synaptic growth and Glutamate receptor composition at Drosophila NMJs
4-1. Henji functions in the postsynapse to regulate GluRIIA abundance
Given that Henji function in postsynapses ie n regulating postsynaptic dPak levels and presynaptic bouton growth, we further tested t , while presynaptic expression of Henji failed to do so. To further test whether postsynaptic Henji activity also regulates NMJ physiology, the GluRIIA abundance were examined in henji mutants with pre- or postsynaptic expression of Henji. The intensity of membrane GluRIIA staining is comparable to wildtype when Henji is overexpressed postsynaptically in henji -/-background. On the contrary, GluRIIA punctae intensity of the presynaptic rescued NMJs is not significantly different from the henji mutant (Figure 1A).
Electrophysiological analysis also shows a restoration of mEJP amplitude recorded from postsynaptically rescued NMJs but not presynaptically rescued ones (Figure 1B).
These data suggest Henji’s role in regulating NMJ growth and development is most importantly at posysynaptic sites.
4-2. Henji regulates GluRIIA abundance via dPak
Our previous data indicates that dPak is a degradation substrate of Henji-Cullin3
ubiquitination E3 ligase complex. In addition, we have shown that Henji regulates presynaptic bouton growth via downregulating dPak (Appendix 9). I further check whether reducing one copy of dpak gene can also rescue GluRIIA accumulation in henji mutant. This speculation comes from the report stating dPak is required for GluRIIA clustering at the NMJs (Albin and Davis, 2004). Similarly, I introduce one copy of dpak6 null mutant allele into henji-/- flies and stained GluRIIA for analysis. Like presynaptic boutons, postsynaptic GluRIIA punctae intensity at the synapse is restored to levels close to wildtype after dpak gene dosage reduction (Figure 2). We then conclude that the elevation of GluRIIA abundance in henji mutants is an outcome of impaired dPak degradation in the lack of Henji and that dPak is an important substrate for Henji to regulate NMJ functions at both sides of the synapse.
4-3. Henji promotes dPak downregulation regardless to its active state
Mammalian Paks can be grouped into two categories, according to their structural homologies. dPak is homologous to type 1 Paks, which is known to form inactive dimmers via the N-terminal autoinhibitory (AI) domain. When type 1 Paks are activated by Cdc42 and Rac, this binding would rearrange local conformations of the AI domain, releasing the activation loop and dissociating the dimer. Considering the drastic conformational change after activation, we wonder if Henji has any preference to target
only certain form(s) of dPak. Three forms of UAS-dPak transgenes are generated by T.
T. Lai according to the structure-based studies. The constitutive active form of dPak contains a point-mutation of T423E, mimicking a constitutively phosphorylation at the first autophosphorylation site. Expression of T423E Pak CA construct is sufficient to promote motility, and anchorage-independent growth via the MAPK kinase pathway in human breast cancer lines (Vadlamudi et al., 2000). On the other hand, the dominant negative form of dPak carries three point-mutation, H83L/H86L/K299R, with the former two mutations disrupting the interaction with Cdc42/Rac1 and the later being a kinase dead mutation. This construct is also proved to inhibit the downstream activation of Ras signaling in mammalian cell lines (Tang et al., 1997). The last construct is made from full-length wildtype dPak. The expression of these constructs is tested by immunoblotting using specific anti-Myc antibody (Figure 3A). Both Myc-tagged dPak CA and dPak DN are successfully overexpressed in muscle cells using C57-GAL4;
however, Myc-dPak wt lines are somewhat problematic. The UAS-Myc-dPak wt construct is not defective in its construct sequence. More lines will be generated in order to obtain strong, proper-expressing ones.
Myc-dPak CA and dPak DN constructs are overexpressed postsynaptically with muscle-specific C57-GAL4 and then GluRIIA intensity is analyzed as a terminal phenotype in response to dPak activity at the synapse. However, the abundance of
GluRIIA in all three forms of dPak overexpression is similar to C57-GAL4, henji +/-heterozygous control (Figure 3B-E), while C57-GAL4, henji2/henji1 mutant still shows an enhanced GluRIIA clustering (Figure3G). Nevertheless, this data is consistent with the previous report that overexpressing myristoylated dPak, mimicking a constitutively active form, in wildtype flies is not sufficient to upregulate GluRIIA abundance at Drosophila NMJs (Albin and Davis, 2004).
