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FMRP activity and control of Csw/SHP2 translation regulate MAPK-dependent synaptic transmission [1]

['Shannon N. Leahy', 'Department Of Biological Sciences', 'Vanderbilt University', 'Medical Center', 'Nashville', 'Tennessee', 'United States Of America', 'Chunzhu Song', 'Dominic J. Vita', 'Kendal Broadie']

Date: 2023-02

Noonan syndrome (NS) and NS with multiple lentigines (NSML) cognitive dysfunction are linked to SH2 domain-containing protein tyrosine phosphatase-2 (SHP2) gain-of-function (GoF) and loss-of-function (LoF), respectively. In Drosophila disease models, we find both SHP2 mutations from human patients and corkscrew (csw) homolog LoF/GoF elevate glutamatergic transmission. Cell-targeted RNAi and neurotransmitter release analyses reveal a presynaptic requirement. Consistently, all mutants exhibit reduced synaptic depression during high-frequency stimulation. Both LoF and GoF mutants also show impaired synaptic plasticity, including reduced facilitation, augmentation, and post-tetanic potentiation. NS/NSML diseases are characterized by elevated MAPK/ERK signaling, and drugs suppressing this signaling restore normal neurotransmission in mutants. Fragile X syndrome (FXS) is likewise characterized by elevated MAPK/ERK signaling. Fragile X Mental Retardation Protein (FMRP) binds csw mRNA and neuronal Csw protein is elevated in Drosophila fragile X mental retardation 1 (dfmr1) nulls. Moreover, phosphorylated ERK (pERK) is increased in dfmr1 and csw null presynaptic boutons. We find presynaptic pERK activation in response to stimulation is reduced in dfmr1 and csw nulls. Trans-heterozygous csw/+; dfmr1/+ recapitulate elevated presynaptic pERK activation and function, showing FMRP and Csw/SHP2 act within the same signaling pathway. Thus, a FMRP and SHP2 MAPK/ERK regulative mechanism controls basal and activity-dependent neurotransmission strength.

To investigate this hypothesis, we utilized the Drosophila neuromuscular junction (NMJ) glutamatergic model synapse with the combined use of NS, NSML, and FXS disease models. We first tested both LoF and GoF conditions in both (1) csw mutants and (2) transgenic human PTPN11 lines. In two-electrode voltage-clamp (TEVC) electrophysiological recordings, all of these mutant conditions elevate synaptic transmission. We next employed cell-targeted RNAi and spontaneous miniature excitatory junction current (mEJC) recordings to find Csw/SHP2 specifically inhibits presynaptic glutamate release probability. We next tested activity-dependent synaptic transmission using high-frequency stimulation (HFS) depression assays to show that the mutants display heightened transmission resiliency, consistent with elevated presynaptic function. We discovered that both LoF and GoF mutations impair presynaptic plasticity, with decreased short-term facilitation, maintained augmentation and post-tetanic potentiation (PTP), supporting altered presynaptic function. Consistent with elevated MAPK signaling in NS, NSML, and FXS disease models, feeding with MAPK-inhibiting drugs (Trametinib and Vorinostat) corrects synaptic transmission strength in mutants. As predicted, we found that FMRP binds csw mRNA and that FMRP loss increases Csw protein levels. Both dfmr1 and csw nulls display elevated phosphorylated ERK (pERK) in presynaptic boutons. Importantly, trans-heterozygous double mutants (csw/+; dfmr1/+) exhibit presynaptic MAPK signaling and neurotransmitter release phenotypes, indicating FMRP and Csw/SHP2 operate to control MAPK/ERK signaling and synaptic function. These discoveries link previously unconnected disease states NS, NSML, and FXS via a presynaptic MAPK/ERK regulative mechanism controlling glutamatergic transmission.

