. 2007). Characterization of tomosyn-1-deficient mice confirmed a role in hippocampal synaptic transmission (Sakisaka et al. 2008), but the in vivo implications are still unknown. Furthermore, although an inhibitory role of tomosyn-2 was suggested by overexpression studies in PC12 cells (Williams et al. 2011) and pancreatic beta cells (Bhatnagar et al. 2011), the neuronal function of tomosyn-2 has not been investigated. To resolve these issues, we generated and characterized tomosyn-2-deficient (Tom2KO/KO) mice. We found that this gene regulates acetylcholine (ACh) secretion at the motor endplate and contributes to overall motor performance. Thus, tomosyn-2 supports motor system functioning by inhibiting spontaneous release and enhancing synaptic strength during sustained activity, revealing a novel evolutionary benefit of this inhibitory mechanism.

Materials and methods Animals A tomosyn-2 knock-in targeting vector (Tom2lox) was constructed by flanking exon 2 (Genbank accession number AY542329) with loxP sites (online resource supplementary Fig. 1). Homologously targeted Tom2lox embryonic stem cells derived from a C57Bl/6 background were injected into wild-type blastocysts and implanted into pseudopregnant females. Chimers were mated with inbred C57Bl/6 mice (Caliper Life Sciences). Tom2KO mice were obtained by mating C57Bl/6 Tom2lox mice with a C57Bl/6 CMV Cre deleter strain (gift from R.J. Pasterkamp) recombining the loxP sites. For colony maintenance, Tom2WT/KO animals were repeatedly backcrossed to inbred C57Bl/6J mice (Charles River Laboratories, L’Arbresle, France); genetically indistinguishable from those obtained from The Jackson Laboratory (Zurita et al. 2011). This breeding strategy ascertained a high degree of allelic homozygosity and precluded effects of spontaneous mutations in our colony. Animals were housed, bred and experimentally used according to Institutional guidelines and Dutch and

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US governmental laws. Mice were housed with their gender-matched littermates in type 2 Makrolon cages on sawdust bedding after weaning. Food and water were provided ad libitum. Temperature (20–22 °C), humidity (70–80 %) and light–dark cycle (7:00 am lights on, 7:00 pm lights off) were controlled. Embryos were obtained by cesarean section of pregnant females from timed matings. Behavior Behavioral tests were performed at an age of 11–16 weeks. Male mice of a pure C57Bl/6 background, as indicated above, that were backcrossed for 2–7 additional generations with C57Bl/6 and interbred for up to four generations to generate homozygous Tom2KO/KO animals were used. Motor performance and motor learning were assessed using an accelerating rotarod (Roto-rod series 8, IITC Life Science, Woodland Hills, CA, USA). Mice were subjected to two habituation trials of 120 s [acceleration from 0 to 20 rounds per minute (RPM) in 120 s] followed by three training trials (acceleration from 0 to 40 RPM in 180 s) on the first day. On day two, mice were subjected to five additional training trials. Previous observations showed that animals never reach the maximum programmed RPM during habituation and training sessions. The maximum RPM reached in each trial was scored. The peak amount of force (N) mice applied upon grasping a bar connected to a force meter (1027DSM Grip Strength Meter, Columbus instruments, Columbus, OH, USA) was scored as another measure of neuromuscular function. Five measures of front paws only were followed by grasping the pull bar five times with front and hind paws. Alternatively, front paw grip strength of mice that were used in NMJ electrophysiology experiments was measured with a 303500 meter in ten repetitive trials (Technical and Scientific Equipment GmbH, Bad Homburg, Germany). The median of all repetitions was taken as grip strength. Reaching reflex was assessed by lifting mice by their tail and slowly lowering them towards a horizontal flat platform, while keeping a distance of at least 3 cm. The number of attempts to reach for the platform in three trials was scored. Diaphragm isolation and staining Diaphragm was dissected from E18 stage mice of pure C57Bl/6 background, backcrossed for two generations and interbred for 5–6 generations to generate homozygous Tom2KO/KO animals. After cesarean section, the embryos were decapitated and the rib cage was dissected out and fixed in freshly prepared 2 % paraformaldehyde (PFA; Merck) in PBS for 90 min at room temperature while

