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Ca2+ release from internal stores is critical for mediating both normal and pathological intracellular Ca2+ signaling. Recent studies suggest that the inositol 1,4,5-triphosphate (IP3) receptor mediates Ca2+ release from internal stores upon cholinergic activation of the neuromuscular junction (NMJ) in both physiological and pathological conditions. Here, we report that the type I IP3 receptor (IP3R1)-mediated Ca2+ release plays a crucial role in synaptic gene expression, development, and neuromuscular transmission, as well as mediating degeneration during excessive cholinergic activation. We found that IP3R1-mediated Ca2+ release plays a key role in early development of the NMJ, homeostatic regulation of neuromuscular transmission, and synaptic gene expression. Reducing IP3R1-mediated Ca2+ release via siRNA knockdown or IP3R blockers in C2C12 cells decreased calpain activity and prevented agonist-induced acetylcholine receptor (AChR) cluster dispersal. In fully developed NMJ in adult muscle, IP3R1 knockdown or blockade effectively increased synaptic strength at presynaptic and postsynaptic sites by increasing both quantal release and expression of AChR subunits and other NMJ-specific genes in a pattern resembling muscle denervation. Moreover, in two mouse models of cholinergic overactivity and NMJ Ca2+ overload, anti-cholinesterase toxicity and the slow-channel myasthenic syndrome (SCS), IP3R1 knockdown eliminated NMJ Ca2+ overload, pathological activation of calpain and caspase proteases, and markers of DNA damage at subsynaptic nuclei, and improved both neuromuscular transmission and clinical measures of motor function. Thus, blockade or genetic silencing of muscle IP3R1 may be an effective and well tolerated therapeutic strategy in SCS and other conditions of excitotoxicity or Ca2+ overload.
Dysregulation of Ca2+ release from internal stores is increasingly recognized as a potential cause of neuronal death or neurodegeneration (Demuro et al., 2010; Bezprozvanny, 2011). Therefore, one potential therapeutic strategy to prevent pathological elevations of Ca2+ may be pharmacological inhibition of internal Ca2+ release, provided that the effect on normal physiological signaling is tolerated. For example, malignant hyperthermia, a condition of ryanodine receptor overactivity and pathological elevation of muscle Ca2+, can be successfully treated by pharmacological blockade of the ryanodine receptor (Kobayashi et al., 2009).
The inositol 1,4,5-triphosphate (IP3) receptor (IP3R) channel initiates a slow wave of internal store Ca2+ release, often at specific subcellular locations, that allows compartmentalization of Ca2+ signals within cells (Marks, 1997). IP3Rs are gated by the combined binding of IP3 generated by the hydrolysis of phospholipids by phospholipase C and by Ca2+, which enters through AChR or voltage-gated channels (Streb et al., 1983; Berridge and Irvine, 1984). Type 1 (IP3R1) is the most abundant and best studied of the three subtypes of IP3R. The gene encoding IP3R1, itpr1, is highly expressed in cerebellar Purkinje neurons and cardiac and skeletal muscle and is highly concentrated at the neuromuscular junction (NMJ) and the subsynaptic nuclei (Furuichi et al., 1990; Ross et al., 1992; Powell et al., 2003).
The discrete localization of IP3R1 near the acetylcholine (ACh) receptors (AChRs) and in postsynaptic areas surrounding NMJ nuclei is striking and suggests a role for IP3R1-mediated Ca2+ release in synaptic gene expression and synaptic physiology in muscle (Powell et al., 2003). Focal iontophoresis of ACh onto endplates of dissociated, fura-2-loaded muscle fibers induces localized increases in NMJ Ca2+ that depend on functioning IP3Rs (Zayas et al., 2007). Therefore, because IP3Rs amplify postsynaptic Ca2+ signals at NMJs and because Ca2+ is critical for postsynaptic changes leading to early development and maturation of the NMJ, we postulate that IP3R1s are required to mediate the effects of cholinergic input on NMJ development *and function (Zhu and Peng, 1988; Megeath and Fallon, 1998).
Ca2+ also mediates the effects of cholinergic excess at the NMJ, such as in myopathy caused by excessive anti-cholinesterase medication (agonist myopathy), congenital myasthenic syndromes, and insecticide poisoning (Kawabuchi, 1982; Engel et al., 2010). The slow-channel congenital myasthenic syndrome (SCS) is caused by mutations in the muscle AChR channel leading to Ca2+ overload and degeneration of the NMJ (Gomez et al., 2002). Elevated postsynaptic Ca2+ in SCS requires entry of Ca2+ through ACh-activated mutant AChR channels and release of Ca2+ from IP3-gated stores (Zayas et al., 2007). We hypothesized that IP3R1 mediates developmental and physiological events localized to the NMJ, as well as pathological sequelae from cholinergic excess at the NMJ. We show that IP3R1s are required to mediate cholinergic signals responsible for AChR cluster dispersal, homeostatic plasticity, and synaptic gene expression at the NMJ. However, blockade of IP3R or specific knockdown of IP3R1 in adult muscle prevents the excitotoxic effects of cholinergic excess and pathological synaptic Ca2+ overload without impacting normal NMJ function. Thus, a therapeutic strategy targeted to block muscle IP3R1 is likely to be well tolerated.
Materials and Methods
Chemical reagents were purchased from Sigma. Cell culture materials were obtained from Invitrogen . Laminin, alpha-bungarotoxin (αBT)-conjugated Texas Red (TxR-αBT), and fluorescence-tagged secondary antibody were purchased from Invitrogen. Secondary antibodies with horseradish peroxidase (HRP) were provided by GE Healthcare Bioscience. GFP-tagged control siRNA was purchased from Santa Cruz Biotechnology Primary antibodies were used against the following targets: neural filament (1:1000; Abcam), phosphorylated H2AX (Ser139; 1:500; Millipore), cleaved caspase 3 and 9 (1:200;Cell Signaling Technology), synaptophysin (1:500; Cell Signaling Technology), S100 (1:100; Abcam), histone deacetylase 4 (HDAC4; 1:500; Abcam), glial fibrillary acidic protein (1:500; Cell Signaling Technology), fibronectin (1:500; Abcam), IP3R1 (1:500; Alomone), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5000; Ambion).
Plasmid and construct assembly.
Mouse INPP5a (inositol polyphosphate-5-phosphatase) cDNA, purchased from Thermo Scientific (Open Biosystems), was cloned into pEGFP-N1 (Clontech).
Cell culture.
C2C12 cells purchased from American Tissue Culture Collection (Manassas, VA) were cultured and differentiated on poly-l-lysine and laminin-coated plates as described previously (Sugiyama et al., 1997). After 72–96 h, C2C12 cells were incubated with calcium (Ca2+; 100 μm), carbachol (CCh; 100 μm), 2-aminoethoxydiphenyl borate (2-APB; 100 μm), or xestospongin C (XS; 10 μm) for 15 min, washed 3× with culture medium, and then incubated at 37°C and sampled for fluorescence at intervals up to 120 min. Cover slides were washed and prepared for cytofluorescence. Cells were transfected with siRNA and scrambled RNA (ctRNA) segments using lipofection (Lipofectamine 2000, Invitrogen).
Animals and tissue preparation.
Four- to six-month-old male wild-type FVB mice and SCS transgenic mice (εL269F) were used in this study (Gomez et al., 1997). All drug treatments and surgical procedures followed the animal care and use protocols established by the Institutional Animal Care and Use Committee (University of Chicago, Chicago, IL). Mice were anesthetized using ketamine and xylazine (Mulder and Mulder, 1979). For denervation, hind limbs of anesthetized mice were shaved to expose skin over the sciatic nerve. Segments of the sciatic nerve (1 cm) were excised, and the two free nerve trunks were physically separated within the muscle before wounds were closed with stainless steel wound clips.
Drug administration.
Neostigmine methylsulfate and 2-APB in saline (USP) were administered intraperitoneally at 250 μg/kg for 36 h and 10 mg/kg/8 h for 9 d, respectively (Carlson and Kriebel, 1985; Bootman et al., 2002). XS (5 nm) in DMSO was injected intraperitoneally into mice every 2 h for 12 h (Galeotti et al., 2006). αBT (1 mg/ml; Tocris Bioscience) in saline was injected intraperitoneally at a dose of 0.5 μg/d for 6 d (Holland and Brown, 1980; Domet et al., 1995). Control tibialis anterior (TA) muscle was treated with DMSO or saline (0.9% w/v; USP) as control.
RNA extraction, cDNA synthesis, and real-time PCR.
TA muscle was homogenized in tri-reagent (Sigma) to extract total RNA, which was used (1 μg) to synthesize cDNA (First-Strand Synthesis Kit; Invitrogen). Assay conditions for real-time PCR were adjusted for each single target using melt curve analysis and gel electrophoresis with specific primers (Table 1) and SYBR Green (Bio-Rad). To minimize variation, all sequences of interest were amplified in parallel from one aliquot of cDNA on the same plate. Real-time PCR was performed on iCycler iQ Real-Time PCR Detection System (Bio-Rad) following the manufacturer's instructions, with GAPDH used as a housekeeping gene. The mean value from triplicate samples generated from the same RNA preparation was used to determine the real-time PCR efficiency (dilution rates: 1:3; 1:9; 1:27; 1:81; 1:243; 1:729) for each gene with the slope of a linear regression model. The data with efficiency (≥98.5%) were selected as valid results.
Western blotting.
