Stress granules assembly does not alter the levels of ATXN2 and ATXN3 proteins
G3BP1 is a core nucleator of SGs, a type of cellular foci formed in response to stress in which mRNAs, translation factors, and RBPs coalesce together to prevent cellular damage31,32. Therefore, we aimed at investigating the impact of SGs assembly in ATXN2 and ATXN3 proteins dynamics, both for their pathological (ATXN2MUT and ATXN3MUT) and non-pathological forms (ATXN2WT and ATXN3WT). For that, in Neuro2a cells expressing ATXN2 (ATXN2WT: pEGFP-ATXN2-Q22 or ATXN2MUT: pEGFP-ATXN2-Q104) or ATXN3 (ATXN3WT: pEGFP-ATXN3-Q24 or ATXN3MUT: pEGFP-ATXN3-Q844), SGs assembly was pharmacologically induced using sodium arsenite (Fig. S1a). As previously reported, ATXN2 was recruited to SGs33, detected as Polyadenylate-binding protein 1 (PABP)-positive foci; however, ATXN3 was not. As expected, the mutant forms of both proteins accumulated in the form of aggregates (Fig. 1a, 1f). However, stimulation of SGs assembly did not alter the number of cells with ATXN2MUT or ATXN3MUT aggregates, compared to the control conditions (ATXN2MUT and ATXN3MUT, respectively), in which the stress stimulus was not induced (Fig. 1c, 1h). While the non-pathological forms of ATXN2 or ATXN3 did not form aggregates, the induction of SGs assembly led to the formation of aggregates-like structures in both the ATXN2WT. and ATXN3WT-expressing conditions (Fig. 1a, 1f).
SGs assembly is accompanied by the phosphorylation of eIF2α, and translation inhibition34, leading to a reduction in overall protein synthesis (Fig. S1b, c). Thus, we next investigated if the levels of ATXN2 and ATXN3 proteins were altered upon SGs assembly, by analyzing their levels through Western blot (Fig. 1b, 1g). In line with the results obtained for the aggregates, we did not observe alterations in the levels of ATXN2 or ATXN3 when SG formation was induced, compared to controls, neither in the non-pathological (Fig. 1d, 1i) nor in the pathological (Fig. 1e, 1j) protein forms.
Altogether, these results show that, although SGs assembly reduces overall protein translation, it does not interfere with the expression of ATXN2 and ATXN3 proteins nor with their aggregation.
G3BP1 overexpression reduces the number of cells with aggregates and the levels of ATXN2 and ATXN3 proteins
As observed for SGs, G3BP1 overexpression also leads to an inhibition of protein synthesis, although at a lower degree (Fig. S1b, d). The increase in G3BP1 protein levels is around 60%, compared to non-transfected controls (Fig. S2a, b). Taking this into consideration, we next aimed to investigate the impact of G3BP1 overexpression in ATXN2MUT and ATXN3MUT proteins aggregation. To achieve this goal, we co-transfected Neuro2a cells with ATXN2MUT or ATXN3MUT and G3BP1, and as controls cells were co-transfected with ATXN2MUT or ATXN3MUT and lacZ or transfected only with ATXN2MUT or ATXN3MUT (Fig. S4). As described above, expression of the mutant forms of both proteins led to the formation of aggregates, which are a hallmark of polyQ diseases (Fig. 2a, 2b). We found that G3BP1 overexpression was able to significantly reduce the number of cells with aggregates of both ATXN2MUT (ATXN2MUT+G3BP1: 0.39±0.0153, versus ATXN2MUT: 0.53±0.036, n=3, P=0.0233) and ATXN3MUT (ATXN3MUT+G3BP1: 0.35±0.036, versus ATXN3MUT: 0.66±0.073, n=3, P=0.0201), compared to control conditions (Fig. 2c, 2d).
