Ras is required for glutamate-mediated activation of PI3K
Glutamate application to filleted third instar Drosophila larvae activates PI3K in motor nerve terminals, as assayed by monitoring levels of phosphorylated Akt (p-Akt), which are increased by PI3K (Colombani et al. 2005; Dionne et al. 2006; Howlett et al. 2008; Palomero et al. 2007). Glutamate activates PI3K via the single metabotropic glutamate receptor DmGluRA: the DmGluRA112b null mutation, or presynaptic RNAi knockdown of DmGluRA, each block this glutamate-evoked PI3K activation (Howlett et al. 2008).
PI3K is activated by either the p85 regulatory subunit or Ras (Fig. 1). To determine if Ras is required for DmGluRA-mediated PI3K activation, we inhibited Ras activity in three ways. First, we used the D42 motor neuron Gal4 driver (Brand & Perrimon 1993; Parkes et al. 1998) to express the dominant-negative RasN17 variant within motor neurons (Lee et al. 1996). This RasN17 mutant sequesters the guanine nucleotide exchange factor, an essential Ras activator, thus preventing activation of the WT Ras (Powers et al. 1989). We term larvae of this genotype ‘D42>RasN17’ to indicate that the D42 Gal4 element drives expression of the UAS-driven RasN17 transgene. Second, we used the Gal4 system to express a Ras-RNAi construct in motor neurons (termed D42>Ras-RNAi). Third, we constructed larvae heterozygous for two chromosomal Ras mutations, Rase2F and Ras12a, which in combination decrease Ras activity sufficiently to confer phenotypes, but retain sufficient activity to maintain viability (Zhong 1995). We found that whereas glutamate application to WT larvae induced a significant increase in p-Akt levels (Fig. 2; t(63) = 3.17, P = 0.002), any of the three methods employed for decreasing Ras activity blocked the ability of glutamate to increase p-Akt levels (Fig. 2; D42>RasN17, t(46) = 1.76, P = 0.092; D42>Ras-RNAi, t(56) = 0.569, P = 0.571; Rase2F/Ras12a, t(58) = 0.436, P = 0.665). These observations suggest that Ras is a critical intermediate in the PI3K activation mediated by glutamate-liganded DmGluRA.
Figure 1. PI3K activation by Ras-dependent and Ras-independent mechanisms. The excitatory neurotransmitter glutamate activates PI3K via the metabotropic glutamate receptors at both mammalian central synapses and the Drosophila nmj, shown here. Many of the neuronal responses to PI3K activation, including increased nerve terminal growth and decreased neuronal excitability, occur via the phosphorylation and activation of the downstream kinase Akt. However, it is not clear if this PI3K activation occurs via Ras-dependent, p85 independent or Ras-independent, p85 dependent mechanisms, or by both mechanisms.
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Figure 2. Ras is required for glutamate-induced p-Akt increases in larval motor nerve terminals. (a) Representative images of larval nmjs of WT, in larvae expressing the UAS-driven transgenes RasN17 and Ras-RNAi in motor neurons (D42>RasN17 and D42>Ras-RNAi), and in Rase2F/Ras12A, PI3KA gen-PI3K+/PI3KA and PI3KA gen-PI3KRBD/PI3KA. Neurons were labelled with anti-p-Akt either without or following a 1 min application of 100 μm glutamate, indicated either as ‘−’ or ‘+’, respectively. Preparations were also labelled with anti-HRP to enable visualization of motor nerve terminals. White arrowheads indicate p-Akt immunoreactivity. Scale bar as indicated. (b) Means ± SEMs of normalized pixel intensities (Y-axis) of the indicated genotypes (X-axis). Nmjs, marked by anti-HRP, were traced using the Image-J (NIH, Bethesda, MD) freehand selection tool and the selection was transferred to the anti-p-Akt image where pixel intensity value was quantified. p-Akt staining intensity was measured either without or following a 1 min application of 100 μm glutamate. The pixel intensity of the anti-p-Akt staining was averaged for each genotype and normalized to the average intensity of the control preparation: values from Rase2F/Ras12A, PI3KA gen-PI3K+/PI3KA and PI3KA gen-PI3KRBD/PI3KA were normalized to WT, whereas values from D42>RasN17 and D42>Ras-RNAi were normalized to D42>+ (data not shown). For all genotypes, n = 30. The following genotypes showed significant differences in basal p-Akt intensity, computed as P-values derived from Student's t-test: WT vs. PI3KA gen-PI3K+/PI3KA, P < 0.001; vs. PI3KA gen-PI3KRBD/PI3KA, P < 0.001. No significant differences in basal p-Akt were found for the following comparisons: D42>+ vs. D42>RasN17, P = 0.936; vs. D42>Ras-RNAi, P = 0.532; WT vs. Rase2F/Ras12A, P = 0.81. Differences in p-Akt intensity prior to and following glutamate application were calculated from a Student's t-test and are shown on the figure. (c) Means ± SEMs of normalized pixel intensities (Y-axis) of the indicated genotypes (X-axis). The pixel intensity of the anti-p-Akt staining was averaged for each genotype and normalized to the average intensity of the control preparation. For all genotypes, n = 30. All data were collected in the absence of glutamate application. The following genotypes showed significant differences in p-Akt intensity, computed as P-values derived from Student's t-test: WT vs. PI3KA/PI3K+, P = 0.028; and vs. PI3KA gen-PI3K+/PI3K+, P = 0.0005. p-Akt intensity was not significantly different between PI3KA/PI3K+ and PI3KA gen-PI3K+/PI3K+ (P = 0.18).
