Phenotypic characterization and behavioral examination of G3BP1 KO mice
Homozygous null mutations of the G3bp1 gene in 129/Sv mice induce embryonic lethality (Zekri et al. 2005). To circumvent the potential influence of genetic background, mice heterozygous for a null mutation in G3bp1 gene were crossed with Balb/c mice, for 10 generations. Resultant heterozygote mice were intercrossed with heterozygote 129/Sv mice. Viable homozygous knockout mice with a mixed genetic background (50% 129/Sv/50% Balb/c: 129/SvBalb/cF1) were thus obtained. Offspring homozygous for the targeted mutation were detected at a lower frequency than predicted by Mendelian laws (33% WT, 58% heterozygous, 9% KO), indicating that G3bp1−/− is lethal during fetal development. Moreover, although KO mice were viable, less than 20% of G3bp1−/− mice lived longer than 2 months (male and female). On average, the body mass of G3BP1-deficient mice was also half to three-fourth that of WT mice, and this growth retardation was increasingly apparent with age (Fig. 1a). In addition, G3BP1 KO mice (male and female) demonstrate a striking akinesia; most KO mice dragged their limbs behind them when walking (Fig. 1b).
Prior to any further behavioral investigation, we examined the general motility of male WT and G3BP1 KO mice in an open-field paradigm. As summarized in Fig. 1c, G3BP1 KO mice showed an important pattern of alterations. G3BP1 KO mice presented a decreased locomotor ability, a diminished locomotor speed and an increased immobility duration. It is important to notice that this diminution in general activity was not restricted to mobility, as decreased number of rearing and grooming behaviors were also measured. Analysis of the locomotor time-course pattern during the 10-min session showed that the cumulative activity, measured in 2-min time intervals, regularly increased for both WT and KO mice, but remained significantly lower in KO mice at all time points (repeated measure anova, p < 0.0001: F(4,49) = 69.1, n = 10, for WT; F(4,59) = 16.8, n = 12, for KO, Fig. 1d). The activity analyzed within 2-min time interval decreased significantly for WT animals (F(4,49) = 6.71, p < 0.001; Fig. 1e), but on the contrary, increased for KO animals (F(4,59) = 6.04, p < 0.001; Fig. 1e), suggesting an alteration of the spontaneous reaction to a novel environment (a putative lack of motivation to explore, for instance).
We have also analyzed the motor functions of G3BP1−/− mice. When suspended by the tail toward the floor (limb clasping test), WT mice (male and female 129/SvBalb/cF1) extend their limbs, whereas G3BP1−/− mice showed paw clasping reflex close to a bat-like posture (Fig. 1f, upper right pictures). This test was performed with limb-paralyzed and non-paralyzed KO mice, to assess if this response was only because of the physical incapacitation of the mice or if it was because of an alteration of the CNS. Quantitation of this behavior in mice ranging from 15 days to 2 years of age by scoring the position of their limbs for 15 s (Fig. 1f) demonstrated that KO mice with paralysis (‘affected’, n = 4) have the highest score, but that ‘unaffected’ KO mice (n = 4) progressively lose normal reflex with age, rapidly acquiring higher scores than WT mice (n = 6). Given that no anomalies were detected in muscles or motor neurons from G3BP1 KO mice (data not shown), it seems more likely that the ataxia-like phenotype is because of a dysfunction of the CNS.
Learning and memory were then analyzed using two tests assessing different memory processes (Maurice et al. 2008): namely, the spontaneous alternation behavior in the Y-maze assessing spatial working memory, and passive avoidance response assessing non-spatial long-term memory. G3BP1 KO mice showed a significant impairment of spontaneous alternation, as compared with WT animals, which showed a correct percentage of alternation (62.6 ± 3.4%, n = 10, for WT mice vs. 46.5 ± 4.9%, n = 11, for G3BP1 KO mice; Fig. 1g, upper panel), suggesting working memory impairment. Interestingly, the total number of arm entries did not differ significantly between WT and KO mice (Fig. 1g, lower panel), suggesting that maze exploration was not significantly affected, contrary to what could be expected from the open-field test results. During the first passive avoidance training session (T1), G3BP1 KO mice did not show a significant difference in shock sensitivity (18 ± 3, n = 11, for WT; 13 ± 2, n = 13, for KO, p > 0.05, Student's t-test), but showed a significantly higher step-down latency (Fig. 1h) that could be related to their decrease in general activity (Fig. 1c). G3BP1 KO mice showed, however, a similar significant increase in latency during T2 (Fig. 1h) and a correct learning, as the retention session latency (Fig. 1h) and percentage of animals-to-criterion (72% in WT and 82% in KO reaching an avoidance criterion, not shown) did not differ among groups.