Next, we investigated whether the observed reduction in aggregation upon G3BP1 overexpression (Fig. S2) could be associated with a reduction in the protein levels of ATXN2MUT and ATXN3MUT (Fig. 2e, 2f). Additionally, we also analyzed the impact of G3BP1 overexpression in the levels of the non-pathological forms of the proteins, respectively ATXN2WT and ATXN3WT (Fig. 2e, 2f). We found that G3BP1 overexpression was able to significantly reduce the expression levels of both ATXN2WT (ATXN2WT+G3BP1: 0.65±0.06, versus, ATXN2WT+lacZ: 0.692±0.08, n=5, P=0.04) and ATXN2MUT (ATXN2MUT+G3BP1: 0.35±0.1343, versus, ATXN2MUT+lacZ: 0.82±0.116 n=5, P=0.0076) (Fig. 2g, 2h). In the same line, we also observed a significant reduction in ATXN3WT and ATXN3MUT levels upon G3BP1 overexpression, compared to control conditions (ATXN3WT+lacZ: 0.608±0.026, versus, ATXN3WT+G3BP1: 0.3±0.071, n=5, P=0.02 and ATXN3MUT+G3BP1: 0.28±0.067, versus, ATXN3MUT+lacZ: 0.95±0.154 n=5, P=0.004, respectively) (Fig. 2i, 2j). However, no alteration was observed in mouse endogenous levels (Neuro2a cells) of Ataxin-2 and Ataxin-3 upon G3BP1 overexpression (Fig. S3). Moreover, in an additional control experiment, GFP levels were not altered when G3BP1 was overexpressed (Fig. S4). Altogether, these results show that G3BP1 overexpression reduces the levels of ATXN2 and ATXN3 proteins and decrease aggregation of their mutant forms.
Overexpression of core components of SGs, including G3BP1, can induce the formation of these foci14,35. However, we observed that, in Neuro2a cells, G3BP1 overexpression alone is less effective at inducing SGs formation, than when combined with a sodium arsenite stimulus (Fig. S5). In the same line, in fibroblasts from SCA2 and SCA3 patients, G3BP1 has a diffuse expression, which is also observed in healthy fibroblasts. On the contrary, G3BP1 condensates in PABP-positive foci upon sodium arsenite treatment (Fig. S6).
The NTF2-like domain is important for G3BP1 action on ATXN2 and ATXN3 mutant proteins
G3BP1 is an RBP with several molecular and biological functions, including mRNA binding, DNA binding36, helicase activity, and has important roles in immune response37. Overall, RBPs, including G3BP1, interact with mRNAs through specific RNA-binding domains16,38. The RNA recognition motif (RRM) of G3BP1 is known for interacting with target RNA sequences39. G3BP1 also harbors a NTF2-like domain that is involved in the nuclear shuttling of proteins through the nuclear pore complex40, facilitates protein-protein interactions41, mediates G3BP1 dimerization, and is important in SGs formation14. Therefore, to better understand G3BP1 action on mutant ATXN2 and ATXN3 aggregation and protein levels, we developed two different forms of the protein, one in which the NTF2-like domain was deleted (G3BP1-ΔNTF2) and the other with a deletion of the RRM domain (G3BP1-ΔRRM) (Fig. 3a, 3b). Next, we co-transfected Neuro2a cells with the ATXN2MUT or ATXN3MUT and either G3BP1-ΔNTF2 or G3BP1-ΔRRM, or, alternatively, with full-length G3BP1 or lacZ, as controls (Fig. 3c, 3d). Co-expression of G3BP1-ΔRRM led to a significant decrease in the number of cells with aggregates of ATXN2MUT and ATXN3MUT, compared to the lacZ control condition (ATXN2MUT+G3BP1-ΔRRM: 55±0.815 versus ATXN2MUT+lacZ: 62±0.814, n=4, P<0.001, and ATXN3MUT+G3BP1-ΔRRM: 66.5±2.305 versus ATXN3MUT+lacZ: 80.7±2.37, n=4, P<0.001, respectively) (Fig. 3e, 3f). However, when compared to the expression of the full-length G3BP1, G3BP1-ΔRRM led to a significant increase in the number of cells with aggregates of ATXN2MUT and ATXN3MUT. On the contrary, the expression of G3BP1-ΔNTF2 led to an increase in the number of cells with aggregates of ATXN2MUT and ATXN3MUT, compared to both lacZ and full length G3BP1 conditions (Fig. 3e, 3f).