Tissue was homogenized in 50 mm Tri-HCl, pH 7.6, 150 mm NaCl, 1% SDS, 1% Triton X-100, 0.1 mm EGTA, and 0.1 mm EDTA supplemented with a (Roche Protease Inhibitor Cocktail. Homogenates were fractionated by SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad). Membranes were blocked for 1 h in TBS-T (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, and 0.05%Tween-20) containing 5% nonfat milk and then incubated overnight with primary antibodies. After 3× wash in TBS-T, the membrane was incubated with HRP-conjugated secondary antibody for 1 h and washed again 3×. Immunolabeled bands were developed using ECL reagents (Ambion) on x-ray film. Reaction product levels were quantified by scanning densitometry using NIH ImageJ (version 5.3).
Cell and tissue staining and imaging.
For cytofluorescence, cells grown on coverslips were fixed in methanol-acetone (−20°C × 20 min) and blocked with blocking solution (3% normal goat serum, 0.3% Triton X-100, 1 h at room temperature). TxR-αBT (1:500; Molecular Probes) was used to detect AChRs. Motor endplates were localized and Ca2+-overloaded endplates were detected in cryosections using histochemical stains for cholinesterase and Ca2+ [glyoxal-bis(2-hydroxyanil) (GBHA)] as described previously (Koelle and Friedenwald, 1949; Kashiwa and House, 1964). For immunohistochemistry, tissue sections were fixed by methanol-acetone at −20°C for 30 min, air dried for 20 min, and incubated with blocking solution for 1 h at room temperature, followed by overnight incubation with primary antibody at 4°C. After washing, the sections were incubated with fluorescent secondary antibody in PBS-T (phosphate buffered saline and 0.05% Tween 20) for 1 h at room temperature. Confocal fluorescence microscopy was carried out under a TCS laser scanning microscope (Leica). Optical sections of 0.5 μm were scanned at the z-axis. ImageJ was used to quantify the percentage of labeling by specific antibody in NMJs of each sample.
Quantitative studies in tissue sections.
Quantitation of the proportion of labeled NMJs in limb muscle cryosections was carried out using sequential alternate sections stained for cholinesterase or test labeled (GBHA, pH2X, and HDAC4) as described previously (Gomez et al., 1997, 2002) employing ImageJ (version 5.3).
Calpain and caspase activity assay.
Calpain, caspase-3, and caspase-9 activities in muscle or cell homogenates were measured using a firefly luciferase-based assay (Calpain-Glo Protease Assay, Caspase-Glo 3/7 Assay, or Caspase-Glo 9 Assay; Promega). Cell and muscle tissue were homogenized as described previously (Lee et al., 2008), except for the addition of 10 mm NH4Cl and 10 mm 3-methyladenine to the homogenization buffer to stabilize lysosomes and the proteosome. Protein samples (20 μg) were analyzed by luminometer (Turner BioSystems) in triplicate.
Intramuscular electroporation-mediated gene delivery.
Electroporation of plasmid and siRNA were carried out as described previously (Schertzer et al., 2006). Briefly, after anesthesia the legs were shaved and the TA muscles were injected with hyaluronidase (30 μl; Sigma-Aldrich) 2 h before performing electroporation. Subsequently, 10 μg of optimal siRNA segments, control RNA, or expression plasmids in saline were injected into the TA muscle. Immediately following injection, 10 separate electrical pulses (10 ms) were delivered through circle electrodes (1 cm diameter) applied to the ventral and dorsal surfaces flanking the TA at 175 V/cm with 1 Hz via a square-wave electroporator (ECM830; Harvard Apparatus).
Electromyography and in vitro electrophysiology.
Compound muscle action potentials (CMAPs) were detected and calculated as described previously using a Nicolet VikingQuest (Nicolet Biomedical) (Groshong et al., 2007).
Voltage-clamp studies.
All experiments were carried out in vitro at room temperature on the sciatic nerve and tibialis muscle preparations of 5–6 week-old mice. For dissection, mice were deeply anesthetized with isofluorane. The tibialis anterior muscle was dissected free from the extensor digitorum longus (EDL) muscle, partially bisected, and folded apart to flatten the muscle before pinning it down. Because the sciatic nerve innervates both EDL and TA muscles, we transected the EDL and posterior portion of the TA muscle to eliminate excessive muscle contraction. To prevent muscle action potentials, μ-conotoxin GIIIB (2 μm) was included in some experiments.
The TA muscle was perfused with Tyrode's solution (137 mm NaCl, 2.8 mm KCl, 1.8 mm CaCl2, 1.1 mm MgCl2, 11.9 mm NaHCO3, 0.33 mm NaH2PO4, and 11.2 mm dextrose, pH 7.4, when bubbled with a mixture of 95% O2 and 5% CO2) at 30°C. Intracellular potentials and currents were measured using an Axoclamp-2A amplifier system and glass microelectrodes filled with 3 m KCl (resistance, 10–15 MΩ). Using the two-microelectrode voltage-clamp system, synaptic currents, including miniature endplate currents (MEPCs) and endplate currents (EPCs), were obtained at −70 mV holding potential. The currents were digitized at 50 μs per point and stored, captured, and analyzed using pClamp9 software (Molecular Devices). EPCs were elicited by stimulating the corresponding sciatic nerve with rectangular pulses of 0.05 ms duration. During single shock stimulation, the nerve was stimulated at 0.2 Hz. The amplitude decay and time to peak of the EPCs and MEPCs were captured and subsequently analyzed with pClamp 9.2 and the Mini Analysis Program (Synaptosoft). Quantal content was determined by dividing MEPC amplitude with EPC amplitude from the same endplate (Bhattacharyya et al., 1997; Wilson et al., 2002; Groshong et al., 2007).
Treadmill gait analysis.
Mouse gait analysis was performed via the DigiGait Imaging System (Mouse Specifics) as described previously (Hampton et al., 2004; Gómez-Sintes and Lucas, 2010). Briefly, mice walked on a motor-driven treadmill with a transparent treadmill belt. A high frame rate camera was focused on the ventral plane of the mice as they walked within an acrylic chambe, ∼5 cm wide by ∼25 cm long. The areas of the approaching or retreating paws relative to the belt and camera were calculated throughout each stride. Plotting the area of each paw in time provides a dynamic gait signal, representing the temporal record of paw placement relative to the treadmill belt. Each gait signal for each limb comprises a stride duration (stride time), which includes the stance duration when the paw of a limb was in contact with the treadmill, plus the swing duration when the paw of the same limb was not in contact with the treadmill belt. Stance duration is comprised of a braking time (increasing paw contact area over time) and propulsion time (decreasing paw contact area over time). Stride frequency was calculated by counting the number of gait signals over time. Stride length was calculated from the equation: speed = stride frequency × stride length. Due to the remarkable reduction of muscle strength in SCS mice, brake time was primarily selected as the most sensitive metric to demonstrate functional improvement of treated muscle in SCS mice. The walking speed was set to 25 cm/s. Approximately 5 s of video were collected for each walking subject to provide ∼20 sequential strides.
Values were represented as the mean ± SD. Data were analyzed using a Student's t test, where appropriate, and a nonparametric Mann–Whitney U test, for comparison of percentages and ratio values, as indicated in figure legends.
IP3R1 mediates cholinergic agonist-induced dispersal of AChR clusters in myogenic cells
In normal muscle cells and C2C12 myogenic cells, stimulation of AChRs with cholinergic agonists causes a transient increase in cytoplasmic Ca2+ levels and dispersal of agrin- or laminin-induced AChR clusters in a process that requires activation of the calcium-activated protease, calpain (Chen et al., 2007). Although fetal AChRs are permeable to Ca2+, calpain remains activated well beyond the interval anticipated for AChR gating, suggesting that the Ca2+ arises from another source (Chen et al., 2007). Because of the proximity of IP3R1 to the mature NMJ, we tested whether persistent calpain activation required release of Ca2+ through IP3Rs in C2C12 cells expressing laminin-induced AChR clusters (Weston et al., 2007). First, we applied the small interference RNA technique to selectively knock down IP3R1, the most abundant IP3R1 in skeletal muscle (Wells, 2004). We compared the effect of six siRNA segments alone or in pairs (see Materials and Methods). We found that two segments, S1 and S2, together resulted in ∼95% suppression of IP3R1 protein by day 3 after transfection of cultures (Fig. 1A, left) with marked reduction of IP3R1-specific immunofluorescence in C2C12 cells (Fig. 1A, right). In our hands the time course of calpain activation in C2C12 cells upon exposure to the cholinergic agonist, carbachol or CCh, was similar to that reported previously (Chen et al., 2007). Calpain levels remained constant in the absence of CCh but began to increase minutes after exposure, reaching a peak twice the basal level at 1 h (Fig. 1B). In the presence of 100 μm Ca2+ added to the medium, the peak calpain reached 2.8 times over baseline during the same time course. The increase in calpain activity induced by CCh was completely blocked by either XS (xestospongin C) or 2-APB (2-aminoethoxydiphenyl borate), both blockers of the IP3 receptor, without apparent effect on viability (Fig. 1B). siRNA knockdown of IP3R1 in C2C12 cells for 3 d caused a slight reduction in the basal activity of calpain without CCh treatment (Fig. 1B). siRNA-mediated IP3R1 knockdown had a nearly identical effect as 2-APB and XS in preventing CCh-induced calpain increases, while control RNA had no effect (Fig. 1B). This indicates that calpain elevation depends on the presence of intact, functioning IP3R1 in muscle cells.