In our hypothesis, Henji is responsible to specifically downregulate dPak at the NMJs.
When dPak CA is overexpressed postsynaptically, Henji rapidly promotes the degradation of the excess dPak protein. Therefore, even though it is the CA form of dPak overexpressed, the functional dPak protein at the synapse is not sufficient to promote additional GluRIIA clustering to the PSDs. In order to prove this hypothesis, these three dPak constructs are overexpressed with C57-GAL4 in henji+/- flies.
Strikingly, GluRIIA punctae intensity is further enhanced in dPak CA overexpressing flies compared with C57-GAL4, henji+/- heterozygous control and even stronger than C57-GAL4, henji2/henji1mutant (Figure 3F-H). On the other hand, GluRIIA clustering is significantly inhibited in dPak DN overexpressing henji+/- NMJs (Figure3F,G and I). To know whether the overexpressed dPak is localized to the synapse and thus perform its function to enhance GluRIIA clustering, anti-Myc antibody (9E10) is employed for staining. When Myc-dPak CA or dPak DN is overexpressed in the muscle, the overall
background signal of Myc staining is elevated in the muscle cell compared with C57-GAL4 control (Figure 3J, K and M). However, when these constructs are
overexpressed in henji+/- flies, strong Myc-positive punctae appear and specifically surround the NMJ (Figure 3L and N). These results indicate a role for Henji to very specifically regulate local dPak protein levels at the PSDs and this regulation is not related to the active states of dPak.
Chapter 5. Discussion
5-1. Bifurcated signaling downstream of /FAK56
Ras/MAPK signaling was shown to specifically downregulate FasII level at the synapse and this negative regulation is well-correlated to bouton addition (Koh et al., 2002). On the other hand, PKA was shown to affect transmitter release from the presynapse (Davis et al., 1998); mutants defects in cAMP production also showed abnormal bouton growth (Cheung et al., 1999). In both cases, the upstream cue modulates activities of Ras or cAMP was not clearly defined. In this study, we described an integrin -mediated ECM signaling, distinct form BMP/Gbb or Wnt/Wg pathways, functions to restrain NMJ bouton growth. This pathway included the activation of FAK56 and further elevation of cAMP signal by NF1 and inhibiton of Ras/MAPK via Vap. These two bifurcated signaling pathways finally converge to regulate synaptic growth.
5-2. Cullin3/Henji E3 complex affects postsynaptic GluRIIA/GluRIIB ratio by
downregulating dPak protein level
During the development of the NMJ, strengthening of both neurotransmission and synaptic structure requires modulations in local protein level and proper localization.
When GluRIIA expression is augmented in the postsynapse by either UAS-GluRIIA
transgene overexpression or genetically reduce glurIIB gene dosage, proper clustering of IIA-containing GluRs were formed at PSDs and the evoked response and synaptic bouton number were significantly elevated (Sigrist et al., 2002). In our experiments, we have identified a Cullin 3-based E3 ligase comples responsible for regulating the GluR subunit composition at the postsynapse. In henji mutant, the GluRIIA/GluRIIB ratio is increased and this alteration corresponded to the enhanced muscle response to the presynaptic spontaneous vesicle release.
The postsynaptic GluRIIA localization requires dPak activity but when constitutively active, membrane-bound dPak transgene was overexpressed postsynaptically, GluRIIA abundance was not further enhanced (Albin et al., 2004). We generated a constitutively phosphorylated dPak transgene and consistently, overexpression of this dPak CA in the muscle did not change GluRIIA. However, when this construct was postsynaptically overexpressed in henji+/- background, GluRIIA clustering at PSDs were dramatically enhanced. Therefore, we propose that Henji is required at the synapse to regulate dPak protein level and further influence local GluRIIA abundance; GluRIIA CA construct expressed in wildtype synapse, this protein would be rapidly downregulated by Henji and thus had no effects on GluRIIA abundance.
5-3. Neuortransmission homeostasis is not disrupted in henji mutant
Brp-positive punctae were quantified and in henji mutant there was a 20% increase in total release sites per NMJ (Appendix 5B). However, EJP remained unaltered in henji mutant regardless to the increase of both release sites and boutons, showing intact homeostatic machinery. The enhanced postsynaptic GluRIIA augments muscle response to neurotransmitter released; to compensate such hyper-sensitivity of the postsynapse and to maintain synaptic homeostasis in neurotransmission, presynaptic vesicle release machinery was attenuated to keep EJP unchanged. The enhanced synaptic structure and expanded AZs were suppressed by this homeostatic compensation.