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To confirm that Ras inhibition prevented p-Akt increases by preventing PI3K activation, rather than via a distinct Ras effector pathway, we tested the prediction that a PI3K variant that cannot be activated by Ras would fail to be activated by glutamate application. This PI3K variant, termed ‘PI3KRBD’, carries a four amino acid substitution in the PI3K Ras binding domain that prevents activation by Ras but permits activation by p85 (Orme et al. 2006; Pacold et al. 2000). This variant was introduced into a genomic clone, which placed this allele under transcriptional control of the PI3K endogenous promoter, and then introduced into Drosophila as a transgene (Orme et al. 2006). A control transgene, encoding PI3K+, was constructed and introduced into Drosophila in parallel. Then the PI3KA deletion, which removes about 300 amino acids (including the kinase domain) from the C terminus of PI3K (Halfar et al. 2001; Weinkove et al. 1999), was introduced into each fly line above by a genetic cross. Thus, the transgene-based PI3KRBD or PI3K+ (called gen-PI3KRBD and gen-PI3K+ to distinguish these transgenes from the endogenous PI3K) were the sole source of active PI3K in these flies. gen-PI3K+ behaves equivalently to endogenous PI3K+ in all assays applied (Orme et al. 2006). Finally, PI3KA, gen-PI3K+ and PI3KA, gen-PI3KRBD flies were each crossed to flies bearing PI3KA, and experiments were performed on the resulting larvae.
We found that flies expressing gen-PI3K+ displayed a robust glutamate-evoked p-Akt increase (Fig. 2, t(113) = 4.79, P < 0.0005). In contrast, glutamate-evoked p-Akt increases were blocked in larvae expressing the gen-PI3KRBD allele (t(77) = 6.31, P = 0.55). These experiments confirm that Ras is a critical intermediate in the activation of PI3K by glutamate.
We also noted that basal p-Akt levels (p-Akt levels prior to glutamate application) were decreased in larvae carrying gen-PI3K+ in the PI3KA background (Fig. 2). The decrease in basal p-Akt in gen-PI3K+ might be a consequence of decreased PI3K gene dosage in these larvae. Alternatively, this decrease could reflect properties of the PI3KA allele: PI3KA is predicted to encode a 668 amino acid protein truncated at the C terminus, which might confer partial dominant-negative properties. To distinguish between these possibilities, we compared basal p-Akt levels in WT larvae, in PI3KA/PI3K+ larvae, and in PI3KA, gen-PI3K+/PI3K+ larvae. We found that larvae expressing PI3KA exhibited significantly less basal p-Akt than WT regardless of whether these larvae simultaneously expressed one dose (PI3KA/PI3K+) or two doses (PI3KA, gen-PI3K+/PI3K+) of PI3K+ (Fig. 2c, WT vs. PI3KA/PI3K+, t(56) = 2.25, P = 0.029; WT vs. PI3KA gen-PI3K+/PI3K+, t(56) = 3.69, P < 0.001.). We conclude that expression of PI3KA decreases basal p-Akt levels, most likely because PI3KA encodes a PI3K fragment with dominant-negative activity.