Since calpain is implicated in AChR dispersal in myoblasts, we explored the role of IP3R1-derived Ca2+ release in this early synaptic developmental event. C2C12 induced to form AChR clusters on laminin was incubated with CCh as described above. AChR clusters were enumerated at intervals up to 2 h after labeling with TxR-αBT. As noted previously, exposure to CCh accelerates the rate of dispersal of AChR clusters in C2C12 beginning within 15 min, particularly in the presence of increased extracellular Ca2+ (Fig. 1C) (Chen et al., 2007). However, when C2C12 were treated with either XS or 2-APB, AChR cluster dispersal was largely prevented (Fig. 1C). Furthermore, we show that IP3R1 silencing had an equivalent effect on preventing AChR cluster dispersal as the drug treatment while control RNA had no effect, indicating a specificity for type 1 IP3R (Fig. 1C). Thus, agonist-induced AChR cluster dispersal, an early event in neuromuscular development, requires the participation of IP3R1, presumably for amplification of internal Ca2+ levels and activation of cytosolic calpain.
Muscle IP3R1 activity influences presynaptic and postsynaptic neuromuscular transmission and presynaptic vesicle recycling in NMJ
Many synapses exhibit the property of homeostatic plasticity, in which reductions in synaptic activity lead to compensatory increases in presynaptic strength, usually observed as an increase in transmitter release (Murthy et al., 2001). In the NMJ, partial blockade of neuromuscular activity by TTX or endplate AChRs by αBT for durations of 8–14 d changes the activity of the presynaptic neuron. TTX blockade leads to an increase in quantal size and probability of release, as well as increased postsynaptic ACh sensitivity. αBT blockade increases quantal content by increasing the number of releasable vesicles, suggesting that the NMJ is governed by tightly controlled homeostatic plasticity (Plomp et al., 1992; Wang et al., 2010). We hypothesized that the effect of cholinergic input in eliciting homeostatic plasticity is mediated through the skeletal muscle IP3R1 receptor. We reasoned that systemic pharmacologic blockade of IP3Rs was likely to have nonspecific effects in an electrophysiological study. Therefore, we set up a system for subacute silencing of IP3R1 in vivo by directly electroporating IP3R1-specific siRNA into TA muscle (Golzio et al., 2005). Preliminary experiments using a GFP reporter plasmid and siRNA specific for the GFP showed that this procedure achieved expression of GFP in 80–90% of muscle fibers (Fig. 2A), and siRNA specific for GFP reduced levels of overexpressed GFP activity to 16% of control after 5 d (Fig. 2A). For in vivo silencing of IP3R1, we employed the same siRNA sequences applied for the C2C12 cells. We found that the IP3R1 levels were decreased to ≤15% of the level seen in untreated TA between days 7–9 postelectroporation and returned to normal levels at day 14 after electroporation of IP3R1-specific siRNAs (Fig. 2B). Immunostaining for IP3R showed that IP3R1 silencing had a pronounced effect on IP3R expression at NMJs. NMJs in both untreated and control RNA-treated muscles had intense labeling of IP3Rs colocalized with AChRs and subsynaptic nuclei (Fig. 2C), while NMJs in siRNA-treated muscle had greatly reduced immunostaining of IP3Rs at both AChRs and subsynaptic nuclei. Figure 2D shows that IP3R silencing by siRNA electroporation was specific for muscle and NMJ. Compared with Figure 4C, IP3R1 did not colocalize with nerve termini labeled for synaptophysin (Fig. 2D, far left panel) or Schwann cell nuclei labeled with S100 (Fig. 2D, middle left panel). We also used GFP-labeled control siRNA to demonstrate the distribution of electroporated RNA in muscle. We found that siRNA was mainly located in the subsarcolemmal region, including NMJs, whereas no siRNA colocalized with Schwann cells marked by S100 protein (Fig. 2D, middle right panel) but did overlap with the endplate regions (Fig. 2D, far right panel). Silencing of IP3R1 had no effect on levels of expression of other muscle proteins, such as RyR1 and L-type calcium channel alpha 1 subunit, at 9 d (data not shown).
We studied the effect of IP3R1 knockdown on neuromuscular transmission in TA muscle pretreated 9 d earlier with IP3R1-siRNA by using the two-electrode voltage-clamp technique. We recorded both spontaneous MEPCs (miniature endplate currents) and EPCs (endplate currents) evoked by direct stimulation of the attached sciatic nerve and characterized both the presynaptic and postsynaptic components of neuromuscular transmission (Ikeda et al., 1984). Figure 3A (left) shows raw data tracings of spontaneous activity for TA muscle treated 9 d previously with ctRNA (scrambled RNA) or IP3R1-siRNA.
Figure 3, B and C, shows that both the amplitude (3.86 ± 0.32 nA vs 2.53 ± 0.29 nA, p < 0.05) and decay phase of MEPCs (1.5 ± 0.08 ms vs 1.14 ± 0.15 ms, p < 0.05) were significantly increased in TA muscle treated with siRNA-IP3R1 (by 52 and 31%, respectively), relative to contralateral control-treated TA. MEPC parameters in control-treated TA were no different from untreated muscle. These findings indicate that IP3R1 plays an important role in modulating the postsynaptic component of neuromuscular transmission. In addition, the frequency of MEPCs (3.11 ± 0.26 s−1 vs 1.4 ± 0.09 s−1, p < 0.001; Fig. 3A, right) more than doubled, suggesting that IP3R1 has an additional role in modulating the presynaptic component of transmission, i.e., on release of neurotransmitter from the presynaptic sites. These combined presynaptic and postsynaptic changes were accompanied by an ∼2-fold increase in the amplitude of the EPC (endplate current) evoked by sciatic nerve stimulation (122.49 ± 12.92 vs 50.21 ± 5.9 nA, p < 0.001; Fig. 3D). Finally, the quantal content (34.2 ± 1.8 vs 26.08 ± 3.96, p < 0.05) increased by 30% (Fig. 3E). These observations are similar to those seen in the TTX and αBT partial blockade models and suggest that IP3R1-mediated Ca2+ release from internal stores in muscle at the NMJ mediates the action of cholinergic input on both presynaptic and postsynaptic aspects of homeostatic plasticity (Wang et al., 2005, 2010).
IP3R1 activity controls synaptic gene expression in muscle
The increase in amplitudes and decay phases of MEPCs induced by IP3R1 silencing suggests that IP3R1 signaling is involved in AChR expression. To test this, we first compared AChR expression by measuring fluorescence intensity of endplates labeled with TxR-αBT in muscle treated by denervation, which is known to increase AChR expression, or by siRNA IP3R1 knockdown. We found that IP3R1 knockdown in TA muscle was associated with a 1.8-fold increase in endplate AChR intensity, a smaller, but similar effect on AChR expression as that seen with denervation (Tsay and Schmidt, 1989; Akaaboune et al., 1999) (Fig. 4A). The change in AChR expression is in good agreement with the 1.5-fold increase in MEPC amplitudes induced by IP3R1 silencing (Fig. 3B).
Since cholinergic input influences both synaptic currents and contractile (electrical) activity, it is difficult to separate the differential effects of these two variables and their modes of coupling to gene expression. Studies have pointed to a role for the L-type Ca2+ channel as the principal intracellular Ca2+ source for signals coupled to electrical activity (Friedrich et al., 2008). Based on the striking colocalization of IP3R1 with AChRs and subsynaptic nuclei at the NMJ, we hypothesized that this channel is a source of internal Ca2+ involved in regulating synaptic muscle gene expression. We studied the effect of IP3R1 silencing on expression of a panel of NMJ and muscle-specific genes. We first investigated the time course of AChR gene expression between 3 and 14 d after knockdown, an interval that spans the duration of the effect of a single siRNA electroporation on IP3R1 expression, and then used quantitative RT-PCR to compare this effect to that of denervation.
In siRNA-treated muscle, expression levels of both α and γ subunit genes began to increase steadily 3 d after siRNA knockdown until reaching peak levels of >10-fold and >60-fold over baseline, respectively, after 9 d (Fig. 4B, right). The sharp peak and decline in AChR gene expression after IP3R1 knockdown in muscle were coincident with the time course of IP3R1 silencing (Fig. 2B). The increase in δ gene expression was more modest, peaking at day 9 (Fig. 4C). IP3R1 had a small but significant effect on ε subunit expression over the same time course as the δ subunit (Fig. 4C). Control RNA had no effect on AChR subunit gene expression. In contrast, as reported previously, after denervation α, γ, and δ subunit expression increased immediately after 3 d and remained significantly elevated for at least 9 d (Tsay and Schmidt, 1989; Akaaboune et al., 1999) (Fig. 4B, left and right). The increases after denervation were of greater magnitude, but the differential effects on α, γ, and δ subunit expression were comparable. However, as reported previously, denervation had little effect on ε subunit expression, particularly at day 3, when expression of the other subunits increases (Fig. 4C) (Martinou and Merlie, 1991). The increases in AChR subunit gene expression, including the appearance of the (embryonic) γ subunit expression (slower channel kinetics), are consistent with the finding that IP3R1 knockdown increased MEPC amplitudes and MEPC decay phase duration (Fig. 3B,C). These studies suggest that the effect of IP3R1 may resemble the effect of denervation on AChR gene expression, although differences in the control of ε subunit expression may point to distinct influences on synaptic genes.