Brain Struct Funct

gently shaking. The PFA was subsequently blocked with 0.1 M glycine in PBS for 20 min. Next, the diaphragm was isolated from the rib cage and incubated in 1 % H2O2 for 30 min at room temperature on a shaker. After washing with PBS plus 0.05 % Tween-20, the tissue was incubated for 1 h with blocking solution (5 % normal goat serum, 2.5 % bovine serum albumin, 0.2 % Triton X-100 in PBS). Then, the sections were incubated with primary antibody (anti-synapsin, E028, 1:1,000 and anti-gap43, 9527, 1:1,000) and Alexa Fluor 488 conjugated a-bungarotoxin (molecular probes, 1 lg/ml) diluted in blocking solution overnight at 4 °C on a shaker. The tissue was rinsed with PBS-Tween again and incubated for 2 h at room temperature with secondary Alexa Fluor conjugated antibody diluted in blocking solution (molecular probes, 1:1,000). After rinsing the sections with PBS-Tween, they were mounted using DABCO-Mowiol. Imaging was performed on a confocal laser scanning microscope (Zeiss LSM510) with a 409 oil objective, NA1.3 and 0.79 mechanical zoom. Maximal projections of z-stacks were generated using ImageJ. After electrophysiological investigations (see below), the diaphragms of adult Tom2WT/WT and Tom2KO/KO mice were fixed in 1 % PFA in PBS at room temperature for *30 min and stored at 4 °C until further processing. For staining of the ACh receptors (AChRs), a *3-mm-wide strip from the most dorsal side of the right hemidiaphragm was cut off and first thoroughly washed with PBS, then incubated for 3 h with 1 lg/ml Alexa Fluor488 conjugated a-bungarotoxin (Invitrogen), followed by thorough PBS wash for 45 min, all at room temperature. Muscle strips were mounted on microscope slides with ProLongGold antifade mounting medium (Invitrogen) and left to harden overnight in the dark at room temperature. The next day, samples were viewed under a Leica TCS STED CW confocal laser scanning microscope using a 409 oil immersion objective. A 387.5 9 387.5 lm overview picture was taken from the NMJ-rich midline zone of each sample. AChR receptor staining area was quantified using the thresholding feature of the ImageJ v1.48k program (http:// rsbweb.nih.gov/ij/) at ten randomly chosen NMJs within each of these pictures. Per muscle sample the mean of these ten NMJ values was calculated and from that group mean ± SEM was calculated. Ex vivo neuromuscular junction electrophysiology Ex vivo NMJ electrophysiology was performed in diaphragm muscles of male Tom2WT/WT and Tom2KO/KO animals (12–15 weeks of age) with a pure C57Bl/6 background, backcrossed for 2–7 generations and interbred for up to five generations. Postsynaptic electrophysiological signals caused by presynaptic ACh release were recorded

with the investigator blinded for genotype. Directly after electromyography (supplementary Materials and methods), still anesthetized mice were killed by CO2 asphyxiation. Diaphragm muscle–nerve preparations were dissected and placed in Ringer’s medium at room temperature (20–22 °C), containing (in mM): NaCl 116, KCl 4.5, CaCl2 2, MgCl2 1, NaH2PO4 1, NaHCO3 23, glucose 11, pH 7.4, bubbled with a 95 % O2/5 % CO2 gas mixture. Intracellular recordings of miniature endplate potentials (MEPPs) and endplate potentials (EPPs) were made in Ringer’s solution at 26–28 °C in phrenic nerve–hemidiaphragm preparations, essentially as described before (Klooster et al. 2012). Muscle fibers were impaled near the NMJ with the tip of a glass micro-electrode (5–20 MX, filled with 3 M KCl) connected to a Geneclamp 500B (Axon Instruments/ Molecular devices, Sunnyvale, CA, USA) for signal amplification and filtering (10 kHz low pass). The signal was digitized using a Digidata 1322A (Axon Instruments/ Molecular Devices) digitizer in combination with the Clampfit 9.2 program (Axon Instruments/Molecular Devices). Off-line analysis was done using Mini Analysis 6.0.3 (Synaptosoft, Fort Lee, USA). Muscle action potentials were prevented by the specific skeletal muscle Na? channel blocker, l-conotoxin GIIIB (3 lM; PeptaNova GmbH, Sandhausen, Germany). For EPP recording, the phrenic nerve was stimulated with electrical pulses from a computer-controlled programmable stimulator (AMPI, Jerusalem, Israel). Mean EPP and MEPP amplitudes at each NMJ were normalized to -75 mV resting potential, with the reversal potential for ACh-induced current assumed 0 mV (Magleby and Stevens 1972). At each NMJ, the quantal content (i.e., the number of ACh quanta released per nerve impulse) was calculated. To this end, the normalized mean amplitude of the 20 EPPs recorded at low-rate (0.3 Hz) stimulation was corrected for non-linear summation (McLachlan and Martin 1981) and then divided by the normalized mean MEPP amplitude (calculated from the MEPPs sampled during a 1 min recording period). At each hemidiaphragm tested, 10–15 NMJs were sampled. For MEPP analysis after high-frequency stimulation, MEPPs were recorded for 5 s directly after cessation of the stimulation. A 30 s resting period was included between different high-frequency stimulations at a single NMJ. Before starting measurements in a next NMJ, a further resting period was adhered to of at least one min. Bath application of 500 mM sucrose Ringer’s medium was used to assess MEPP frequency upon depletion of the RRP. The recordings were started after one min sucrose incubation. In each muscle, 15 NMJs were sampled quickly after one another during a total period of *8 min. Overall mean muscle values of the investigated parameters were calculated per mouse from NMJ mean values. Mean values for groups of mice were calculated after deblinding for genotype.