Thus, G3BP1 appears to contribute to spatial working memory, but not to long-term memory as assessed in the passive avoidance procedure. However the general physical ability of KO mice rendered difficult a more detailed analysis of their long-term memory ability in the water-maze test.
G3BP1 localization in mouse brain
To further investigate the role of G3BP1 in different structures of the CNS, we established its expression profile by immunohistochemistry. We first confirmed its expression in WT brain and its absence in KO by western blotting (Fig. 2a), and observed that the hippocampal and cerebellar structures were maintained in the akinetic KO (Fig. 2b). In the cerebellum, however, Purkinje cells (PCs) appear affected in G3BP1 KO mice, as there was a slight Purkinje cells degeneration (assessed visually and by counting the number of Purkinje cells, PCs, by μm, with a p-value of 0.1) (Fig. 2b, lower right panel). Given that no anomalies were detected in muscles or motor neurons from G3BP1 KO mice and that PCs are the sole output of the cerebellum involved in the control of motor activity (Cheron et al. 2008), the ataxia-like phenotype might be linked to cerebellum dysfunction. However, other motor-related brain regions may also be involved in this phenotype and need to be further studied.
Figure 2. Distribution of G3BP1 in mouse brain. (a) Western blots and immunoblotting by anti-G3BP1 antibody confirm the absence of the protein in KO brain extracts and a decrease in heterozygous extracts. Anti-β-actin antibody is used as a loading control. (b) Histological staining shows conserved anatomy of hippocampus in wild-type (WT) and akinetic G3BP1 KO mice (top panels). The cerebellum architecture is also conserved in the akinetic KO (lower panels) even if there is some loss of Purkinje cells [stained with an antibody against PCP-2 (Purkinje cell protein 2)] (lower panel, right). Scale bars = 100 μm.. The box plot represents the number of Purkinje cells per micrometer, with + the median and − the mean. ml: molecular cell layer, gl: granule cell layer, PC: Purkinje cells layer. (c) G3BP1 distribution in the hippocampus (i), with magnifications of CA1 (ii) and CA3 areas (iii), in the frontal cortex (iv), and the cerebellum (v–vi, with magnification in vi). Vibratome slices of 2-month-old WT mice, 100 μm thick, were labeled with anti-G3BP1 (green). DNA was counter-stained in blue with Hoechst. Scale bars represent 50 μm (i and v), 20 μm (ii–iv), and 25 μm (vi). (d) G3BP1 is present in stress granules formed in neurons under arsenite treatment. MAP2 (microtubule-associated protein 2) staining confirms the neurons identity. Caprin-1 and T cell internal antigen 1 (TIA-1) colocalization with G3BP1 confirms that G3BP1-containing granules are SGs. Concordant with previous studies, TIA-1 is cytoplasmic and nuclear. Scale bar = 10 μm.
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Indeed, G3BP1 is found in different parts of the brain in WT, with high expression in the cerebellum and hippocampus, but also the cortex (Fig. 2c, panels a–f). G3BP1 demonstrated strong expression in the soma of the dentate granule cell layer and CA pyramidal regions of hippocampal formation (Fig. 2c, panel a, b, and c). In the cerebellum, it is predominantly found in the Purkinje cells layer (Fig. 2c, panel e–f).