To test whether IP3R1 silencing influences a wider array of NMJ and muscle-specific genes similar to the role of cholinergic input, we compared the effect of muscle IP3R1 silencing (day 9) with the effect of denervation (day 3) on the expression of a panel of other muscle-specific genes known to be influenced by cholinergic activity. Figure 4C showed that, like denervation, IP3R1 silencing led to increased expression of rapsyn and the neuregulin receptor ErbB2. Curiously, as with the AChR genes, the effect of IP3R1 silencing on rapsyn was of smaller magnitude than denervation, but the effect of IP3R1 silencing on ErbB2 expression was >2-fold that of denervation. Moreover, IP3R1 silencing did not alter muscle-specific receptor tyrosine kinase (MuSK) or acetylcholinesterase (AChE) expression, while denervation increased MuSK and decreases AChE expression. For more widely expressed genes, IP3R1 silencing had no effect on extrajunctional muscle genes such as the glucose transporters glut1 and glut4, while denervation increased expression of glut1 and decreased expression of glut4 (Fig. 4D) (Jones et al., 1998). These findings indicate that IP3R1 activity has a distinct influence on the expression of synapse-specific genes.
Modulation of some NMJ-localized genes and other extrajunctional muscle genes by denervation, but not by IP3R1 silencing, is in agreement with observations that expression of these genes is under control of multiple factors, including electrical activity, and peptide modulators such as neuregulin and agrin (Lømo et al., 1985; Goldman et al., 1988; Tsay and Schmidt, 1989; Yang et al., 1991; Castelló et al., 1993; Jones et al., 1998; Moore et al., 2001; Lebrasseur et al., 2003). The fact that IP3R1 silencing, which leaves muscle innervation and contraction intact, also increases expression of some NMJ genes, particularly ErbB2, and selectively increases expression of the ε subunit, suggests that there are additional overlapping factors that influence synaptic gene expression (Lebrasseur et al., 2003; Sunesen and Changeux, 2003). Therefore, we explored the effect of combined IP3R1 silencing and denervation. Nine days before harvesting muscle, the TA was electroporated with siRNA for IP3R1 or control RNA. Three days before harvest, TA muscles were denervated. Figure 4, C and D, compares the effect of denervation alone, of IP3R1 silencing alone and of denervation following IP3R1 silencing for 9 d on muscle gene expression. IP3R1 silencing had no greater effect on expression of glut1 and glut4 even when combined with denervation. However, we found that expression of both the δ subunit and rapsyn, which were increased by IP3R1 silencing alone, were further increased by denervation, greater than by denervation alone. In addition, while IP3R1 silencing had no effect on expression of MuSK or AChE, the combined effect of IP3R1 silencing and denervation significantly increased MuSK expression and eliminated the effect of denervation on AChE expression. More importantly, while the effect of IP3R1 silencing was to increase expression of ε subunit and that of ErbB2 greater than the effect of denervation, the combined effect of siRNA and denervation was significantly greater than the effect of IP3R1 silencing or denervation alone. One interpretation of these findings is that IP3R1 activity, and presumably synaptic Ca2+ fluxes, exert distinct localized control on NMJ gene expression.
To investigate whether overlapping influences on muscle gene expression between cholinergic input and IP3R1 activity can be explained by changes in transcription or protein expression, we studied the expression of the HLH (helix-loop-helix) family of transcription factors involved in muscle gene expression. We found that both denervation and IP3R1 silencing led to increases in myogenin and HDAC4 and reduced expression of Dach-2 within muscle fibers (Fig. 4E), suggesting that both treatments equally influenced expression of these transcription factors at the gene level. These findings are consistent with the observation that many downstream muscle genes are influenced by both denervation and IP3R1 silencing. We also detected expression of this panel of genes in muscle with 2-APB-mediated blockade of IP3R and selective elimination of muscle IP3 by INPP5a, and the results were similar as with IP3R1 silencing (data not shown).
Changes in skeletal muscle gene expression mediated by muscle electrical activity and Ca2+ fluxes are partly regulated by the CaMKII-dependent nucleocytoplasmic shifts of the transcription protein HDAC4 and direct binding and reciprocal regulation of MEF2 transcription factor activity (Tang et al., 2004; Cohen et al., 2007). Because IP3R1 mediates localized Ca2+ fluxes at the NMJ that can activate CaMKII and phosphorylate HDAC4, we compared the effect of denervation, known to increase myonuclear translocation of HDAC4, with the effect of siRNA silencing of IP3R1 in vivo on myonuclear translocation of HDAC4. Denervation of TA muscle was associated with a 3-fold increase in the proportion of extrajunctional myonuclei labeling for HDAC4, from 2.34 ± 0.3% in innervated muscle to 7.46 ± 0.3% after denervation (HDAC-EN; Fig. 4F; n = 3, p < 0.05). Looking specifically at subsynaptic nuclei in TA muscle fibers, we found that denervation caused a 4-fold shift in the number of HDAC4-positive subsynaptic nuclei (10.88 ± 0.8%, HDAC4-SN; n = 3, p < 0.05) compared with innervated TA muscle (2.56 ± 0.2%; Fig. 4F). In comparison, siRNA-IP3R1 treatment of TA muscle caused a nearly 3-fold increase (7.36 ± 0.3%; n = 3, p < 0.05) in HDAC4-positive subsynaptic nuclei but had no effect on HDAC-positive extrajunctional nuclei (2.33 ± 0.2%, HDAC4-EN; Fig. 4F; n = 3, p > 0.87). Control RNA had no effect on HDAC4 labeling of TA muscle nuclei in either compartment (Fig. 4F). The changes in NMJ nuclear expression of HDAC4 are highly consistent with the selective changes in NMJ gene expression and suggest that Ca2+ released through IP3R1 at the NMJ acts via synaptic myonuclear HDAC4 to regulate NMJ-specific gene expression. The results point to a role for IP3R1 in targeting the synaptic effects of cholinergic input to mediate localized gene expression through an HDAC4 mechanism.
IP3R1 silencing prevents pathological Ca2+ overload, cysteine protease activation, and degeneration of NMJs
Numerous pathological processes, such as seizure activity, excitotoxicity, organophosphorus poisoning, or excessive anti-cholinesterase medication, lead to deleterious increases in cytoplasmic Ca2+, synaptic degeneration, and neuronal death (Ariens et al., 1968; Choi, 1992, 1995; Kobayashi et al., 2009; Thiermann et al., 2009). Agonist myopathy is an acute model of cholinergic excitotoxicity induced by excessive inhibition of anti-cholinesterases as used in therapy for myasthenia gravis (Leonard and Salpeter, 1979; Kawabuchi, 1982). It is associated with pathological accumulation of Ca2+ and degeneration of the NMJ. SCS is a genetic disorder in which mutations in the AChR cause prolonged AChR gating and overload of the NMJ with Ca2+, resulting in degeneration of the NMJ (Engel et al., 1996; Gomez et al., 1997, 1998). In a mouse model for SCS in which the clinical, pathological, and electrophysiological features of the disease are reproduced by muscle expression of the AChR subunit bearing SCS mutations such as εL269F, fura-2 imaging studies suggested that Ca2+ overload in this disorder depends on IP3 receptor-gated stores rather than Ca2+ entry through L-type channels or ryanodine receptors (Gomez et al., 1997; Zayas et al., 2007).
We hypothesized that IP3R blockade or selective reduction of IP3R1 by siRNA knockdown in vivo would prevent pathological accumulation of postsynaptic Ca2+ at the NMJ. TA muscles of wild-type (WT) mice were pretreated by electroporation with siRNA-IP3R1 or control RNA in contralateral hind limbs. Nine days later mice were injected intraperitoneally with the cholinesterase inhibitor neostigmine (0.25 mg/kg), a dose previously established to cause NMJ Ca2+ overload based on the GBHA histochemical stain and degeneration of NMJ (Leonard and Salpeter, 1979; Kawabuchi, 1982). After 36 h, endplate Ca2+ deposits were identified at ∼39.8 ± 4.5% of endplates in nonpretreated mice, which was reduced to 22.1 ± 5.5% in mice pretreated with siRNA-IP3R1 (Fig. 5A; n = 7, p < 0.001). Control RNA had no effect on neostigmine-induced NMJ Ca2+ overload (37.6 ± 3.1%; Fig. 5A). Blockade of IP3 receptors by systemic pretreatment of mice for 9 d with the nonspecific blocker 2-APB or XS also reduced the proportion of GBHA-labeled endplates to 28.9 ± 3.6% and 26.2 ± 4.6%, respectively (Fig. 5A; n = 7, p < 0.001).
Muscle from both SCS patients and SCS transgenic mice bearing different AChR mutations have Ca2+-overloaded NMJs proportional to the severity of the effect of the mutation on the prolongation of synaptic currents (Gomez et al., 1997, 2002; Zayas et al., 2007). In this study we employed the SCS mouse line expressing εL269F, which has the most extensive endplate Ca2+-overload of a series of five lines studied in detail (Gomez et al., 1997, 2002; Zayas et al., 2007). We found that electroporation of siRNA-IP3R1 into TA muscle of SCS mice completely eliminated the Ca2+ overload of NMJs at 9 d (Fig. 5A; n = 7), while control RNA had no effect (33.4 ± 3.1%; Fig. 5A; n = 7, p > 0.91). Also, treatment of SCS mice with the IP3R blocker 2-APB or XS for 2 d significantly reduced the proportion of GBHA-labeled endplates to 25.5 ± 4.4 and 22.9 ± 5.3%, respectively (Fig. 5A; n = 7, p < 0.001). These findings suggest that pathological elevation of Ca2+ at the intact neuromuscular synapse in vivo requires participation of IP3R1-mediated Ca2+ release in skeletal muscle.