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Brain Struct Funct

Hippocampal autapse electrophysiology For autapse electrophysiology, dissociated hippocampal neurons were plated at a density of 2 K/well (12-well plate) on microdot islands of glia cells. Microislands were prepared by plating 6 K/well freshly prepared rat glia on etched glass coverslips, coated with 0.15 % w/v agarose and dots of 0.1 mg/ml poly-D-lysine (Sigma)/ 0.7 mg/ml rat tail collagen (BD Biosciences) substrate (adapted from Segal and Furshpan 1990). Hippocampi were isolated from E18 stage mouse brains (backcrossed for 2 generations and interbred for 2–4 generations) in Hanks Buffered Salt Solution (HBSS; Sigma) plus 10 mM HEPES (Invitrogen) at room temperature. After removal of the meninges, hippocampi were incubated in HBSS/HEPES plus 0.25 % trypsin (Invitrogen) for 20 min at 37 °C. The hippocampi were washed in HBSS and triturated with a fire-polished pipette tip to obtain a cell suspension. Cultured neurons were maintained in neurobasal medium supplemented with 2 % B-27, 1.8 % HEPES, 0.25 % glutamax and 0.1 % penicillin/streptomycin (all from Invitrogen). Whole-cell voltage clamp electrophysiology was performed with borosilicate glass pipettes (2–4 MX) on autaptic hippocampal neurons that had been in culture for 14–18 days. Extracellular solution contained 10 mM HEPES, 10 mM glucose, 140 mM NaCl, 2.5 mM KCl, 4 mM MgCl2 and 4 mM CaCl2. Intracellular solution contained 125 mM K?-gluconic acid, 10 mM NaCl, 4.6 mM MgCl2, 4 mM K2-ATP, 15 mM creatine phosphate, 10 U/ml phosphocreatine kinase and 1 mM EGTA. The pH of both solutions was set to 7.3 and buffers were ensured to have an osmolarity of 300 mOsm. Cells were stimulated and signals were recorded at room temperature with a Multiclamp 700B amplifier and Digidata 1440A (Axon Instruments). Electrical stimulation was done by depolarization from -70 to 30 mV for 0.5 ms. In sucrose experiments, 75 % series resistance compensation was used. Sucrose stimulation was done by bath application of 500 mM sucrose. Only cells with a series resistance below 10 MX, a leak current below 300 pA and excitatory postsynaptic current (EPSC) amplitudes of at least 300 pA were used for analysis. Analysis was done with Clampfit 10 (Axon Instruments/Molecular Devices), Mini Analysis (Synaptosoft) and Matlab (Mathworks). Statistical analyses Statistical analysis was performed using SPSS (Version 20.0, Armonk, NY: IBM Corp.). Kolmogorov–Smirnov was used to test normality of data distribution. A Mann– Whitney test for two independent samples was applied

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Table 1 Mendelian skew in Tom2WT/KO interbreedings Genotype

Expected (%)

Observed E18 (p [ 0.30) (%)

Observed P21 (p \ 0.0001) (%)

Tom2WT/WT

25

19.8

35.0

WT/KO

Tom2

50

56.5

56.1

Tom2KO/KO

25

23.6

8.9

when groups were not normally distributed. Else, independent sample Student t tests were performed, taking into account Levene’s test for equality of variances. Repeated measures or two-way ANOVA tests were used when indicated. A significance level of p \ 0.05 was used. Supplementary materials and methods are available as online resource.

Results Generation of Tom2KO/KO mice Homozygous tomosyn-2-deficient (Tom2KO/KO) mice were generated from embryonic stem cells with C57Bl/6 background and validated as shown in supplementary Fig. 1 (online resource). In these mice, tomosyn-2 exon 2 was deleted using flanking loxP sites, introducing an early stop codon by frameshift. No protein product was detected by immunoblotting using a tomosyn-2 specific antibody that recognizes an epitope coded by exon 22–23. Upon interbreeding of Tom2WT/KO mice, the genotype distribution was as expected with 23.6 % Tom2KO/KO at late embryonic stage E18 [v2(2) = 4.7, p [ 0.05; n = 237; Table 1]. At 3 weeks of age the number of Tom2KO/KO animals was reduced to only 8.9 % [v2(2) = 18.5, p \ 0.0001; n = 123]. This was likely due to abnormalities already evident at E18. Approximately one-third of Tom2KO/KO embryos were less motile and had a white appearance often combined with hemorrhages in the head or abdomen and degeneration of the nervous system (online resource supplementary Fig. 2). After postnatal day 21 (P21) no additive loss of Tom2KO/KO animals was observed. Importantly, the embryonic lethal phenotype was observed only in a subset of Tom2KO/KO animals originating from initial Tom2WT/KO interbreedings (2–4 generations of backcrossing) and was no longer observed after seven generations of backcrossing and interbreeding of early generation Tom2KO/KO survivors, indicative of a phenotypic shift related to unidentified compensational processes. Such phenotypic heterogeneity observed in Tom2KO/KO mice is striking, but not unique as it was also observed in tomosyn1 mutant mice (Sakisaka et al. 2008).