Fragile X syndrome (FXS) is similarly well characterized by hyperactivated MAPK signaling within neurons [ 13 ], and the causal Fragile X Mental Retardation Protein (FMRP) RNA-binding translational regulator is proposed to directly bind PTPN11/SHP2 mRNA [ 14 , 15 ]. FMRP also binds many other neuronal transcripts [ 16 ] and could interact with SHP2 in multiple ways to coregulate the MAPK pathway. Moreover, like the NS and NSML disease states, FXS is likewise a cognitive disorder and the leading heritable cause of intellectual disability [ 16 ]. Like NS and NSML, the Drosophila FXS disease model also manifests strongly impaired LTM consolidation [ 17 , 18 ]. Mechanistically, MAPK signaling is well known to modulate glutamatergic synaptic neurotransmission strength via the control of presynaptic vesicle trafficking dynamics and glutamate neurotransmitter release probability [ 19 ]. Consistently, FMRP is also well characterized to regulate glutamatergic synaptic neurotransmission, including presynaptic release properties and activity-dependent functional plasticity [ 20 ]. Importantly, treatment with the MAPK inhibitor Lovastatin corrects hippocampal hyperexcitability in the mouse FXS disease model and ameliorates behavioral symptoms in human FXS patients [ 21 , 22 ]. In the Drosophila FXS disease model, dfmr1 null mutants show elevated presynaptic glutamate release underlying increased neurotransmission strength [ 17 ], as well as activity-dependent hyperexcitability and cyclic increases in glutamate release during sustained high-frequency stimulation trains [ 23 ]. Based on this broad foundation, we hypothesized that FMRP regulates PTPN11 (SHP2)/Csw translation to modulate presynaptic MAPK signaling, which, in turn, controls presynaptic glutamate release probability to determine both basal neurotransmission strength and activity-dependent synaptic plasticity.

Noonan syndrome (NS) is an autosomal dominant genetic disorder caused by mutations in the mitogen-activated protein kinase (MAPK) pathway [ 1 , 2 ]. Missense mutations within the protein tyrosine phosphatase non-receptor type 11 (PTPN11) gene account for >50% of all disease cases [ 3 ]. In both patients and disease models, the MAPK pathway is hyperactivated by NS gain-of-function (GoF) mutations that disrupt the auto-inhibition mechanism between the catalytic protein tyrosine phosphatase domain and N-Src homology-2 (SH2) domain of the PTPN11 encoded SH2 domain-containing protein tyrosine phosphatase-2 (SHP2; [ 4 , 5 ]). In the NS with multiple lentigines (NSML) disease state, PTPN11 loss-of-function (LoF) mutations decrease protein tyrosine phosphatase domain catalytic activity, but the mutants nevertheless maintain a more persistently active enzyme state with temporally inappropriate SHP2 function, causing elevated MAPK pathway hyperactivation similar to the GoF disease condition [ 6 ]. Consequently, NS and NSML patients share a great many symptoms associated with elevated MAPK signaling, including cognitive dysfunction (approximately 30% of cases) as well as long-term memory (LTM) impairments [ 7 , 8 ]. The Drosophila NS (GoF) and NSML (LoF) disease models from mutation of the corkscrew (csw) homolog likewise both increase MAPK activation, with GoF and LoF also phenocopying each other [ 9 , 10 ]. Drosophila LTM training generates repetitive waves of csw-dependent neural MAPK activation, with the LTM spacing effect misregulated by csw manipulations [ 11 ]. PTPN11 GoF and LoF mutations from human patients transgenically introduced into the Drosophila model provide a powerful new means to compare with csw GoF and LoF mutants in the dissection of conserved neuronal requirements [ 12 ].

Results

Corkscrew/PTPN11 regulates high-frequency stimulation synaptic depression To further investigate how csw/PTPN11 affects presynaptic neurotransmission strength, we stimulated at a heightened frequency that has been shown to cause synaptic depression over a time course of several minutes [34–36]. Synaptic depression occurs when HFS causes synaptic vesicles to be released at a faster rate than they can be replenished in presynaptic boutons [34,37]. Based on published HFS protocols for the Drosophila NMJ [34,36,38], we compared the genetic background control (w1118), csw null LoF mutant (csw5), and patient-derived PTPN11N308D GoF mutant (elav-Gal4>PTPN11N308D) with a HFS paradigm. To determine the baseline EJC amplitudes, we first stimulated for 1 minute under basal conditions (0.5 ms suprathreshold stimuli at 0.2 Hz in 1.0 mM external [Ca2+]). We then stimulated at 100X greater frequency (20 Hz) for 5 minutes while continuously recording EJC responses. This sustained HFS train causes progressively decreased neurotransmission over time (depression). HFS transmission was quantified to analyze the synaptic vesicle readily releasable pool (RRP) and paired-pulse ratio (PPR) release probability. Representative HFS recordings and quantified results are shown in Figs 3 and S7. PPT PowerPoint slide