To assess the formation of G3BP1-positive stress granules in neurons, we analyzed the intracellular localization of endogenous G3BP1 by performing immufluorescence in cultured hippocampal neurons. In untreated neurons, G3BP1 is distributed in large granular structures in the soma (Fig. 2d). However, when the neurons are stressed with arsenite treatment for 1h, G3BP1 becomes localized in discrete cytoplasmic structures that resemble previously described G3BP1-positive SGs (Fig. 2d). Furthermore, these granules colocalize with Caprin-1, partner of G3BP also involved in SGs assembly, and with TIA-1, a known marker of SGs. G3BP1, which is present in cells important in plasticity like in the hippocampus, might be an actor in the processes of learning/memory in mouse.
Synaptic plasticity in hippocampal CA1 neurons of mice lacking G3BP1
In view of the spatial memory impairment of the KO mice, we next characterized synaptic transmission by evoking excitatory synaptic transmission between Schaffer collaterals and CA1 neurons in 1- to 2-month-old KO mice (presenting an akinetic phenotype) and their WT littermate controls (129/Sv/Balb/cF1).
First of all, basal synaptic transmission was examined. The Input–Output curve was constructed by plotting the fEPSP amplitude obtained at various stimulation amplitudes as a function of the corresponding fiber volley. It appears that in KO mice, the Input–Ouput curve had a greater slope than that obtained with WT [5.26 ± 0.46 (r2 = 0.98; n = 4) vs. 4.98 ± 0.49 (r2 = 0.99; n = 4); p < 0.001 (anova)] (Fig. 3a). This indicates a facilitatory effect of basal transmission in KO mice.
Figure 3. Synaptic plasticity is modified in G3BP1 knockout (KO) mice. (a) Input–output relationship in wild-type (WT) and akinetic KO mice. Graphs were generated by plotting the field excitatory post-synaptic potential (EPSP) amplitude against the corresponding fiber volley amplitude obtained for different stimulation values. Data are averages of both fEPSP and fV amplitudes. (b) Short-term synaptic plasticity in KO and WT mice. Paired-pulse facilitation was elicited by pairing stimulation pulses with an interval ranging from 25 to 500 ms. In the graph, data are means ± SEM of four independent experiments conducted with akinetic KO mice and five independent experiments with WT mice. Samples traces extracted from recordings of synaptic facilitation obtained at an interpulse interval of 300 ms in WT and KO mice are shown. (c) Depression was not sustained in WT or KO mice when stimulations of 1 Hz were done during 15 min. (d) However, paired-pulse stimulations induced a robust long-term depression (LTD) in KO mice, not observed in WT. This LTD was abolished by 2-Methyl-6-(phenylethynyl)pyridine, an inhibitor of metabotropic glutamate receptors.
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Short-term synaptic plasticity was then studied, by applying paired stimuli with increasing interval from 25 to 500 ms. Paired-pulse facilitation ratio was significantly higher in KO mice (n = 4) than in WT (n = 5) at all the intervals considered (anova, p < 0.001) (Fig. 3b), demonstrating an enhancement of short-term synaptic plasticity. Thus, the invalidation of G3BP1 gene leads to a pre-synaptic facilitatory effect on synaptic transmission.
Long-lasting synaptic changes were then studied. We first examined the induction of long-term potentiation (LTP) by one train at 100 Hz (1 s) and four trains (100 Hz, 1 s) with 5-min interval between trains. However, both protocols led to transient potentiations in both WT and KO mice. This negative result can be attributed to the mouse strain used here. Indeed, although there are no data available regarding synaptic plasticity in the mouse strain Balb/c backcrossed with 129/Sv, different recent reports indicate that 129/Sv mice are unable to produce conventional LTP (Nguyen et al. 2000; Blackshaw et al. 2003). However, a substantial synaptic enhancement can be obtained by delivering a 4 × 100 Hz stimulus protocol with a 3-s interval between each 100-Hz train (Nguyen et al. 2000). We have tried such a protocol with this mouse strain: it consistently led to the occurrence of epileptic-like activity in both WT and KO mice (data not shown). LTD was also examined. In both WT and KO, conventional LTD elicited by 900 shocks delivered at 1 Hz for 15 min was not observed (Fig. 3c). However, applying paired-pulse shocks at 1 Hz for 15 min (paired-pulse Low-Frequency Stimulation, ppLFS), which did not trigger any LTD in WT (n = 6), surprisingly elicited a consistent depression in KO (n = 5) (Fig. 3d). This paired-pulse protocol is thought to involve the activation of the group 1 metabotropic glutamate receptors mGluR1/5, mGluR1 in the cerebellum and mGluR5 in the hippocampus (Collingridge et al. 2010; Lüscher and Huber 2010). The use of 2-Methyl-6-(phenylethynyl)pyridine, a specific antagonist of mGluR5, abolished the LTD observed in KO (Fig. 3d), confirming the involvement of mGluR5 in this significant induction of LTD. Thus, in absence of G3BP1, a mechanism involved in the expression of a form of LTD dependent on mGluR is exacerbated in CA1 area of the hippocampus.