Day 1, trial # 1-5

16

**

12 8 Tom2WT/WT; n=8 Tom2KO/KO; n=12

4

b

20 16

0 2

3 4 Trial #

e

**

1.2 1.0 0.8 0.6 0.4 0.2 17 0.0

12 8 Tom2WT/WT; n=8 Tom2KO/KO; n=12

4

WT

21 KO

6

5

Grip strength all paws (N)

Grip strength front paws (N)

d

***

0 1

7

8 9 Trial #

10

f

*

2.5 2.0 1.5 1.0 0.5 0.0

c

Day 2, trial # 6-10

17

21

WT

KO

Learning effect on RPM (Day2-Day1)

20

Percentage of mice

Maximum RPM

a

Maximum RPM

Brain Struct Funct

2.5 2.0 1.5 1.0 0.5 7

12

WT

KO

0.0

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

Reaching attempts

0/3 1/3 2/3 3/3

WT

KO

Fig. 1 Reduced motor coordination, muscle strength and reaching reflex in Tom2KO/KO mice. a The maximally reached RPM of Tom2KO/KO mice on an accelerating rotarod was lower than Tom2WT/ WT on day 1. No significant effect of genotype on RPM increase by repeated testing was found. b Poor motor performance of Tom2KO/KO mice compared to Tom2WT/WT mice was also observed on day 2. c The rotarod learning effect was determined by subtracting the average RPM on day 1 from day 2. No differences were found.

Muscle strength was reduced in Tom2KO/KO mice, in measurements of (d) forepaws only or measurements of (e) all paws. f Reaching reflex was scored as the number of attempts out of three trials to reach for a grid. A subset of Tom2KO/KO mice performed worse than Tom2WT/WT mice on this task. n = 8 for Tom2WT/WT and n = 12 for Tom2KO/KO mice. Bars represent mean ± SEM. Significance is indicated as *p \ 0.05, **p \ 0.01, ***p \ 0.001

Tom2KO/KO mice showed reduced sensorimotor gating and impaired motor performance

coordination and motor learning on an accelerated rotarod at 3 months of age, Tom2KO/KO mice reached lower maximal RPM, both on day 1 and 2 [Fig. 1a, b; repeated measures ANOVA for genotype F(1,18) = 9.093, p = 0.007 and F(1,18) = 21.98, p \ 0.001, respectively]. The animals were capable of learning this task, since repeated testing on day 1 caused similar RPM improvements in Tom2KO/KO and Tom2WT/WT mice [Fig. 1a; repeated measures ANOVA F(4,72) = 0.693, p = 0.6]. Furthermore, the learning effect, calculated as the average score on the second day minus that of the first day, was unaffected (Fig. 1c). The impaired performance on the rotarod was accompanied by reduced grip strength [Fig. 1d, e; forepaws only: t(36) = 3.241, p \ 0.01; all paws: t(36) = 2.538, p \ 0.05]. Also, the number of reaching reflexes displayed by a mouse upon lowering it to the edge of a grid was reduced in a subset of Tom2KO/KO mice (Fig. 1f). This could involve a vision component. In the open field test, Tom2KO/KO mice traveled a larger distance [129 %, t(18) = -2.856, p = 0.01; online resource supplementary Table 1]. Of note, in this set of animals the body weight of Tom2KO/KO mice was slightly reduced [Tom2WT/WT 26.00 ± 0.40 g; Tom2KO/KO 24.33 ± 0.35 g; t(18) = 3.071, p = 0.007]. Taken together, these data

To assess the role of tomosyn-2 in the adult nervous system, we performed a broad behavioral analysis on surviving Tom2KO/KO mice. Overall, they showed normal body weight development (online resource supplementary Fig. 3a). Nesting behavior, learning and memory as well as most measures of anxiety were also normal (online resource supplementary Table 1). Moreover we assessed sensorimotor gating by pre-pulse inhibition (PPI) of an acoustic startle reflex (Swerdlow et al. 2000). Whereas the acoustic startle response upon a single stimulus was not affected (online resource supplementary Fig. 3b), indicative of normal hearing and startle reflexes, the startle response of Tom2KO/KO mice was significantly less inhibited by a pre-pulse preceding the startle stimulus (online resource supplementary Fig. 3c, d). This suggests that tomosyn-2 regulates sensorimotor gating control. Tomosyn-2 may function in motor performance by its expression in spinal cord and cerebellar granule cells (Groffen et al. 2005; Lein et al. 2007; online resource supplementary Fig. 1c). Indeed, when tested for motor