Previous studies have shown that Ca2+ overload in NMJs leads to activation of the Ca2+-activated protease calpain and, indirectly, that of the caspase proteases caspase-3 and caspase-9, which contribute to NMJ degeneration (Leonard and Salpeter, 1979; Vohra et al., 2004, 2006). We found that genetic inhibition of calpain improved endplate ultrastructure, neuromuscular transmission, and muscle strength in SCS mice, but also found evidence for a significant role for caspases (Vohra et al., 2004, 2006; Groshong et al., 2007). Since siRNA-IP3R1 reduced or eliminated Ca2+ accumulation at endplates with either Neostigmine treatment or in SCS, we tested whether IP3R1 silencing would prevent increases in calpain activity in neostigmine-treated WT mice and SCS transgenic mice. To measure calpain activity in muscle homogenates, we used a highly sensitive luciferase-based calpain assay that employs a luminogenic calpain substrate (Calpain-Glo, Promega). Calpain activity in both neostigmine-treated WT and SCS mouse TA muscle was 3.3–4.2 times the level in untreated WT muscle (Fig. 5B). siRNA knockdown of IP3R1 in TA muscles 9 d before neostigmine treatment or in SCS TA muscles significantly reduced calpain activity to 1.4–1.9-fold of WT (Fig. 5B; n = 7, p < 0.005). Furthermore, treatment of SCS mice with IP3R inhibitors 2-APB (n = 7, p < 0.005) or XS (n = 7, p < 0.005) significantly reduced muscle calpain activity in SCS mice, although levels still remained 3.0- and 2.7-fold greater than WT, respectively (Fig. 5B).
Caspase activation at the NMJ of SCS patients presumably occurs via the intrinsic pathway when Ca2+ overload provokes mitochondrial stress and release of cytochrome c (Boehning et al., 2003). Activated forms of caspases are detected at NMJs of SCS patients and SCS transgenic mice (Vohra et al., 2004). Using antibodies specific for activated caspases, the proportions of active caspase-labeled endplates are roughly equivalent to the proportions of Ca2+-overloaded endplates in these mice. In mice treated with neostigmine 36 h before detection, >25% of endplates were labeled intensely for active caspase-9 and active caspase-3 (Fig. 5C). In εL269F SCS mice, ∼45% of endplates were labeled with active caspase-9 and active caspase-3, as reported previously (Fig. 5C) (Vohra et al., 2004; Groshong et al., 2007).
Caspase activity can also be characterized using a luciferase-based luminogenic caspase substrate assay (Caspase-Glo 9 and Caspase-Glo 3/7, Promega) to study TA muscle homogenates. Caspase-9 activity in neostigmine-treated WT mice was 1.6-fold over the untreated level and was 2-fold over normal levels in SCS mouse TA muscle (n = 5, p < 0.005). Caspase-3 activity was 2.5-fold over the untreated levels in both neostigmine-treated WT and SCS mice (Fig. 5B; n = 5, p < 0.005).
Treatment of TA muscle with siRNA-IP3R1 by direct electroporation in vivo before neostigmine treatment significantly reduced the fraction of endplates labeled with activated caspase-9 and caspase-3 to 14 and 15% (Fig. 5C; n = 5, p < 0.005), respectively. Silencing of IP3R1 also completely prevented the increase in both caspase activities induced by neostigmine treatment, while control RNA had no effect. In SCS mice IP3R1 knockdown reduced the proportion of active caspase-9 and caspase-3-positive endplates >2-fold (Fig. 5C; n = 5, p < 0.005). Similarly, siRNA-IP3R1-treated SCS mice significantly decreased the activity of both caspases (Fig. 5B; n = 5, p < 0.005), whereas control RNA treatment had no effect in preventing caspase accumulation at endplates. Lastly, treatment of SCS mice with a nonspecific IP3R inhibitor, either 2-APB or XS, 2 d prior significantly diminished the accumulation of caspase-9 and caspase-3 (Fig. 5C; n = 5, p < 0.005) and diminished caspase-3 and caspase-9 activity (Fig. 5B; n = 5, p < 0.005, p < 0.05) at endplates. These findings demonstrate that in two models of synaptic Ca2+ overload in muscle, acute pharmacological inhibition of acetylcholinesterase and the SCS mouse model, elevated caspase-3 and caspase-9 activity and accumulation of activated caspase-3 and caspase-9 at NMJs depend on the presence of skeletal muscle IP3R1, which presumably amplifies internal release of Ca2+. Thus, in addition to its physiological role, IP3R1 plays a key role in pathological elevations of proteases at synapses.
NMJ regions of SCS mouse muscle fibers have evidence of DNA damage best seen by immunostaining for phosphorylated H2AX (pH2AX) at Ser139, a histone modification that initiates DNA damage repair (Rogakou et al., 2000) (Fig. 5D). Figure 5D shows that immunolabeling of pH2AX was seen selectively in subsynaptic nuclei of SCS mice labeling ∼37% of NMJs, similar to the numbers seen for GBHA and active caspase labeling and as compared with extensive pH2AX labeling detected in widespread apoptosis-induced staurosporine treatment (Belmokhtar et al., 2001). IP3R1 knockdown significantly reduced the proportion of pH2AX-labeled NMJs to 11.4 ± 3.6% in SCS TA muscle (Fig. 5D; n = 5, p < 0.005). Control RNA did not reduce pH2AX-labeled endplates (35.6 ± 6.5%; n = 5; p > 0.96). Treatment of mice with the nonspecific IP3R inhibitors 2-APB and XS also effectively decreased the proportion of pH2AX-labeled endplates in SCS mice to 24.2 ± 1.7 and 20.5 ± 3.2% (Fig. 5D; n = 5, p < 0.005), respectively. Collectively, these findings demonstrate that the IP3R1-mediated Ca2+ release plays a significant role in mediating acute and chronic forms of synaptic Ca2+ overload and the resulting activation of cysteine proteases at endplates and DNA damage in subsynaptic nuclei. Blockade or elimination of IP3R1 protects NMJs from these processes.
IP3R1 silencing improves neuromuscular transmission and functional recovery in SCS transgenic mice
Weakness and fatigability in SCS and other NMJ disorders arise from impaired synaptic transmission manifested by decremental CMAP (compound muscle action potential) amplitudes with repetitive stimulation electromyography (rsEMG) and by combinations of presynaptic and postsynaptic defects as assessed using two-electrode voltage-clamp analysis of intact muscle in vitro. We performed rsEMG to investigate the role of IP3R1 in the impaired neuromuscular transmission and weakness induced in WT mice by anti-cholinesterase excess or that develops in untreated SCS mice (Laskowski and Dettbarn, 1979; Gomez et al., 1997). In mice with neuromuscular disease, decremental responses in the rsEMG are only detectable over the flexor digitorum brevis (FDB) muscle, presumably because of a high safety factor in most murine skeletal muscles (Schieppati et al., 1995; Corna et al., 1996). Thirty-six hours after intraperitoneal administration of neostigmine to WT mice, rsEMG revealed a mean decremental response of 10.6 ± 1.1% (Fig. 6; n = 5). However, when the FDB muscles on one side were pretreated by siRNA-mediated IP3R1 knockdown 9 d earlier, there was significant protection from neostigmine-induced decremental responses in rsEMG (5.6 ± 0.7%; n = 5, p < 0.005), while the treatment of contralateral FDB with control RNA had no effect on decremental responses (11.8 ± 1.2%; n = 5, p > 0.95). Similarly, untreated SCS mice showed mean decremental responses of 14.8 ± 1.4% (Fig. 6; n = 5) in the rsEMG. siRNA-mediated IP3R1 knockdown in FDB muscle diminished the decrement to 7.5 ± 1.1% (n = 5; p < 0.005), while control RNA had no effect (16.0 ± 1.0%; n = 5, p > 0.83). Lastly, treatment of SCS mice with the IP3R blocker 2-APB for 2 d significantly diminished decremental responses in SCS mice (8.0 ± 2.1%; n = 5, p < 0.005).
SCS mice have severely reduced MEPC amplitudes, MEPC frequency, and quantal content of evoked responses (Gomez et al., 1997). To investigate the physiological basis for the improvement in neuromuscular transmission induced by IP3R1 silencing in SCS mice, we used voltage-clamp analysis of endplate currents in excised TA muscle of SCS mice electroporated 9 d earlier with IP3R1-siRNA or control RNA (Fig. 7). As before, we found that both the amplitude and frequency of MEPCs were reduced, as was quantal content, in SCS mice (Gomez et al., 1997). Due to the expression of the mutant εL269F AChR subunit, MEPC decay phases were also prolonged, a finding that underlies the ionic overload leading to the disease process (Gomez et al., 1997). Surprisingly, upon treatment of TA muscle with IP3R1-siRNA we found that the MEPC amplitudes increased by 50% (1.63 ± 0.19 pA vs 1.1 ± 0.12 pA, p < 0.03; Fig. 7B), and the MEPC frequency was increased by >2-fold (1.75 ± 0.1 s−1 vs 0.78 ± 0.04 s−1, p < 0.001), and restored to the normal values (Fig. 7A, right). In contrast, MEPC amplitude and frequency in control RNA-treated SCS muscle remained reduced to levels similar to those seen in untreated SCS mice (Fig. 7A,B) and significantly lower than that seen in WT mice (Fig. 3A,B). In addition, in TA muscle the amplitude of EPCs evoked by stimulating the corresponding tibialis nerve more than doubled in IP3R1-silenced SCS muscle (45.59 ± 6.45 vs 13.72 ± 2.8, p < 0.003) with RNA-treated or untreated SCS mice (Fig. 7D). The computed quantal content in IP3R1-silenced SCS TA muscle (Fig. 7E) was 28.03 ± 4.6, more than double that of control RNA-treated SCS muscle (12.55 ± 2.32, p < 0.03) and similar to normal TA muscle. Not surprisingly, MEPC decay time remained prolonged due to the continued expression of the mutant ε subunit (Gomez et al., 1997) (Fig. 3C). These results suggest that there is an overall restoration of both the presynaptic and postsynaptic defects in neuromuscular transmission in SCS mice by knockdown of the IP3R1 internal Ca2+ store, which plays a key role in mediating the pathophysiological effects of the mutant AChRs in SCS. The fact that these parameters are improved without correcting the underlying kinetic defect in neuromuscular transmission indicates that the IP3R1 plays a key role in mediating the pathogenic effects of the mutant AChRs.