Next, we analyzed the levels of ATXN2MUT and ATXN3MUT upon expression of both truncated forms of G3BP1 (Fig. 3g, 3i). We found that G3BP1-ΔRRM led to a significant reduction of the levels of ATXN2MUT and ATXN3MUT, compared to control (ATXN2MUT+G3BP1-ΔRRM: 0.48±0.035 versus, ATXN2MUT+lacZ: 0.64±0.013, n=4, P<0.001, and ATXN3MUT+G3BP1-ΔRRM: 0.725±0.001 versus, ATXN3MUT+lacZ: 0.93±0.012, n=4, P<0.001, respectively) (Fig. 3h, 3j). Conversely, the expression of G3BP1-ΔNTF2 led to a significant increase in the levels of ATXN2MUT and ATXN3MUT proteins (Fig. 3h, 3j).
Altogether, these results point to a relevant role of the NTF2-like domain in the molecular mechanism that mediates G3BP1 action on mutant ATXN2 and mutant ATXN3 protein levels and propensity to aggregate.
The Ser149 phosphorylation site is important for G3BP1 action on ATXN2 and ATXN3 mutant proteins
The NTF2-like domain of G3BP1 is in the close vicinity of the S149 phosphorylation site, which has been described to have an important functional role15–17. Since we observed that G3BP1-ΔRRM was able to reduce the levels and aggregation of ATXN2MUT and ATXN3MUT, although to a lesser extent than the full length G3BP1, we next aimed to investigate whether S149 modification was also important in this context. For that, we developed two phosphomutants of G3BP1, a phosphomimetic S149D mutant and a nonphosphorylatable S149A mutant (Fig. S7). Neuro2a cells were co-transfected with ATXN2MUT or ATXN3MUT and with G3BP1 (S149D) or G3BP1 (S140A). Using confocal imaging, we observed that cells expressing wild-type G3BP1 displayed no aggregates of ATXN2MUT or ATXN3MUT (Fig. 4a, 4b; white arrows). The same pattern was observed upon expression on the phosphomimetic G3BP1 (S149D) mutant. On the contrary, we observed aggregates of ATXN2MUT and ATXN3MUT in cells expressing the phospho-dead G3BP1(S149A) mutant (Fig. 4a, 4b; white arrow heads).
Next, we investigated the impact of the expression of the two phosphomutants on the protein levels of ATXN2MUT and ATXN3MUT (Fig. 4c, 4d). We found that the protein levels of ATXN2MUT were significantly increased with G3BP1 (S149A) expression (ATXN2MUT + G3BP1: 0.24±0.026 versus ATXN2MUT + G3BP1(S149A): 0.37±0.028, n=3, P<0.05) (Fig. 4e). On the other hand, the protein levels of ATXN2MUT were similar between cells co-expressing wild-type G3BP1 or the phosphomimetic G3BP1 (S149D) mutant (Fig. 4e). Consistently, the protein levels of ATXN3MUT are increased upon G3BP1(S149A) expression, compared to the wild-type G3BP1 and G3BP1(S149D) expressing conditions (Fig. 4g). The expression of G3BP1(S149D) also led to a significant reduction in the levels of ATXN3MUT, as compared to the wild-type G3BP1 condition (ATXN3MUT + G3BP1: 0.36±0.03 versus ATXN3MUT + G3BP1(S149D): 0.25±0.01, n=3, P<0.05).