123

Brain Struct Funct

reveal a role for tomosyn-2 in the in vivo nervous system as a regulator of motor performance. Normal neuronal morphology in Tom2KO/KO mice Affected sensorimotor gating and motor performance in Tom2KO/KO mice may be caused by defective synaptic transmission (Baba et al. 2005; Chen et al. 2011; Gracheva et al. 2006; McEwen et al. 2006; Sakisaka et al. 2008; Yamamoto et al. 2010b) or impaired neurite outgrowth, wiring and general nervous system development (Kraut et al. 2001; Sakisaka et al. 2004). To investigate the latter, individual animals that exhibited motor impairments (Fig. 1) were examined for histological hallmarks of central or peripheral neurodegeneration. We did not observe any tissue abnormalities in the brain, spinal cords, muscle or optic tract of these mice, nor did we find gross deviations in other tissues (online resource supplementary Fig. 4a–d). E18 diaphragm preparations were stained with pre- and postsynaptic markers to assess development of the peripheral nervous system in more detail (Fig. 2a, b). Quantification showed that the amount (Fig. 2c) and distribution (Fig. 2d) of AChR clusters were similar in Tom2WT/WT and Tom2KO/KO animals. Also the overlap of pre- and postsynaptic puncta was normal in Tom2KO/KO diaphragm (Fig. 2e), suggesting that neuromuscular synaptogenesis was unaffected in these animals. In adult diaphragm NMJs the AChR cluster area was unaffected too (Fig. 2f, g). To further corroborate these findings, spinal cord motor neurons were taken in primary culture and their morphology was analyzed quantitatively. Similar to cultured hippocampal neurons and cerebellar granule cells, neuronal development was unaffected (online resource supplementary Fig. 4e–j; online resource supplementary Table 2). Thus, both in vivo and in vitro, tomosyn-2 was not essential for neuronal morphology or neurite outgrowth. Abnormal transmitter release at the Tom2KO/KO neuromuscular junction The motor performance impairment of Tom2KO/KO mice could be caused by aberrant neurotransmission in the motor innervation pathway. To investigate this, we recorded EPPs in Tom2KO/KO and Tom2WT/WT diaphragm muscle upon stimulation of the phrenic nerve. Spontaneous ACh release, measured as MEPP frequency, was considerably increased at Tom2KO/KO NMJs [by 66 %, t(10) = -5.551, p \ 0.001; Fig. 3a, b]. MEPP amplitude (Fig. 3b, c) and kinetics were unchanged, suggesting normal postsynaptic ACh receptor density and function. Stimulation of the phrenic nerve always evoked an EPP in all tested muscle fibers, indicating intact motor nerve innervation and proper

123

axonal action potential conduction. At low-rate (0.3 Hz) nerve stimulation, the EPP amplitude and kinetics were similar in both genotypes (Fig. 3d, e). The quantal content (i.e., number of ACh quanta released per nerve impulse) was *45 in both genotypes [t(10) = -1.250, p = 0.24; Fig. 3f]. Interestingly, when the nerve was stimulated at a higher, more physiological rate (40 Hz; Eken 1998), the EPP amplitude at Tom2WT/WT NMJs ran down to 84 % of the first EPP, while those at Tom2KO/KO NMJs decreased significantly further to 76 % [t(10) = 7.374, p \ 0.001; Fig. 3g–i]. Also paired pulse facilitation was reduced in Tom2KO/KO NMJs [12.5 ms interval t(10) = 2.983, p = 0.014; 25 ms interval t(10) = 3.002, p = 0.013; 50 ms interval t(10) = 2.911, p = 0.016; Fig. 3j, k]. The lower EPP amplitudes upon repetitive stimulation were not accompanied by changes in the frequency of MEPP events, which remained elevated in Tom2KO/KO NMJs to the same extent as before high-frequency stimulation [206 %; t(10) = -3.287, p = 0.008; Fig. 3l, m; online resource supplementary Fig. 5g]. The MEPP amplitude before and after the repetitive stimulation was similar [t(10) = 1.574, p = 0.147; Fig. 3n], indicating that the EPP rundown was not caused by receptor desensitization via putative postsynaptic tomosyn (Barak et al. 2010). Stimulation at 20 and 80 Hz yielded similar results (online resource supplementary Fig. 5). Furthermore, application of 500 mM sucrose to deplete the readily releasable pool (RRP; Rosenmund and Stevens 1996) enhanced MEPP frequency in Tom2KO/KO and Tom2WT/WT NMJs to a similar extent [t(8.234) = 0.646, p = 0.536; Fig. 3o, p]. The decreased EPP amplitudes did not affect the compound muscle action potential (CMAP) amplitude recorded in vivo with electromyography in calf muscle upon repetitive stimulation of the sciatic nerve (online resource supplementary Fig. 6). The lack of a direct effect of reduced synaptic efficacy on CMAPs is readily explained by the high fidelity of neuromuscular transmission due to a large safety margin (Wood and Slater 2001). Of note, in this small subset of mice no significant impairment of grip strength was found [Tom2WT/WT 93.3 ± 9.83 g; Tom2KO/KO 102.2 ± 6.28 g; t(10) = -0.757, p = 0.47]. Thus, tomosyn-2 deficiency leads to a combination of increased spontaneous neurotransmitter release frequency and, when the synapse is repetitively stimulated, reduced EPP amplitudes. Interestingly, this indicates a beneficial effect of basal tomosyn-2 mediated release inhibition upon sustained stimulation in the mammalian NMJ. Normal neurotransmitter release at hippocampal Tom2KO/KO synapses Transmission at hippocampal synapses has been shown to be regulated by tomosyn-1 (Sakisaka et al. 2008). To assess

Brain Struct Funct

AChR

Synapsin

Overlay

AChR

Synapsin

Overlay

Tom2WT/WT

a

17

18

WT

KO

Tom2WT/WT; n=17 Tom2KO/KO; n=18

0

10

20

30

40

50

60

70

80

n.s.