Altered intracellular calcium in G3BP1 KO neurons
Synaptic transmission involves calcium transients in pre-synaptic and post-synaptic sites. We measured intracellular calcium concentration ([Ca2+]i) in primary cultures of WT and KO hippocampal neurons (from E18.5 embryos) after 12 days of culture, using the Fura-2 probe. [Ca2+]i was higher in G3BP1-deficient cells than in WT, with half of the values above 112 nM (median) for KO, against 83 nM for the WT (mean ± SEM: 98.34 nM ± 5.85 in WT vs. 167.1 nM ± 15.68 in KO) (Fig. 4a, left panel). Consistent with the fact that calcium homeostasis is disturbed in aged neurons in the hippocampus (Hajieva et al. 2009), basal [Ca2+]i was higher at later stages of in vitro development [17 days in vitro (DIV)], and the increase in calcium in KO neurons compared with WT was even more important when considering only the measurements performed at this stage (Fig. 4a, right panel).
Figure 4. Intracellular calcium is affected in G3BP1-deficient neurons. (a) Intracellular calcium concentration in cultured hippocampal neurons (from E18.5 embryos), calculated from Fura-2 emission ratio (F340/F380) at 510 nM when exciting at 340 and 380 nM, using the equation of Grynkiewicz. Knockout (KO) neurons have higher basal [Ca2+]i than wild-type (WT) neurons (left), even more in older neurons (17 DIV, 17 days in culture) (right). (b) When stimulated with (S)-3,5-dihydroxyphenylglycine (DHPG), KO hippocampal neurons elicit a higher calcium release from intracellular stores than WT neurons, as shown by the ratio of emissions of Fura-2 when excited at 340 and 380 nm (F340/F380) (top left). A typical response to DHPG in WT (top middle) and KO (top right) neuron. The increase in response is even more significant at 17 DIV (bottom). Data are represented as box plots depicting the four quartiles. n: total number of neurons measured, from at least four different embryos from three different litters. **p ≤ 0.005, ****p ≤ 0.00005, Mann–Whitney test.
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Thus, in the absence of G3BP1, calcium homeostasis is altered in hippocampal neurons. Pre-synaptic Ca2+ concentration directly regulates basal transmission and short-term plasticity by modulating glutamate release. The defects in basal synaptic transmission and STP observed in KO can therefore be associated to this altered calcium homeostasis.
LTD induced in KO hippocampal slices was dependent on mGluR activation. In neurons, DHPG application induces the production of inositol-1,4,5-triphosphate (IP3) under mGluR activation, followed by a release of calcium from internal stores after binding of IP3 to inositol-1,4,5-triphosphate receptors (ITPR or IP3R) on internal compartments membrane. Indeed, DHPG treatment (Fig. 4b, top panels) induced an increase in the ratio of emission fluorescence (F340/F380). Interestingly, this increase tended to be significantly higher in KO neurons compare to WT, with half of the values between 1.3 and 2.1 times increase of the basal ratio for the KO, compared with WT neurons where the increase never exceeds 1.59 times. This suggests an increase in ITPR response to DHPG treatment. Again, the increase in calcium release was even more significant at 17 DIV (Fig. 4b, bottom panel).