Mice with neuromuscular disease have impaired motor function, strength, and fatigue, as assessed by a variety measures of motor behavior (Gomez et al., 2002; Groshong et al., 2007). To investigate whether the single limb treatment paradigm led to improved motor performance, we used a computerized, video-assisted treadmill (DigiGait, MouseSpecifics), which allows side-by-side comparison of limb motor behavior using several hindlimb performance parameters including "brake time," "stride length," and "stride frequency" (defined in Materials and Methods). Each of these was significantly prolonged in SCS mice (Fig. 8A). However, treatment of one TA with siRNA-IP3R1 and the opposite TA with control RNA led to a significant improvement in all three parameters in siRNA-treated SCS limb compared with the control RNA-treated SCS limb (Fig. 8A). In untreated SCS mice, stride length was 4.5 cm compared with 3.7 cm in WT mice (Fig. 8B; n = 11, p < 0.001). In the siRNA-IP3R1-treated side the mean stride length was 3.6 cm while the control RNA-treated side was 4.6 cm (n = 1,; p < 0.001). Mean stride frequency in SCS mice was 5.2 s−1 compared with 6.4 s−1 in WT mice (Fig. 8B; n = 11; p < 0.001). In siRNA-treated hindlimb, the mean stride frequency was 6.7 s−1 compared with 5.1 s−1 in control RNA limb (n = 11, p < 0.001). These findings further demonstrate the importance of the IP3R1 in mediating the full spectrum of histochemical, pathological, electrophysiological, and clinical consequences arising from pharmacological or genetic perturbation of AChR gating in neuromuscular disorders.
In skeletal muscle fibers many developmental and physiological processes depend on Ca2+ fluxes associated with cholinergic input. The NMJ is the site of transient increases in sarcoplasmic Ca2+ distinct from other Ca2+ fluxes in muscle that, until now, were attributed to the permeability of the AChR to Ca2+ (Miledi et al., 1980; Goldman et al., 1988; Decker and Dani, 1990; Villarroel and Sakmann, 1996; Fucile et al., 2006). We previously used living, fura-2-loaded muscle fibers focally activated at the NMJ with iontophoresed ACh to show that localized increases in NMJ Ca2+ to both normal and pathological levels could be inhibited by blocking IP3Rs or IP3 production (Zayas et al., 2007). Initial increases in Ca2+ still presumably occur by entry through WT or mutant AChRs (Zayas et al., 2007). IP3 is produced by the action of phospholipase C activated by G protein-coupled receptors such as muscarinic receptors (Furlan and Godinho, 2005; Garcia et al., 2005; Wright et al., 2009). In the current study we identified the responsible IP3R subtype as IP3R1 and demonstrated for the first time that IP3R1s are required for critical developmental and physiological processes at the NMJ, ranging from the earliest events of AChR cluster dispersal in developing myotubes to homeostatic plasticity and the control of synaptic gene expression in innervated fibers. Furthermore, we found that in adult fibers IP3R1s also underlie pathological conditions of focal synaptic Ca2+ overload, which are prevented by IP3R blockade or IP3R1 knockdown.
The findings in this study are significant for several reasons. First, they reveal a novel role for IP3R1 in enabling the signaling required for early maturation of the NMJ, presumably by amplifying agonist-evoked Ca2+ signals at the AChR clusters to accelerate cluster dispersal. No comparable role for IP3R1 in other neuronal cell types has been demonstrated. Second, the findings in mature NMJs suggest that this novel role is expanded in formed synapses to provide an additional mechanism by which localized neural signals may be transduced efficiently into Ca2+ store release. This expanded role influences presynaptic and postsynaptic homeostatic synaptic plasticity and changes in synaptic gene expression, yet remains compartmentalized at the muscle fiber synapse. Third, they show that this internal Ca2+ release store is a significant source of pathological elevations of Ca2+ and therefore a potential target for therapy in disorders mediated by Ca2+ overload, such as excitotoxicity, without apparent adverse effects in the fully formed synapse.
The importance of IP3R1 in early NMJ development is consistent with the previous observation that ACh mobilizes internal Ca2+ in C2C12 cells and the critical finding that agonist-mediated cluster dispersal depends on activation of calpain (Giovannelli et al., 1995; Chen et al., 2007). IP3R1 expression appears in embryonic development and, based on the presence of an E-box motif in the ITPR1 gene promoter, likely exhibits coordinate regulation with other muscle genes by myogenic regulatory factors (Konishi et al., 1997; Yafe et al., 2008). The basis for its predominant localization at the NMJ has yet to be explained.
Increases in quantal transmitter release and AChR expression in innervated muscle fibers induced by partial blockade of neuromuscular transmission reflect the property of homeostatic plasticity (Gallego et al., 1979; Domet et al., 1995; Wang et al., 2005, 2010). Our findings that these changes can be mimicked by IP3R1 silencing suggest that they may occur through the intracellular Ca2+ release via IP3R1.
The selective role of IP3R1 activity on gene expression at the NMJ can be partially explained by the localized effect of IP3R1 silencing or inhibition on HDAC4 translocation into myonuclei at the NMJ (Cohen et al., 2007). Reducing intracellular Ca2+ by siRNA-mediated IP3R1 knockdown may diminish Ca2+-dependent CaMKII activity, which leads to reduction in CaMKII-mediated phosphorylation of HDAC4. HDAC4 must be phosphorylated to be exported from nuclei, and thus accumulates in nuclei (Backs et al., 2006; Cohen et al., 2007). This is consistent with our immunostaining results of intense localization of HDAC4 in subsynaptic myonuclei in IP3R1 knockdown in TA muscle, which was similar to but more localized than the widespread accumulation of HDAC4 in myonuclei after denervation. Many of the effects of muscle denervation on gene expression may act through HDAC4, which suppresses the transcriptional corepressor Dach2 and allows expression of myogenin and downstream E-box-containing NMJ genes (Tang et al., 2004). Thus, the changes in gene expression after siRNA silencing of IP3R1 may result from a similar although more NMJ-localized action of HDAC4 on gene expression. The increased expression of ErbB2, the neuregulin receptor in intact synapses, may provide an additional positive feedback for increased AChR expression (Méjat et al., 2003).
Denervation and IP3R1 silencing do not have identical effects on NMJ gene expression. Clearly, numerous other neural factors such as agrin and neuregulin, as well as sarcolemmal electrical activity, may also play roles in modulating gene expression and are likely to persist in intact NMJs after IP3R1 silencing (Goldman et al., 1988; Lebrasseur et al., 2003; Weston et al., 2007). In some cases, combined denervation and IP3R1 silencing were similar or additive to single treatments, suggesting similar pathways, while other cases, such as the greater effect of denervation on rapsyn and the opposing effect on AChE expression, suggest that polypeptide neural factors may play a greater role in controlling expression of these genes. On the other hand, IP3R1 silencing had a greater effect on expression of the ε subunit and particularly ErbB2, suggesting that either the expression of these genes is coordinately regulated or that they act in the same pathway.
These studies provide a detailed description of the role of muscle IP3R1 and compartmentalized Ca2+ signals at the NMJ in coordinating synaptic processes. Given the differences in expression patterns and levels of IP3R in different cells and the diverse and severe phenotypes of spontaneous and induced mutations, it is likely that IP3Rs have very cell-specific functions (Matsumoto et al., 1996; Acharya et al., 1997; Inoue et al., 1998; Itoh et al., 2001). Thus, these findings will require further study in other systems to be able to extrapolate to other cell types.
While our findings in developing muscle suggest that elimination of IP3R1 in embryonic muscle might adversely affect the development of synapses, we find that IP3R1 antagonism in adult innervated muscle, while playing a key role in function, may only qualitatively affect synaptic function through refining of synaptic gene expression and homeostatic regulation of synaptic transmission. Thus, therapy directed at reducing IP3R1 activity may be well tolerated as a therapeutic strategy for excitotoxic conditions or conditions of Ca2+ overload, such as muscular dystrophy (Mondin et al., 2009).