90 100 +

AChR cluster distance from midline (µm) Tom2KO/KO

AChR

Tom2WT/WT

30 25 20 15 10 5 0

AChR clusters with synapsin co-localiztion (%)

d

n.s.

e

100

n.s.

80 60 40 20 0

6

8

WT

KO

g AChR area (µm2)

f

0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00

% of all AChR clusters

c

# AChR clusters/µm midline

Tom2KO/KO

b

300 250 200 150 100 50 0

n.s.

6

6

WT

KO

Fig. 2 Normal morphology and connectivity of Tom2KO/KO diaphragm NMJs. Synapses were stained with Alexa Fluor 488 conjugated a-bungarotoxin (green) and synapsin antibody (red) in E18 diaphragm preparations from a Tom2WT/WT and b Tom2KO/KO mice. c A line was drawn through the highest cluster density to assess the amount of postsynaptic AChR clusters per lm midline. This was similar for both genotypes. d The AChR cluster distribution relative to the midline was also unaffected in Tom2KO/KO mice. e Furthermore,

AChR clusters were found to overlap with presynaptic synapsin, similar to Tom2WT/WT diaphragm. f Alexa Fluor 488 conjugated a-bungarotoxin staining was performed in the diaphragm preparations which were also investigated electrophysiologically from adult (12–15 weeks old) Tom2WT/WT and Tom2KO/KO mice. g Adult NMJ AChR area was not affected by knock-out of tomosyn-2. Bars represent mean ± SEM with the number of animals depicted in the bars

whether tomosyn-2 also regulates transmitter release at hippocampal synapses, we investigated synaptic function in cultured Tom2KO/KO hippocampal neurons. EPSCs in these neurons had a normal amplitude and kinetics

(Fig. 4a–c) and paired pulse ratios were unaffected (Fig. 4d). Frequency (Mann–Whitney U = 2,023, z = -1.939, p = 0.052, r = -0.16; Fig. 4e, f), kinetics and amplitudes of spontaneous release events were also normal.

123

1.0

Tom2WT/WT

KO

. Tom2KO/KO

EPP amplitude (% first)

.

g

n.s.

Tom2WT/WT

30 20

Tom2KO/KO

10

40 Hz

WT

j

KO

Tom2WT/WT

100 90 80

Tom2KO/KO Tom2WT/WT Tom2KO/KO

60 0 1

5

10 15 20 25 30 35

12.5 ms 25 ms

Tom2KO/KO

100 ms

Tom2WT/WT

Tom2KO/KO

123

Sucrose-enhanced MEPP frequency (/s)

p

1 mV

o

post 40Hz stimulation MEPP frequency (/s)

1 mV

Tom2WT/WT

15 10 5 0

WT

KO

***

100 75 50 25 0

WT

*

*

KO

8 7 6 5 4 3 2 1 0

*

1.00 0.95

0

10 20 30 40 50 60

pulse interval (ms)

**

WT

KO n.s.

80 70 60 50 40 30 20 10 0

WT

Tom2WT/WT Tom2KO/KO

1.05

n

m 0.5 s

1.10

50 ms

EPP#

l

20

k

5 ms

70

25

h

40

0

n.s.

30

KO

100 ms

50

10 mV

10 mV 1 ms

110

WT

Tom2KO/KO

f

Tom2WT/WT

0.0

EPP#2/EPP#1

WT

0.5

10 mV

0.0

1.0

KO

post 40Hz stimulation MEPP amplitude (% of value before stimulation)

0.5

d

EPP amplitude (mV)

100 ms

n.s.

EPP rundown level (mean 21st-35th EPP as % first)

0.5 mV

1.5

e

i

c

MEPP amplitude (mV)

b

***

2.0

Quantal content

a

MEPP frequency (/s)

Brain Struct Funct

n.s.

100

50

0

WT

KO

Brain Struct Funct b Fig. 3 Increased spontaneous ACh release and more pronounced

rundown of high-rate evoked release at Tom2KO/KO diaphragm NMJs. a Increased spontaneous MEPP frequency in Tom2KO/KO. b Representative MEPP recordings, ten superimposed traces of 1 s duration. c No change in MEPP amplitude was seen. d Similar EPP amplitudes at 0.3 Hz nerve stimulation. e Representative EPP recording traces; a black dot indicates the moment of nerve stimulation. f Similar quantal content at 0.3 Hz nerve stimulation. g Representative traces of EPP trains recorded at 40 Hz nerve stimulation. h Tom2KO/KO NMJs showed a deeper EPP rundown level, i.e., the mean amplitude of the 21st–35th EPP recorded expressed as percentage of the first EPP amplitude in the train. i More pronounced Tom2KO/KO EPP rundown at 40 Hz nerve stimulation. j Representative paired pulse recording traces at 12.5, 25 and 50 ms interstimulus interval. k Reduced paired pulse facilitation was seen in Tom2KO/KO NMJs. l Representative post 40 Hz stimulation MEPP recordings. These traces start with the ‘feet’ of the last few high-frequency stimulation-evoked EPPs. m Increased spontaneous MEPP frequency after 40 Hz stimulation in Tom2KO/KO. n Similar post 40 Hz stimulation MEPP amplitudes, normalized to pre-stimulation amplitudes, in Tom2WT/WT and Tom2KO/KO NMJs. o Representative MEPP recording traces upon 500 mM sucrose application. p Sucrose-enhanced MEPP frequency was unaffected. All data are from n = 6 mice per genotype group (10–15 NMJs sampled per mouse). Bars represent mean ± SEM. Significance is indicated as *p \ 0.05, **p \ 0.01 and ***p \ 0.001