Upon wild-type G3BP1 expression, we observed that there was a significant reduction in the mRNA levels of ATXN2MUT and ATXN3MUT, compared to control conditions (Fig. S8). However, no differences were observed in the mRNA levels of ATXN2MUT and ATXN3MUT upon the expression of the two phosphomutants, compared to the wild-type G3BP1 condition (Fig. 4f, 4h). Altogether, these results suggest that the S149 phosphorylation site is important for G3BP1 modulatory activity on the aggregation and expression of mutant ATXN2 and mutant ATXN3.
G3BP1 mRNA and protein levels are reduced in SCA2 and SCA3, whereas silencing G3BP1 increases protein aggregation in the mouse brain
Previous studies have reported that mutant polyQ proteins can dysregulate the expression of several genes1,42. In fact, we have shown that the expression of mutant ATXN3 drives an abnormal reduction of wild-type ATXN2 levels43. Taking this into consideration, we next assessed whether the levels of G3BP1 were altered in samples from SCA2 and SCA3 patients and disease models. In post-mortem brain samples of SCA2 patients, we detected a reduction in the immunodetection of G3BP1 comparing with healthy individuals, both in the striatum and the cerebellum (Fig. S9). Furthermore, in fibroblasts from SCA2 patients, we detected a significant reduction in the protein (Fig. 5a, 5c) and mRNA levels (Fig. 5d) of G3BP1, compared to fibroblasts from healthy controls. Similarly, in fibroblasts from SCA3 patients we observed a decrease in G3BP1 protein (Fig. 5b, 5e) and mRNA levels (Fig. 5f), compared to fibroblasts form healthy controls. The same reduction was also observed in cerebellar samples from a transgenic mouse model of SCA3, which we used in this study (Fig. 5g-i). G3BP1 protein and mRNA levels were significantly reduced in the transgenic SCA3 animals, compared to wild-type C57BL/6 mice. This transgenic mouse expresses a truncated form of ataxin-3 with 69 glutamines in the Purkinje cells of the cerebellum. In fact, microscopy analysis of cerebellar sections demonstrated that the reduction of G3BP1 in the transgenic animals was particular evident in these cells (Fig. S10).
To investigate the functional impact of G3BP1 levels reduction on mutant ATXN2- or mutant ATXN3-induced pathology, we next injected lentiviral vectors encoding a validated shRNA targeting murine G3bp1 (shG3bp1) (Fig. S11) into the lentiviral models of SCA2 and SCA344,45 (Fig. 5i, 5l). Briefly, one hemisphere of the striatum was co-injected with lentiviral vectors encoding for ATXN2MUT (or ATXN3MUT) and shG3bp1, while in the contralateral hemisphere, as control, we injected ATXN2MUT (or ATXN3MUT) and a scramble shRNA (shScr). At 4 weeks post-injection we sacrificed the animals, and the striatum was histologically analyzed for the presence of aggregates of ATXN2MUT and ATXN3MUT (Fig. 5j, 5m). We found that the silencing of G3bp1 led to a significant increase in the average number of aggregates of ATXN2MUT (ATXN2MUT + shG3bp1: 434±55.62 versus ATXN2MUT + shScr: 228±98.85, n=4, P<0.01) and ATXN3MUT (ATXN3MUT + shG3bp1: 390±26.89 versus ATXN3MUT + shScr: 290±22.37, n=3, P<0.05) (Fig. 5k, 5n).
Altogether, these results highlight that G3BP1 mRNA and protein levels are reduced in the context of SCA2 and SCA3, and that its decrease may play a role on disease pathogenesis.