Materials and methods
Fly stocks
All flies were reared in standard conditions at 25℃. We use w1118 from Bloomington stock center as wild-type in this study. Mutant flies integrin1 and 2 have been described previously (Devenport and Brown, 2004). N30 and K24 deletions of FAK56 were generated from KG00304 P-element insertion as previously described as Tsai et al., 2008. The transgenic lines elav-GAL4 (X) and elav-GAL4 (III) were obtained from the Bloomington stock center. MHC-GAL4. vap1 and vap2 mutant flies were kind gifts from Dr. S. Schneuwly’s lab and the alleles were as described (Botella et al., 2003). NF1E2, w;
iso2;iso3, hs-NF1 wt, hs-NF1 KA, hs-NF1 RP, UAS-NF1 GRD, UAS-NF1 GRD KA, UAS-NF1 GRD RP and UAS-NF1 ΔGRD transgenic flies were generated by Dr. J.
Walker. The nature of NF1-related mutant alleles were as previously described (Walker et al., 2006).
Immunostaining
Wandering third instar larvae were dissected for analysis of NMJ phenotypes in all experiments. Dissected larval fillets were incubated in fixative solution (4%
formaldehyde in 1× phosphate-buffered saline) for 20 minutes. The following primary antibodies were used for immunostainig: anti-synaptotagmin (mouse, 1:25; DSHB),
anti-synapsin (3C11, 1:100; DSHB), HRP conjugated with TRITC (rabbit, 1:100;
Jackson ImmunoResearch), anti-FasII (1D4, 1:100; DSHB), anti-Brp (Nc82, 1:100;
DSHB), anti-Dlg (mouse, 1:100; DSHB), anti-dGluRIIA (mouse, 1:100; DSHB), anti-Futsch (22C10, 1:100; DSHB) and anti-GFP (chicken, 1:1000; Abcam). Alexa 488-, FITC-, TRITC-, Cy3- and Cy5-conjugated secondary antibodies and FITC-phalloidin were used (Jackson ImmunoResearch).
Image processing and presentation
Confocal images were acquired using Zeiss LSM 510 Meta or Zeiss LSM 710. Images were processed using Adobe Photoshop CS. Bouton number and muscle area were quantified with Zeiss LSM 510 image examination software. For comparison and quantification of signal intensity at NMJs, larval fillets were stained in the same tube and the images were acquired under the same scanning parameters. NMJ silhouettes were outlined and the signal intensity was calculated by histogram analysis using Adobe Photoshop CS.
Transgenes
UAS--GFP was made by cloning the full-length insert from cDNA library into pTWG vector (DGRC) using the Gateway recombination cloning system (Invitrogen).
The resulting construct carries a C-terminal GFP tag. UAS-GFP-vap wt and UAS-GFP-vap R695K were cloned from cDNA library; Missense point mutation of
R695K was made by PCR-based mutagenesis. Both inserts were cloned into pTGW vector from DGRC and the Gateway recombination cloning system was also used. All constructs were checked by sequencing prior to embryo microinjection. Transgenic flies were generated by standard procedures.
Electrophysiological recording
For sample preparation, dissected larval body walls (including the central nervous system and motor axons) were exposed in cold (4°C) HL3.1 Ca2+ free saline (70 mM NaCl, 5 mM KCl, 4 M MgCl2, 10 mM NaHCO3, 5 mM trehalose, 115 mM sucrose, 5 mM HEPES pH 7.2) [66]. Experiments were performed on muscle 6 of segment A3 in late third instar larvae. The segmental nerve was cut near the ventral ganglion.
Preparations were then incubated in HL3.1 saline containing 0.2 or 1 mM CaCl2 for electrophysiological experiments at room temperature (22°C). For stimulation and recording, a glass microelectrode (30–50 MO in resistance) filled with 3 M KCl was
impaled in the sixth muscle of the third abdominal segment to record the EJPs. The mEJPs occurring in the background within 200 seconds were obtained without any stimulation on the segmental nerve. To evoke an EJP, the segmental nerve was stimulated every 30 seconds through the cut end with a suction electrode with 0.1 ms of pulse duration at 2 times the threshold voltage. Once the threshold voltage was reached, the size of EJPs remained unchanged despite the increase in stimulating voltage. Signals were digitized at 64 KHz by a PCI-6221 data acquisition card (National Instrument, Austin, Texas, USA), and saved on an IBM compatible PC for analysis.