Deranged intracellular Ca2+ release through IP3Rs has been implicated in the pathogenesis of other muscle and neuronal degenerative disorders (Monnier et al., 2000; Bezprozvanny, 2011). We studied two neuromuscular conditions whose pathogeneses are directly related to synaptic (NMJ) Ca2+ overload, anti-cholinesterase excess, and the slow-channel myasthenic syndrome (Engel et al., 1977, 1998; Gomez et al., 1997). As suggested by our previous in vitro single muscle cell study, IP3R1 silencing in vivo completely eliminated synaptic Ca2+ overload in both conditions. Subacute blockade of IP3R in SCS transgenic mice using XS and 2-APB was also effective at eliminating NMJ Ca2+ overload. Moreover, these treatments were effective at interrupting the pathological cascade in the NMJ that includes activation of cysteine proteases, damage to subsynaptic double strand DNA, and physiological and clinical impairment of neuromuscular transmission. Thus, IP3R1 antagonism in this study had a dramatic effect at reducing pathologic changes and protecting the NMJ in two conditions of cholinergic excess. These studies suggest that studies directed at modulating this intracellular Ca2+ release pathway in neurodegenerative or excitotoxic conditions may be neuroprotective and well tolerated.
This work is supported by NIH Grant RO1 NS33202 (C.M.G.).We thank Katherine Hekman for helpful comments in preparing this manuscript.
The authors declare no competing financial interests.
- Correspondence should be addressed to Dr. Christopher M. Gomez, Department of Neurology, Albert Merritt Billings Hospital S237, MC2030, University of Chicago Medical Center, 5841 South Maryland, Chicago, IL 60637. gomez001{at}uchicago.edu


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thanksgiving easy storyA collection of kids Thanksgiving short stories and tales from Children s Literature refer to this flavorful and foolproof method for roasting your turkey and breathe easy. Thanksgiving stories for children show your love for mickey with this easy diy frame our thanksgiving menus make it easy to build the best-ever feast for your friends and family this year. Five myths about Thanksgiving travel we have complete menu ideas for a traditional thanksgiving. The good news is that Turkey Day may not be as bad as you think browse through these simple thanksgiving recipes by type to find old favorites and new takes on classic dishes. THE PLYMOUTH THANKSGIVING STORY When the Pilgrims crossed the Atlantic Ocean in 1620, they landed on the rocky shores of a territory that was begin a wonderful thanksgiving food tradition by making these easy gratitude rolls. THE REAL STORY OF THANKSGIVING read the true story of the first thanksgiving feast. by Susan Bates what did they eat? who attended? learn when it took place and whether it was a holiday or gathering. Most of us associate the holiday with happy Pilgrims and Indians sitting down to a big feast thanksgiving stories - listen online! family friendly stories and thanksgiving day prayers - gather round and listen to stories that illustrate the true meaning of. And that did happen - once teach your kids about turkey day! the thanksgiving story for kids. Check out our easy, kid-friendly Thanksgiving craft ideas and art chances are, your kids (and maybe even you!). 10 Easy Thanksgiving Crafts and Activities for here, the thanksgiving story for kids. never miss out on a must-read story written by ann mcgovern, it shares in simple, easy language that modern-day kids can understand. Sign it's a good pick for kids ages four and up. Fun Thanksgiving Crafts for Children of all ages the thanksgiving story. Great for preschool, elementary and homeschool children these beautiful diy projects will have your home looking cute and crafty this thanksgiving. Every year on the fourth Thursday of November, Americans celebrate with a Thanksgiving meal today s top stories. But what is the origin of this tradition? Did it really begin when the 40 easy thanksgiving crafts both kids and. Wonderful collection for Kids Thanksgiving Stories inspirational, Short Thanksgiving Story for an indian, who is also a school teacher, thanksgiving was never an easy holiday for me to deal with in class. On the occasion of Thanksgiving Day, TheHolidaySpot thanks all its the indian hero of the thanksgiving story. Homebound But Hopeful shares 3 fun, easy, and quick ideas for The 12 Days Of Thanksgiving story time flex your creative muscle this thanksgiving by making these amazing gratitude crafts that are a wonderful activity for the whole family and a perfect reminder of what.
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Underrated Coasters?What coasters don't get the attention they deserve?
Users viewing this topic: No registered users and 0 guests
Discuss theme parks, roller coasters, and mules!
Underrated Coasters?
Some people have got bad comments on Coasters I like... example Xpress (Superman:The Ride) @ Walibi World, it's an outdoor RNRC, but it's verry smooth for a Vekoma and the launch is really great (Ok, I haven't done TTD or Kingda Ka) but it's has some good G's.
And the outside coaster part is good, because it's a little bit above water (and concrete) and that gives a free feeling.
Some people think the ride is a "real Vekoma"..painfull, but I think it's not, and also some people think it's boring because it's not indoors, well RNRC in Paris is better, but it's only because of the theming... that always is a + by me.
Have u got some coasters that you think of that they are underrated?
(oh yeah, i'm sorry if you have got difficulties with reading this, i'm from Holland and my English is a bit messed-up)
Last edited by robbalvey on Sun Nov 25, 2012 5:47 pm.
^ I wouldnt NM Rattler, since the only reason it doesn't get many rave reviews is because not many have ridden it.
I'd say:
Disaster Transport
Thunder Road
BTR @ SFGAm
Laser @ Dorney Park. Although is ride is VERY short, it has strong forces and, now the best part, only a lapbar!
You are talking about Scorpion at BGT right? That and Python are two of my favs. Short but sweet. (I do like Scorpion a bit better tho.)
Blue Streak at CP.
This ride has no line (garenteed rerides) and has awsome lap bars that dont close all of the way. This ride also delives TONS of airtime in the rain. The best seat is 1:3. There old buzz bars were awsome!
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These pages are in no way affiliated with nor endorsed by SeaWorld Parks & Entertainment, Cedar Fair, Legoland, Merlin Entertainment,
All onride
photos and videos on this website were taken with the permission of the park by
a professional ride photographer.
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Goalless draws for Liverpool and Arsenal ensured ground was lost in the top four fight, as the Premier league returned for another round of midweek fixtures. Following a disappointing derby day defeat Manchester United recovered to beat Bournemouth 1-0, table toppers Manchester City brushed past Swansea 4-0 and Everton kept up their good run of form, securing a 1-0 victory …Read More »
Tag Archives: Hector Bellerin
Tottenham's rich vein of form pleases fantasy managers
Manchester United's Marouane Fellaini and Ben Davies of Tottenham Hotspur were the surprise top scorers in this week's fantasy football. Off the back of an impressive 4-0 away win at Huddersfield Town, Spurs boast three of the top five scorers in gameweek seven, with defender Davies, who now sits fourth in overall points scored, joined by Kieran Trippier and the …Read More »


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Spartan colors finish off golden homecoming week
Lynn Reynolds, Production Manager
September 28, 2019
On Friday, students dressed up in school colors for Blue and Gold day.
[PHOTO GALLERY] Annual lip sync competition brings audience to their feet
Elizabeth Trevathan, RubicOnline
September 27, 2019
The audience cheered and applauded, with several standing ovations, after lip sync performances.
A blast from the past: Decades Day revives fashion trends
Zekiah Juliusson, RubicOnline
September 26, 2019
The third day of Homecoming brought retro outfits and the dodgeball tournament.
Liminal SPAces: Diving into spooky spots
Melissa Nie, RubicOnline Editor
March 14, 2019
Melissa Nie explores spaces that give off strange vibes around the school.
PHOTO GALLERY: What's behind the door?
Mimi Geller, RubicOnline Editor
March 31, 2017
The new wall blocking off the construction zone for the new math and science building invites student and faculty creativity.


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Were you always planning to be an accountant and why?
Yes, industry allows for a wide range of avenues to follow (not limited to specific services or specific industry's)
When meeting with clients do they mostly come to your own premises or do you mostly go their theirs?
50/50 between the above
What is your preferred mode of communication with your clients?
Email, Phone
Have you ever done an online video call with a client?
What do you love most about accounting?
Interacting and dealing with various client across various industries
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Annual Financial statements, taxation
Describe your average client:
How far away from your premises is your furthest client?
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No having a CA(SA) on board + cash flow planning
What accounting software do you like best?
Sage one
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Pastel, Xero, Quickbooks, VIP
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No, it will allow for more focused approach to be taken - higher end service offerings
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Havent had any dealings with it, probably where we are headed - would not necessarily be against it
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Euro trip
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Great website for accountants - quality leads received. Leads in currently have slowed down vs 2-3 years back (frequency) - most likely due to increased accountants registered.


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LONELY PLANET, Starring Arnie Burton & Matt McGrath, Enters Final Weeks Off-Broadway
A friendly reminder that this month is your last chance to see Steven Dietz's landmark play Lonely Planet, directed by Keen Artistic Director Jonathan Silverman (The Tempramentals) and starring Arnie Burton (The Government Inspector) and Matt McGrath (The Legend of Georgia McBride).
Performances for this limited Off-Broadway engagement at The Clurman Theatre at Theatre Row (410 West 42nd Street) will continue through November 18th only. The performance runs 1 hour and 45 minutes (including intermission).
Mr. Dietz's play is a testament to the value of friendship during a time of crisis. Keen Company creates theater that provokes identification, reflection, and emotional connection. In intimate productions of plays and musicals, we tell wholehearted stories about people striving to do their best. Keen has been honored with eleven Drama Desk Nominations, two Drama Desk Awards, two Drama League Nominations, and two Obie Awards.