Basal p-Akt levels were even further decreased in gen-PI3KRBD larvae (Fig. 2), suggesting that PI3K activity in motor neurons is chronically decreased when activation by Ras is prevented. A similar decrease in basal p-Akt levels and a block in glutamate-evoked p-Akt increases were reported when either of two additional molecules, the Calcium/Calmodulin-dependent kinase II (CaMKII) and the non-receptor tyrosine kinase focal adhesion kinase (DFak), were inhibited (Lin et al. 2011).
The ability of PI3K to decrease motor neuron excitability requires Ras
In addition to blocking glutamate-evoked increases in p-Akt, the DmGluRA112b null mutation also increases motor neuron excitability (Bogdanik et al. 2004). This increased excitability results from an inability to activate PI3K: transgene-induced PI3K inhibition in motor neurons, accomplished by expressing the dominant-negative PI3KDN (Leevers et al. 1996), increases motor neuron excitability similarly to DmGluRA112b, whereas transgene-induced PI3K activation, accomplished by expressing the constitutively active PI3K-CAAX, decreases excitability (Howlett et al. 2008). Furthermore, PI3K activation is completely epistatic to DmGluRA112b for the control of neuronal excitability: excitability in motor neurons expressing PI3K-CAAX is unaffected by the DmGluRA genotype (Howlett et al. 2008).
If Ras is required for the DmGluRA-dependent activation of PI3K, as the experiments shown in Fig. 2 suggest, then preventing Ras from activating PI3K would similarly be predicted to increase motor neuron excitability. Thus we tested the prediction that Ras inhibition, or the presence of gen-PI3KRBD, would increase neuronal excitability in a manner similar to DmGluRA112b or PI3KDN.
To measure neuronal excitability, we used the same readout as was employed to measure neuronal excitability in DmGluRA112b and in PI3KDN: the rate of onset of a phenomenon termed ‘long term facilitation’ (LTF) (Jan & Jan 1978). LTF is a form of synaptic plasticity induced when a larval motor neuron is subjected to a train of repetitive nerve stimulations at a low external [Ca2+]. Because Ca2+ is essential for neurotransmitter release to occur, nerve stimulation at this low external [Ca2+] initially induces little neurotransmitter release, and thus low amplitude depolarizations (termed excitatory junctional potentials, or EJPs) in the target muscle. However, as repetitive stimulation continues, at a certain point during the stimulus train a threshold is reached, and subsequent stimulations elicit EJPs greatly increased in amplitude and duration. These increased EJPs occur as a consequence of increased and asynchronous neurotransmitter release, which in turn results from prolonged depolarization of the nerve terminal (Jan & Jan 1978) associated with the occurrence of supernumerary action potentials within the nerve (Stern et al. 1990; Stern et al. 1995; Howlett et al. 2008).
The number of stimulations required to reach this LTF threshold (LTF onset rate) is decreased by genetic conditions that increase neuronal excitability either by increasing sodium currents or decreasing potassium currents (Jan & Jan 1978; Mallart et al. 1991; Parker et al. 2011; Poulain et al. 1994; Schweers et al. 2002; Stern & Ganetzky 1989; Stern et al. 1990; Stern et al. 1995). Both the DmGluRA112b null mutation and PI3KDN expression greatly decrease the number of nerve stimulations required to reach LTF (Bogdanik et al. 2004; Howlett et al. 2008), which indicates increased excitability in these genotypes.
The observation that Ras is required for DmGluRA-dependent PI3K activation predicts that Ras inhibition would increase the LTF onset rate in a manner similar to DmGluRA112b and PI3KDN expression. To test this prediction, we measured LTF onset rate in larvae expressing the dominant-negative RasN17 in motor neurons, and in the Rase2F/Ras12a heterozygous combination. We found that as predicted, both genotypes significantly increased LTF onset rate (Fig. 3; One-way anova and Fisher's LSD gave the following differences at 3 Hz, 5 Hz, 7 Hz and 10 Hz, respectively: For D42>+ vs. D42>RasN17, F1,22 = 6.34, P = 0.02; F1,23 = 11.9, P = 0.002; F1,23 = 14.8, P = 0.0008; F1,23 = 8.99, P = 0.006; vs. Rase2F/Ras12A, F1,16 = 4.11, P = 0.06, F1,16 = 10.7, P = 0.005, F1,16 = 10, P = 0.006, F1,16 = 7.18, P = 0.016).