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TIFF original image Download: Fig 3. HFS transmission depression ameliorated in csw nulls. Prolonged HFS drives progressive synaptic amplitude depression over several minutes of continuous recording at 20 Hz (1mM Ca+2). (A) Representative nerve-stimulated EJC traces at the basal frequency (t = 0) and indicated time points during the HFS train for genetic background control (w1118, top), csw null (csw5, middle), and PTPN11N308D GoF mutant (elav-Gal4>PTPN11N308D; bottom). (B) Quantification of cumulative EJC amplitudes over the first 100 stimulations via nonlinear regression exponential for each pair tested using extra sum-of-squares F tests. (C) Quantification of the RRP of w1118 and csw5 (two-sided t test) and elav-Gal4/w1118 and PTPN11N308D (Mann–Whitney). (D) Quantification of the PPR of w1118 and csw5 (two-sided t test) and elav-Gal4/w1118 and PTPN11N308D (Mann–Whitney). Scatter plots show all data points and mean ± SEM. N = number of NMJs. Significance: p < 0.05 (*), p < 0.001 (**), p < 0.001 (***), and p < 0.0001 (****). The data underlying this figure can be found in S1 Data. csw, corkscrew; EJC, excitatory junction current; HFS, high-frequency stimulation; NMJ, neuromuscular junction; PPR, paired-pulse ratio; RRP, readily releasable pool. https://doi.org/10.1371/journal.pbio.3001969.g003 During HFS, w1118 controls exhibit a steady decrease in EJC amplitudes throughout the train (Fig 3A, top). The PTPN11N308D GoF mutants and csw5 LoF nulls show stronger maintained EJC amplitudes over time and prolonged resistance to depression (Figs 3A and S7A). RRP size was calculated by dividing the cumulative EJCs during the first 100 responses by mean mEJC amplitudes [39]. There is a sustained elevated response in both LoF and GoF mutants (Fig 3B). When compared with nonlinear regression and extra sum-of-squares, the stimulation train profiles are significantly greater for both LoF (p < 0.0001, F (2,1296) = 1064) and GoF (p < 0.0001, F (2,1996) = 705.5; Fig 3B) mutants, indicating increased resiliency to depression. The RRP size of csw5 nulls is significantly increased compared to w1118 background controls (p = 0.001, two-sided t test; Fig 3C, left). Similarly, PTPN11N308D GoF mutants exhibit an increased RRP compared to transgenic elav/+ neuronal driver controls (p = 0.047, Mann–Whitney; Fig 3C, right). PPR analyzed for both mutants shows no in change in csw5 nulls (p = 0.865, two-sided t test; Fig 3D, left) or PTPN11N308D GoF mutants (p = 0.941, Mann–Whitney; Fig 3D, right) compared to their respective controls. The depression resistance continues for 5 minutes of continuous stimulation (S7B Fig). Taken together, these results indicate mutants maintain transmission better with a HFS challenge. We therefore next turned to examining changes in activity-dependent synaptic function under both LoF and GoF mutant conditions.