Restoring G3BP1 levels alleviates neuropathology in SCA2 and SCA3 lentiviral mouse models
The expression of ATXN2MUT and ATXN3MUT mediated by lentiviral vectors leads to the formation of intraneuronal aggregates and to the loss of neuronal markers44,45, which are neuropathological signs also found in post-mortem SCA2 and SCA3 brain tissue46–48. Thus, we next aimed to investigate whether restoring G3BP1 levels could improve abnormalities induced by ATXN2MUT and ATXN3MUT expression in vivo. For that, we co-expressed lentiviral vectors encoding ATXN2MUT (or ATXN3MUT) and human G3BP1 in one hemisphere of the striatum and, as a control, in the contralateral hemisphere, we injected lentiviral vectors encoding ATXN2MUT (or ATXN3MUT) (Fig. 6a, 6b). At 12 weeks post-injection for the SCA2 lentiviral mouse model, and at 4 weeks post-injection for the SCA3 lentiviral mouse model, animals were sacrificed, and the striatum was histologically analyzed. In both models, the expression of G3BP1 was able to significantly reduce the number of mutant protein aggregates (ATXN2MUT+G3BP1: 1466±31.13, n=5, versus ATXN2MUT: 2131±71.04, n=5, P=0.0002; ATXN3MUT+G3BP1: 6066±1958, versus ATXN3MUT: 30076±2717, n=7, P<0.0001) (Fig. 6c-d, 6f-g). The mRNA and soluble protein levels of ATXN2 and ATXN3 were also analyzed in a group of animals at 4 weeks post-injection (Fig. S12). In the SCA2 lentiviral model, no significant differences were observed in mRNA and protein levels of ATXN2MUT upon G3BP1 expression (Fig. S12a-b). On the other hand, in the SCA3 lentiviral model, there is a robust reduction in ATXN3MUT protein levels in the hemisphere expressing G3BP1 (ATXN3MUT+G3BP1: 0.285±0.04, versus ATXN3MUT: 0.413±0.08, n=4, P=0.054) (Fig. S12d-e). No alterations in ATXN3MUT mRNA levels were observed between hemispheres (Fig. S12c, S12f).
Consistently, G3BP1 expression led to a preservation of the neuronal marker dopamine- and cAMP-regulated neuronal phosphoprotein 32 (DARPP-32) in both models, as compared to the control hemispheres (ATXN2MUT+G3BP1: 0.02±0.0078, versus ATXN2MUT: 0.08±0.0078, n=5, P=0.001; ATXN3MUT+G3BP1: 0.19±0.0291, versus ATXN3MUT: 0.45±0.0647, n=7, P=0.0072) (Fig. 6c, 6e, 6f, 6h). Altogether, these results show that G3BP1 expression in the striatum promotes neuroprotection in the context of ATXN2- and ATXN3-induced neuropathology pathology, reducing protein aggregation and loss of neuronal markers.
Overexpression of G3BP1 in the brain of wild-type mice did not produce neuronal loss or astrogliosis
We next aimed to evaluate if G3BP1 expression in the brain could have a detectable negative impact, translated in the form of neuronal loss or astrogliosis. For that, lentiviral particles encoding G3BP1 were injected in one hemisphere of the striatum of wild-type C57BL/6 mice, while the contralateral hemisphere was injected with PBS, as control (Fig. 7a). At 4 weeks post-injection, the loss of the neuronal marker DARPP-32 (Fig. 7b) in the hemisphere injected with G3BP1 was significantly lower comparing to the control hemisphere injected with PBS (G3BP1: 0.003±0.0014, versus PBS: 0.01±0.0011, n=4 Student’s t-test, P=0.035) (Fig. 7b, 7c). In fact, in the G3BP1-injected animals the lesion area was restricted to the injection site. Astrocytes activation was analyzed by detection of the GFAP marker and compared between the G3BP1-injected hemisphere and the PBS-injected control hemisphere (Fig. 7d). No differences were found in the immunoreactivity of GFAP between hemispheres (Fig. 7e). Altogether, these results suggest that G3BP1 overexpression in the normal brain does not produce gross histological changes.