Figure 3. Preventing Ras-dependent PI3K activation increases neuronal excitability. The larval neuromuscular preparation (Jan & Jan 1976) was used for all recordings. (a) Representative traces showing increased rate of onset of LTF in D42>+ larvae, in larvae expressing the UAS-driven transgenes RasN17 (D42>RasN17) in motor neurons, as well as in PI3KA gen-PI3K+/PI3KA, and PI3KA gen-PI3KRBD/PI3KA larvae. The arrowhead indicates the increased and asynchronous EJPs, indicative of onset of LTF. The bath solution contained 0.15 mm [Ca2+] and 100 μm quinidine. Nerves were stimulated at a frequency of 10 Hz for each trace. (b) (Top panel) Geometric means ± SEMs of the number of stimulations required to evoke LTF (Y-axis) at the indicated stimulus frequencies (X-axis) for the following genotypes: D42>+, D42>RasN17, Rase2F/Ras12A. From left to right, n = 12, 13, 6. (Bottom panel) Geometric means ± SEMs of the number of stimulations required to evoke LTF (Y-axis) at the indicated stimulus frequencies (X-axis) for the following genotypes: D42>+, PI3KA gen-PI3K+/PI3KA and PI3KA gen-PI3KRBD/PI3KA. From left to right, n = 12, 6, 7. One-way anova and Fisher's LSD gave the following differences at 3 Hz, 5 Hz, 7 Hz and 10 Hz, respectively: For D42>+ vs. D42>RasN17, P = 0.02, 0.002, 0.0008, 0.006; vs. Rase2F/Ras12A, P = 0.06, 0.005, 0.006, 0.016; vs. PI3KA gen-PI3K+/PI3KA, P = 0.902, 0.355, 0.449, 0.827; vs. PI3KA gen-PI3KRBD/PI3KA, P = 0.403, 0.29, 0.006, 0.002. For PI3KA gen-PI3K+/PI3KA vs. PI3KA gen-PI3KRBD/PI3KA, P = 0.612, 0.245, 0.037, 0.002.
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To confirm that Ras inhibition increases excitability by preventing PI3K activation, rather than via a distinct effector pathway, we compared LTF onset rate in larvae carrying the control gen-PI3K+ with larvae carrying gen-PI3KRBD. As shown in Fig. 3, LTF onset rate was significantly greater in gen-PI3KRBD than in gen-PI3K+ at the two higher stimulus frequencies (Fig. 3; PI3KA gen-PI3K+/PI3KA vs. PI3KA gen-PI3KRBD/PI3KA, F1,11 = 5.6, P = 0.037 and F1,11 = 17.1, P = 0.002 at 7 Hz and 10 Hz, respectively) which indicates increased motor neuron excitability in gen-PI3KRBD. The observation that an increased LTF onset rate was not observed at the lower stimulus frequencies is consistent with previous reports suggesting that LTF onset rates are not as affected at low stimulus frequencies in hyperexcitable genotypes (Howlett et al. 2008; Lin et al. 2011). We conclude that the ability of PI3K to decrease neuronal excitability requires Ras. This conclusion is consistent with the previous experiments suggesting that ligand-activated DmGluRA requires PI3K activity to decrease motor neuron excitability (Howlett et al. 2008), and that the ability of ligand activation of DmGluRA to activate PI3K requires Ras (Fig. 2).
Ras activity is not required for PI3K-mediated increases in bouton number
Bouton number at the larval nmj is regulated by PI3K activity: transgene-induced PI3K pathway inhibition, accomplished by expressing either the dominant-negative PI3KDN or PTEN, which encodes the phosphatase that opposes the effects of PI3K, decreases bouton number about twofold. In contrast, transgene-induced PI3K activation increases bouton number almost threefold (Howlett et al. 2008; Martín-Peña et al. 2006). The PI3K activators important for bouton number were not identified. However, the observation that DmGluRA112b decreases bouton number only modestly (Bogdanik et al. 2004) suggests that PI3K activators distinct from glutamate and DmGluRA might be necessary for nerve terminal growth.