Lonely Planet is the story of friendship in a time of crisis. Set in a small map store on the oldest street in an American city, Lonely Planet is an intimate portrait of two friends at the height of the HIV/AIDS epidemic. Shop owner Jody becomes increasingly fearful of the world outside and the dangers it poses, refusing to leave his shop. Meanwhile Carl, his spirited friend, begins filling the store with a variety of mysterious chairs. Funny, moving, and deeply human, Steven Dietz's landmark play examines how we all navigate troubled times. 25th anniversary production!
Arnie Burton's Broadway credits include Machinal, Peter and the Starcatcher, The 39 Steps, The New Yorkers (Encores!), A Free Man of Color, Amadeus. Off-Broadway credits include The Government Inspector (Red Bull Theater); Mystery of Irma Vep (Drama League Nomination); Talespin!;, Lives of the Saints; The Tempamentals (Drama Desk Award, outstanding ensemble); Peter and the Starcatcher (NYTW); The Merchant of Venice/The Jew of Malta (TFANA); Mere Mortals (Primary Stages and commercial run); The Last Sunday in June (Rattlestick and commercial run); The Venetian Twins, The Cherry Orchard (Pearl Theatre) UK: The Merchant of Venice (RSC, Stratford) Regional highlights include The Confederacy of Dunces (Huntington Theater), Big Sky (Geffen Playhouse), How to Succeed in Business... (TUTS), I Am My Own Wife (Kevin Kline Award, best actor, St Louis; Helen Hayes Nomination best actor, DC); All in the Timing ( Geffen Playhouse LA); The Santaland Diaries, The Seagull, The Taming of the Shrew (Old Globe) Mystery of Irma Vep (Berkeley Rep) Also The Alley Theatre, Philadelphia Theatre Co, The Huntington, Berkshire Theatre Fest. TV: "Elementary," "Jessica Jones," "Flesh and Bone", "Frasier," "Law and Order" (also "Criminal Intent"), "White Collar," "Blue Bloods," "Life on Mars," "6 Degrees," "Hope and Faith," "Caroline in the City." Film: The Greatest Showman, The Invention of Lying, Igby Goes Down, The Private Lives of Pippa Lee, Game 6.
Matt McGrath recently received the Lucille Lortel Award for his performance as Miss Tracy Mills in The Legend Of Georgia McBride at MCC (DCPA, Geffen Playhouse). He starred as Wilhelm in Robert Wilson's The Black Rider at the Ahmanson in Los Angeles, San Francisco, London and Sydney. Broadway: Cabaret (Emcee), A Streetcar Named Desire. Off-Broadway: Steve (The New Group), Verite (LCT3 - Lincoln Center), Hedwig and the Angry Inch, A Fair Country (Lincoln Center), Collection/A Kind of Alaska, Minutes from the Blue Route, The Dadshuttle (Atlantic), Fat Men in Skirts, The Old Boy, Life During Wartime, Amulets Against the Dragon Forces, Dalton's Back (Drama Desk nomination). Regional: Black Stach in Peter and the Starcatcher, Putting It Together, Ridiculous Fraud and Raised in Captivity (SCR); His Girl Friday (La Jolla Playhouse); The Rocky Horror Show (Old Globe); Romance, Japes and Bell, Book and Candle (Bay Street Theatre); Caroline in Jersey, Loot (Williamstown Theatre Festival); Distant Fires (LA Weekly Award), and Snakebit. Films: Full Grown Men, Boy's Don't Cry, The Notorious Bettie Page, The Anniversary Party, The Broken Hearts Club, The Impostors. Television: "Modern Family" "Law & Order: Criminal Intent," "Little Britain USA," "Cruel Doubt."
Steven Dietz's thirty-plus plays have been seen at over one hundred regional theatres in the United States, as well as Off-Broadway and in over twenty countries internationally. Mr. Dietz received the 2016 Steinberg New Play Award Citation for Bloomsday (ACT-Seattle, world premiere), having previously been a finalist for Last of the Boys and Becky's New Car. Other recent premieres include This Random World, presented at this year's Humana Festival of New American Plays at Actor's Theatre of Louisville, Kentucky; and the thriller, On Clover Road, seen at multiple U.S. theatres as part of a National New Play Network rolling world premiere. Other awards include the Kennedy Center Fund for New American Plays Award for both Fiction and Still Life with Iris; the Edgar Award for Best Mystery Play for Sherlock Holmes: The Final Adventure; the PEN USA West Award in Drama for Lonely Planet; and the 1995 Yomuiri Shimbun Award (the Japanese "Tony") for his adaptation of Shusaku Endo's novel Silence. Other widely produced plays include Rancho Mirage, Yankee Tavern, Shooting Star, Jackie and Me (from Dan Gutman), Inventing van Gogh, God's Country, Private Eyes, Dracula (from Bram Stoker), and The Nina Variations. Mr. Dietz and his family divide their time between Seattle and Austin, where he teaches playwriting and directing at the University of Texas.


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Ultra Street Fighter II: The Final Challengers releases on Nintendo Switch on May 26, 2017, and here at T3 Towers we've being playing the heck out of an advanced press copy of the game.
This new version of the classic fighter - maybe the best versus fighter ever made - is far from a lazy, cash-grab port too, with a massively engorged character roster, game modes, multiplayer match-ups, options, extras and customisations available, meaning that you get a hell of a lot of bang for your buck.
What follows are 10 of the things we like most about Ultra Street Fighter II: The Final Challengers. Before you get to those, however, check out the game's action-filled trailer to get a taste for how it runs and what it delivers:
1. You can switch graphical and audio style modes
One of the coolest things you must check out immediately in Ultra Street Fighter II: The Final Challengers is the multiple style modes, which allow you switch between the game's original graphics and sound and the new iterations. Neat touches like the original graphics maintaining a 4:3 ratio (rather than the new style's 16:9) and an arcade-style surround, as well as the original end boss instead of the new style's mean both offer a genuinely different experience.
2. The character roster is immense
A whopping nineteen characters pack Ultra Street Fighter II: The Final Challengers' roster. That's not at the dizzy heights of say The King of Fighters '98: Dream Match Never Ends, however, it is very large and includes all of your old favourite Street Fighter characters (and a few new ones too; see below). As such, no matter if you were great with Guile, kicked ass with Ken, or liked to roll with your homeboy E. Honda, you're well catered for here.
3. Gameplay mechanics are as slick as ever
One of the reasons why Street Fighter II took over the world in the arcades was its simple and super slick control mechanics, with each character boasting a series of super and special moves that were intuitive and fun to execute. Nothing has changed. Throwing Ryu's Hadouken or launching Ken's Shoryuken is simple and fast, meaning that it only takes players a short amount of time to create fluid and exhilarating match ups.
4. Game modes are varied and interesting
If you're concerned about longevity of gameplay in Ultra Street Fighter II: The Final Challengers then don't be as not only does the game come with Arcade, Versus and Training modes, but also the brand new Buddy Battle mode where two human players can team up against the CPU in a 2-on-1 battle, as well as the super exclusive Way of the Hado mode that allows players to take on the role of Ryu in first person and use motion controls to dispatch enemies.
5. There's a new colour editor and replay system
Ah now this is cool. A unique new colour editor has been added in Ultra Street Fighter II: The Final Challengers that allows you to customise the colour scheme of any character - if you want T. Hawk to wear nothing but pink then all power to your elbow! Also, you can now relive your greatest victories - or defeats - with a cool new replay system that allows you to rewatch a fight at any time.
6. Beginners are well catered for
While the control scheme and game mechanics remain as approachable as ever, real newbies to versus fighters will be pleased to hear that there is a new "Lite" control scheme included in Ultra Street Fighter II: The Final Challengers. This works a little like the on-screen controls in Super Street Fighter IV 3D Edition on Nintendo 3DS, allowing players to execute special moves with just the press of a button or tap of the touchscreen.
- Best Nintendo Switch accessories buying guide: T3's official list of top Nintendo Switch peripherals
7. Iconic background stages remain faithful
It's not just the characters who have benefited from the new style's fresh art direction, but stage backgrounds too. These look crisper and more vibrant than ever, however, they also remain faithful to the originals as well, with background characters performing the same actions and any paraphernalia (random crates anyone?) in the same locations. So whether you liked fighting in Vegas, in front of F-16 fighter jet, or on the roof of a dōjō, you'll be happy.
8. The gallery mode is stunning
This was one of our favourite new features here at T3 Towers. Ultra Street Fighter II: The Final Challengers comes with a simply stunning gallery mode that allows you to access over 1,400 illustrations taken from the superlative art book SF20: The Art of Street Fighter. The calibre of the art is top draw and really helps brings the characters and fiction alive.
9. Evil Ryu and Violent Ken are fun to play as
17 of Ultra Street Fighter II: The Final Challengers' roster are returning characters, with the other 2 being the new additions of Evil Ryu and Violent Ken. Evil Ryu first appeared in the series in Street Fighter Alpha 2, while Violent Ken has never actually being in a Street Fighter game before now, with the character originating in the crossover fighting game SNK vs. Capcom: SVC Chaos. Each character has their own unique special and super moves too.
10. Local and online multiplayer is supported
Lastly, this is without doubt the most connected version of Street Fighter II ever, with The Final Challengers allowing you play against your friends and family both in Versus mode local multiplayer, as well as online against players from around the world in either ranked or causal matches. The fact too that the Nintendo Switch is so portable, with both handheld and tabletop modes to rely on, means that squeezing in a few matches on a commute or at lunch has never been easier.
For more information about Ultra Street Fighter II: The Final Challengers then check out the game's official website.


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