Corkscrew/PTPN11 enables short-term plasticity facilitation, augmentation, and potentiation Presynaptic activity drives numerous forms of short-term plasticity dependent on release mechanisms [40,41]. In high external [Ca2+], strong stimulation results in neurotransmission depression as above, but with reduced external [Ca2+], many forms of release strengthening are revealed, including short-term facilitation and maintained augmentation during stimulation trains, and PTP following the train [42–44]. Based on published Drosophila plasticity protocols [23], we compared genetic background controls (w1118 or elav-Gal4/w1118), csw LoF nulls (csw5), and PTPN11 GoF animals (elav-Gal4>PTPN11N308D) with the stimulation paradigm illustrated in Fig 4A. To determine baseline EJC amplitudes, we stimulated at the basal frequency (0.5 ms suprathreshold stimuli/0.2 Hz in 0.2 mM [Ca2+]). We then applied a 10-Hz train for 1 minute, before returning to 0.2 Hz for PTP analyses (Fig 4A). In controls, this paradigm drives strong short-term facilitation during the initial stimuli of the train, followed by maintained transmission augmentation for the full duration of the train [42]. Following return to the basal stimulation frequency (0.2 Hz), heightened EJC amplitudes persist during the PTP period (Fig 4B; [42]). We normalized EJC amplitudes during and after the 10-Hz train to the initial mean EJC amplitude to show only transmission changes in response to stimulation. Quantified analyses on w1118 control, csw5 LoF, and PTPN11N308D GoF mutants were done for facilitation (<1 second), augmentation (>5 seconds), and PTP (following the HFS train). Representative short-term plasticity recordings and quantified results are shown in Fig 4. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Activity-dependent synaptic plasticity repressed in csw/PTPN11 mutants. Synaptic plasticity during and following a short-term stimulation train to measure facilitation, augmentation, and PTP. (A) Stimulation paradigm: 1 minute at 0.2 Hz (0.2 mM Ca2+), followed by 1 minute at 10 Hz, and then a return to 0.2 Hz. (B) Sample EJC traces at indicated time points during and following the 10 Hz train for control (w1118, top), GoF PTPN11N308D (elav-Gal4>PTPN11N308D; middle), and csw null (csw5, bottom). (C) Quantification of EJC amplitude during the 10-Hz train normalized to basal EJC amplitude for each genotype. The nonlinear regression exponential for each pair tested using extra sum-of-squares F test. (D-F) Quantification of facilitation (1 second, D) and augmentation (30 seconds, E) during the 10-Hz train, and PTP (10 seconds following train, F) normalized to the basal EJC amplitude for each genotype using Mann–Whitney/two-sided t tests. Scatter plots show all data points and mean ± SEM. N = number of NMJs. Significance: p < 0.05 (*), p < 0.001 (**), and p < 0.0001 (****). The data underlying this figure can be found in S1 Data. csw, corkscrew; EJC, excitatory junction current; GoF, gain-of-function; NMJ, neuromuscular junction; PTP, post-tetanic potentiation; PTPN11, protein tyrosine phosphatase non-receptor type 11. https://doi.org/10.1371/journal.pbio.3001969.g004 Controls exhibit robust synaptic plasticity, including short-term facilitation (<1 second), maintained augmentation (>5 seconds), and persistent PTP (Fig 4C, top two blue lines). With HFS, w1118 controls exhibit a >3-fold amplitude increase in <5 seconds, which strengthens to a 4-fold increase by 30 seconds. After the HFS train, control animals PTP at >2-fold basal transmission. In contrast, this short-term plasticity is strongly repressed in both the csw5 LoF and PTPN11N308D GoF mutants (Fig 4C, bottom two red lines). When quantified via nonlinear regression and extra sum-of-squares, stimulation train profiles significantly differ for both LoF (p < 0.0001, F (2,662) = 38.95) and GoF (p < 0.0001, F (2,374) = 25.85; Fig 4C). During initial short-term facilitation (1 second), w1118 controls show much stronger strengthening normalized to basal amplitude (2.15 ± 0.19, n = 16) compared to csw5 LoF (1.52 ± 0.14, n = 21; p = 0.005, Mann–Whitney) and a trending decrease in PTPN11N308D GoF (1.44 ± 0.16, n = 12; p = 0.229, two-sided t test; Fig 4D). With maintained augmentation during the HFS train (30 seconds), w1118 controls are highly elevated (4.27 ± 0.70, n = 16) compared to csw5 LOF (2.67 ± 0.53, n = 21; p = 0.009, Mann–Whitney) and PTPN11N308D GOF (2.91 ± 0.53, n = 12; p = 0.015, Mann–Whitney; Fig 4E). At peak PTP after the HFS train, w1118 controls exhibit a significant increase (3.02 ± 0.45, n = 16) compared to csw5 LoF (1.63 ± 0.16, n = 21; p = 0.003, Mann–Whitney; Fig 4F). Likewise, the PTPN11N308D GoF (2.58 ± 0.33, n = 11) shows significantly decreased PTP compared to elav-Gal4/w1118 controls (4.55 ± 0.5, n = 9; p = 0.003, two-sided t test; Fig 4F). These results show a role in presynaptic release dynamics, with altered responses to evoked stimulation. To understand the mechanism of these changes, we next turned to testing the role of MAPK/ERK signaling.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001969

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