It was previously reported that Ras regulates larval bouton number (Koh et al. 2002). We confirmed these findings: inhibiting Ras activity in motor neurons by expressing either the dominant-negative RasN17 or Ras-RNAi or by introducing the Rase2F/Ras12a alleles, significantly decreased bouton number (Fig. 4b, D42>+ vs. D42>RasN17, F1,49 = 6.47, P = 0.014; vs. D42>Ras-RNAi, F1,29 = 14.4, P < 0.001; WT vs. Rase2f/Ras12A, F1,45 = 31.4, P < 0.001). The larval muscles that were analysed are innervated by two motor axons that form boutons of different sizes, termed 1b and 1s (Atwood et al. 1993), and certain mutations can differentially affect 1b vs. 1s numbers (Romero-Pozuelo et al. 2007; Bhogal et al. 2011). We similarly found that altered Ras and PI3K differentially affect 1b and 1s numbers. In particular, PI3K pathway inhibition conferred by PTEN overexpression decreased each bouton type by about twofold (Fig. 4c, F1,23 = 44.3, P < 0.001; Fig. 4d, F1,23 = 11.36, P = 0.003), whereas decreased Ras activity also decreased 1b number by about twofold (Fig. 4c), but did not significantly affect 1s number (Fig. 4d). Thus, 1s bouton number is less sensitive than 1b number to decreased Ras activity.
Figure 4. Ras inhibition decreases 1b bouton number but not 1s bouton number. (a) Representative confocal images of nmjs from larvae of the indicated genotypes labelled with an antibody against HRP. Motor neurons innervating muscles 6 and 7 from segments A3 or A4 are shown. D42>PTEN, D42>RasN17 and D42>Ras-RNAi indicate larvae expressing the indicated UAS-driven transgene in motor neurons under control of the D42 Gal4 driver. (b) Mean number ± SEM of boutons (Y-axis) from nmjs of the indicated genotypes (X-axis). Number of nmjs analysed: for WT, n = 12; for D42>+, n = 20; for D42>PTEN, n = 17; for D42>RasN17, n = 31; for D42>Ras-RNAi, n = 11; for Rase2F/Ras12A, n = 27. Significant differences in bouton number among genotypes (from one-way anova and Fisher's LSD or Student's t-test, as appropriate) are indicated on the figure. (c) and (d) Mean number ± SEM of 1b boutons (c) and 1s boutons (d) (Y-axis) from nmjs of the indicated genotypes (X-axis). 1b and 1s boutons were distinguished as described previously (Atwood et al. 1993). Briefly, 1b boutons are larger (3–6 µm vs. 2–4 µm) than 1s boutons and because the two bouton types arise from distinct motor axons. Significant differences in bouton number among genotypes (from one-way anova and Fisher's LSD or Student's t-test, as appropriate) are indicated on the figure.
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Koh et al. (2002) reported that Ras activation was not sufficient to activate PI3K in motor nerve terminals. To determine if Ras activity is necessary for the PI3K-dependent increase bouton number, we compared bouton number in larvae carrying the control gen-PI3K+ with larvae carrying gen-PI3KRBD. We found WT bouton numbers in both of these genotypes (Fig. 5a,b). This observation suggests that the ability of PI3K to increase bouton number is Ras-independent. In contrast, motor neuron expression of the dominant-negative RafDN transgene decreased bouton number to a value similar to those conferred by Ras inhibition (Fig. 5a,b; D42>+ vs. D42>Raf DN, t(24) = 2.7, P = 0.012). In addition, Raf inhibition, like Ras inhibition, significantly decreases 1b bouton number (to 33 ± 7.6 vs. 51.5 ± 3.2, F1,16 = 10.6, P = 0.005) but not 1s bouton number (F1,16 = 0.59, P = 0.45;data not shown). We conclude that Ras regulates bouton number via Raf, as was reported previously (Koh et al. 2002), and that Ras activity is not required for PI3K to increase bouton number.
Figure 5. Ras regulates synaptic bouton number via Raf, not PI3K. (a) Representative confocal images of nmjs from larvae of the indicated genotypes labelled with an antibody against HRP. Motor neurons innervating muscles 6 and 7 from segments A3 or A4 are shown. D42>RafDN indicate larvae expressing the UAS-driven RafDN transgene in motor neurons under control of the D42 Gal4 driver. (b) Mean number ± SEM of boutons (Y-axis) from nmjs of the indicated genotypes (X-axis): WT, D42>+, PI3KA gen-PI3K+/PI3KA, PI3KA gen-PI3KRBD/PI3KA and D42>RafDN. From left to right, n = 12, 20, 10, 9 and 6. Differences in bouton number between genotypes (from one way anova and Fisher's LSD or Student's t-test, as appropriate) are indicated on the figure.
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