Western blots
Third instar larvae or adult heads of different genotypes were homogenized in in RIPA lysis buffer. Equal amounts of lysates were separated in 7.5% or 8% SDS-PAGE gel.
Blots were probed with anti-dPak (rabbit, 1:10,000), anti-Myc (9E10, 1:1000, Santa Curz), anti-GFP (mouse, 1:1000, Invitrogen), anti-a tubulin (mouse, 1:200,000, Sigma) and HRP conjugated secondary antibodies.
Dibutyl cAMP/forskolin feeding
Adult NF1E2 flies were put into vials containing standard medium mixed with 10nM of di-cAMP or forskolin. After 6 hours all adult flies were transferred into another vial
filled with standard medium and the eggs laid on media mixed with di-cAMP or forskolin were reared until late third instar when they were dissected and NMJ morphology analyzed.
Electron Microscopy
For ultrastructural NMJ studies larval fillets were dissected at room temperature in calcium free medium and subsequently fixed overnight in 4% paraformaldehyde /1%
glutaraldehyde/0.1 M cacodylic acid (pH 7.2). Microwave irradiation (MWI) with the PELCO BioWave® 34700 laboratory microwave system was used for subsequent EM processing steps. After overnight fixation, the fixed fillets were additionally post-fixed with 1% aqueous osmium tetroxide 2x at 90W for 2minON-2minOFF-2minON under vacuum and placed on ice in between changes with additional 1 hour incubation on rotator, dehydrated in increasing ethanol concentrations(50%,70%,80%,90%,100%) 1x at 150W for 40s each,. Samples were gradually infiltrated with increasing resin to propylene oxide ratio up to full resin 2x at 250W for 3min each under vacuum. The samples were embedded in flat silicone mold with EMBED-812 resin and cured in the oven at 60℃
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Figure 1. mutant displays overgrowth NMJ phenotype.
(A) (B) Third instar larvae were dissected and co-immunostained with HRP and Syn.
NMJ6/7 from A3 segment were photographed. 1 mutant has overgrown NMJ phenotype (B) as compared to wildtype (A). (C) Bouton number is significantly increased in1 mutant. Triple-asterisks indicate significance by Student's t test (p <
0.001) and error bars represent the standard error of the mean (SEM). (D)(E) Electron micrographic sections of type I bouton in 1/+ heterozygous control (D) and 1 larvae (E). Lower panels show electron-densed active zones with a clear T bar. Though sample number is not large enough for meaningful quantification, no significant alterations in SSR morphology, bouton size, active zone length, vesicle size and number,
Figure 2. is required presynaptically to regulate bouton growth.
The same UAS--GFP line is used in this experiment to compare the expression level of this transgene. Bar 3 and bar 4 differ statistically significant from w1118 control shown
by bar 1 (both p < 0.001). elav>-GFP rescued the bouton-overaddition phenotype in
1/2 mutant and their bouton number is not significantly different from wildtype control (p > 0.05).
Figure 3. GRD of NF1 is both required and sufficient to rescue reduced NF1
mutant body length.
(A) Isogenic fly iso2; iso3 were used to generate the NF1E2 null mutant; therefore this line was used as a control. Pupal length was measured with a caliper gauge with resolution for 0.01mm. Triple-asterisks indicate significance by Student's t test (p <
0.001). Asterisk represents significance with p < 0.05. (B) All the flies, including iso2;iso3 control and NF1E2 were put into the same heat-shock procedure at the same time. Embryos of 24hours AEL (after egg-laying) were heat-shocked in 37℃ water bath for 30 minutes. This transient heat-shock was repeated every 24 hours until late pupal stage. Then the pupal were picked out and their lengths measured under a dissecting microscope.
Figure 4. GRD of NF1 is not essential for the regulation of bouton addition.
Neuronal expression of wildtype NF1 GRD failed to rescue NF1E2 NMJ overgrowth phenotype (p=0.26). Similarly, NF1 GRD RP also failed to restore NF1E2 NMJ.
Figure 5. andNF1 regulates bouton number through cAMP-dependent pathway.
(A) w1118 wildtype larvae were reared in food containing DMSO, forskolin, distilled
(A) w1118 wildtype larvae were reared in food containing DMSO, forskolin, distilled