Furthermore, EPSC amplitude depression upon prolonged high-frequency stimulation in Tom2KO/KO cells was highly similar to Tom2WT/WT cells (average Tom2KO/KO trace shown in Fig. 4g). A normal size of the RRP was observed as measured by back extrapolation during a 40 Hz train (Fig. 4h; Schneggenburger et al. 1999) and by application of 500 mM sucrose (Mann–Whitney U = 1,113, z = -1.535, p = 0.125, r = -0.15; Fig. 4j, k; Rosenmund and Stevens 1996). Recovery from these stimulation paradigms was normal too (Fig. 4i, m). Finally, vesicular release probability was normal in Tom2KO/KO neurons (Fig. 4l). Thus, despite high tomosyn-2 expression in the hippocampus (Groffen et al. 2005), Tom2KO/KO hippocampal autaptic neurons showed normal synaptic transmission, suggesting that tomosyn-2 is not required for general synaptic transmission.

Discussion Taken together, we conclude that tomosyn-2 is essential for survival in a subset of animals and contributes significantly to motor coordination in adult mice. Therefore it is a novel candidate gene for involvement in neuromuscular disorders. On the cellular level, tomosyn-2 inhibits basal ACh release from axon terminals of motor neurons and helps to avoid synaptic fatigue upon repetitive stimulation. This is in line with an inhibitory role for homologous tomosyn genes in neurotransmitter release (Baba et al. 2005; Chen et al. 2011; Gracheva et al. 2006; McEwen et al. 2006;

Sakisaka et al. 2008; Yamamoto et al. 2010b). Since the amount of transmitter released at NMJs is usually much larger than required for action potential triggering (Wood and Slater 2001), it is not surprising that the reduced EPP amplitudes observed at 40 Hz stimulation in Tom2KO/KO mice did not immediately lead to CMAP decrement. Presumably, the mild motor impairments observed in Tom2KO/KO mice were caused by a decreased safety margin, which may lead to increased failures under suboptimal conditions in vivo. Furthermore, it cannot be excluded that postsynaptic tomosyn (Barak et al. 2010) further affects motor function by a yet unknown mechanism independent of its role in synaptic transmission. All in all, it is likely that the synaptic defects contributed at least partly to the overall motor impairments. The increased spontaneous ACh release frequency in Tom2KO/KO NMJs, persisting after RRP depletion using high-frequency stimulation, suggests that tomosyn inhibits vesicular release willingness. Release willingness is defined as an intrinsic property of the vesicular fusion machinery (Lou et al. 2005), affecting the probability of basal and activity-induced vesicular release. An increased release probability causes faster depletion of the RRP during repetitive stimulation, thus providing a good explanation for the faster depression of EPP amplitudes. In line with this, paired pulse facilitation was reduced in Tom2KO/KO NMJs. Steady-state vesicle recruitment and fusion upon hypertonic shock were similar at Tom2WT/WT and Tom2KO/KO NMJs, implying that replenishment of the RRP was unaffected. Taken together, the simplest explanation for all synaptic changes is the hypothesis that tomosyn-2 inhibits the release willingness of ACh vesicles, thereby favoring sustained release at the cost of spontaneous ACh secretion. Release willingness could be mediated by tomosyn-dependent regulation of the number of mature SNARE complexes per vesicle contributing to fusion (Mohrmann et al. 2010). Also, tomosyn may induce conformational changes in SNARE complex zippering, involving layers ?1 through ?8 of the four-helical SNARE bundle (Sutton et al. 1998), since these are conserved in the secretion-enhancing yeast tomosyn homolog Sro7p (Pobbati et al. 2004). Possibly, engagement of tomosyn along the full length of the t-SNARE complex inhibits fusion, whereas disengagement of the -7 through -1 layers may promote N-terminal entry of synaptobrevin and promote fusogenic SNARE zippering (Pobbati et al. 2006; Yamamoto et al. 2010a). A third putative mechanism to mediate release willingness by tomosyn is to control SNARE complex accessibility to SNARE-interacting proteins, (e.g. complexin; Pobbati et al. 2004). Of note, the normal first EPP amplitude in Tom2KO/KO NMJs suggests that the RRP size is somewhat reduced, possibly by homeostatic compensation for the enhanced release

123

1.0

1.0 0.8 0.6 0.4 0.2 0.0

155

159

WT

KO

f

e

0.9

Tom2WT/WT

0.8

Tom2KO/KO 100 pA

Tom2WT/WT; n=93 Tom2KO/KO; n=83

0.0 0.0 0.2 0.4 0.6 0.8 1.0 Paired pulse interval (s)

1000 pA

40 Hz

0.5 s

i

40 Hz 2000

Tom2WT/WT; y=439x + 476 Tom2KO/KO; y=484x + 495

1500 1000 500

Tom2WT/WT; n=44 Tom2KO/KO; n=37

0 0.0 0.5 1.0 1.5 2.0 2.5

40 Hz

15 10 5 0

2000 1500 1000 500 45

60

0 KO

willingness. Thus, the suggested role for tomosyn in release willingness is likely to be underestimated here. Neurotransmission in cultured hippocampal Tom2KO/KO neurons was unaffected. It has been reported that tomosyn-

138

144

WT

KO

n.s.

5 4 3 2 1 64

78

WT

KO

0.2 Hz

1.2 1 0.8 0.6 0.4 0.2 0

Tom2WT/WT; n=35 Tom2KO/KO; n=26

0

10

l

20

30

40

50

m

n.s.

0.30

1.4

0.25 0.20 0.15 0.10 0.05 45

60

0

Sucrose recovery ratio

n.s.

WT

123

20

Time (s)

Vesicular release probability

1000 pA

1s

500 mM sucrose charge (pC)

k 500 mM sucrose

25

6

Time (s)

j

30

0

h Cumulative total charge (pC)

g

0.5 s

Normalized EPSC amplitude

0.7

90-10% decay time (ms)

1000 pA

Paired pulse ratio

d

40 ms

c

1.2

Spontaneous release frequency (Hz)

b

a

Normalized EPSC amplitude

Brain Struct Funct

1.2 1 0.8 0.6 0.4 0.2

39

51

WT

KO

0 WT

KO

2 expression is abundant in layer CA2 specifically (Groffen et al. 2005). Potential involvement of a specific subset of tomosyn-2 expressing hippocampal neurons in sensorimotor gating cannot be excluded. Furthermore, tomosyn-2

Brain Struct Funct b Fig. 4 Electrophysiology of Tom2KO/KO autaptic hippocampal neu-

rons was unaffected. a Average single EPSC traces show normal b EPSC amplitude and c decay time in Tom2KO/KO neurons. Other kinetic parameters of single evoked currents were also unchanged. d Paired pulse ratios at various interval times were unaffected in Tom2KO/KO neurons. e Representative spontaneous release traces are shown. f Spontaneous release frequency was unaffected in Tom2KO/KO cells. g Average trace of current released during a 2.5 s 40 Hz train stimulation in Tom2KO/KO neurons. h Cumulative charge released during the stimulation in g, with back extrapolation of the last 40 stimuli to estimate RRP size (y-axis intercept) and pool replenishment (slope), which were unaffected in Tom2KO/KO neurons. i Recovery from this 40 Hz stimulation protocol was unchanged in Tom2KO/KO neurons. j Typical responses to 500 mM sucrose as a measure of RRP size are shown (Rosenmund and Stevens 1996). k Sucrose stimulation-estimated RRP sizes were not significantly different between the genotypes, determined by the charge released until a steady state is established. l Vesicular release probability was unaffected, calculated from the sucrose-evoked charge and the charge of a single EPSC. m Recovery of the vesicular pool released upon sucrose stimulation was assessed by paired sucrose application with a 4 s interval. Bars represent means ± SEM with the number of cells depicted in the bars

expression in many other brain regions may affect sensorimotor gating control (Swerdlow et al. 2000). Clearly, the lack of tomosyn-2 does not cause a general defect in synaptic transmission, possibly by (partial) redundancy with tomosyn-1. In tomosyn-1-deficient mice, hippocampal mossy fiber short-term plasticity and the early stage of long-term potentiation are also affected (Sakisaka et al. 2008). Our observations in mutant mice indicate that tomosyn-2 regulates synaptic strength at motor endplates. Restriction of release willingness could thus be a conserved mechanism for both tomosyn isoforms, particularly important in heavyduty synapses where efficient release during high-frequency firing is important (Mistry et al. 2011; Wood and Slater 2001). Acknowledgments For excellent technical support we would like to thank Jurjen Broeke, Niels Cornelisse, Joost Hoetjes, Hilde Hopman, Hans Lodder, Rolinka van der Loo, Chris van der Meer, Frank den Oudsten, Desiree Schut, Sabine Spijker, Aafje Vossenaar, Ruud Wijnands, Joke Wortel at the VU and VUmc, as well as staff of the AvL laboratory for Experimental Animal Pathology. We thank Annelies van der Laan and Joop Wiegant for excellent help with laser scanning confocal microscopy at the microscopy facility of the Molecular Cell Biology Department of the LUMC. This study was supported by the EU Eurospin project Health-F2-2009-241498, Synsys project HealthF2-2009-242167 and CMSB2 project 3.3.5.

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