Restoring G3BP1 levels in the cerebellum mitigates behavior deficits and neuropathological abnormalities in a SCA3 transgenic mouse model
PolyQ SCAs are characterized by progressive neuronal loss and motor dysfunctionality. Thus, to model this phenotype, we used the above-mentioned transgenic mouse line expressing a truncated form of mutant ATXN3 with 69 glutamines in cerebellar Purkinje cells, which displays severe motor dysfunctions and neurodegeneration with an early onset49. This line can also be envisioned as a relevant general model of polyQ diseases, considering that it only contains a small region of the ataxin-3 protein and a significant tract of glutamines, sufficient for causing pathology as observed in other polyQ diseases1,49. We aimed to investigate the impact of G3BP1 expression in this transgenic mouse model, which we determined to present reduced levels of G3BP1 (Fig. 5g-i). For that, at 4 weeks of age, animals were stereotaxically injected into the cerebellum50 with lentiviral vectors encoding for G3BP1, while control animals were injected with lentiviral vectors encoding for GFP. A third group of non-injected animals was also included in the experiments.
Animals were subjected to a battery of behavior tests every 3 weeks, until 9 weeks post-injection. We observed that, at this final timepoint, G3BP1-injected animals stayed for longer times in the rotating rotarod, comparing to the control animals, thus showing an amelioration of motor deficits (G3BP1: 1.45±0.0124, n=7, versus NI: 0.84±0.1082, n=7, P=0.0254) (Fig. 8a). Consistently, at 9 weeks post-injection, compared to control animals G3BP1-injected animals took less time to complete a swimming test, in which the mice had to cross a pool to reach a safe platform, (G3BP1: 0.55±0.0974, n=7, versus NI: 0.99±0.173, n=7, P=0.0476) (Fig. 8b). Finally, in a footprint patterns test that analyses diverse gait parameters extracted from the prints that mice leave when crossing a white sheet tunnel with their paws painted, the animals injected with G3BP1 had a better footprint overlap, as compared to control animals, which suggests a mitigation of motor deficits (G3BP1: 1.06±0.1081, n=7, versus NI: 1.62±0.1997, n=7, P=0.0297) (Fig. 8c). Altogether, these results show that G3BP1 expression in the cerebellum can ameliorate motor deficits.
Neuropathologically, this mouse model is characterized by the formation of aggregates in cerebellar Purkinje cells, a severe reduction in the number of these cells and a marked disarrangement of cerebellar layers architecture49,51. Thus, we next aimed to evaluate the impact of G3BP1 expression on these neuropathological abnormalities (Fig. 8d). In agreement with the improvements observed in motor deficits, we found that animals injected with G3BP1 exhibited a significantly reduced number of pathological aggregates (HA-tagged), compared to controls (G3BP1: 63.12±10.17, n=6, versus NI: 101.2±15.29, n=6, P=0.0397) (Fig. 8e). We also found that, compared to controls, G3BP1-injected animals showed a preservation in the number of Purkinje cells, imaged using calbindin as a marker (G3BP1: 1.62±0.2405, n=6, versus NI: 0.91±0.1904, n=6, P=0.0437) (Fig. 8f). Importantly, in non-transduced lobes, no differences were found between the experimental groups regarding the number of pathological aggregates (G3BP1: 44.45±7.169, n=6, versus NI: 49.7±9.385, n=6) or the number of cerebellar Purkinje cells (G3BP1: 1±0.1457, n=6, versus NI: 0.97±0.1988 n=6) (Fig. S13). As these transgenic animals present a strong atrophy of the cerebellum, we next analyzed the thickness of cerebellar layers. We found that the molecular layer thickness of transduced lobules (II/III) was significantly wider in G3BP1-injected animals than non-injected controls (G3BP1: 64.99±3.189, n=6 versus NI: 56.03±1.824, n=6, P<0.0118), while no difference was found in non-transduced lobules (Fig. S14).
Altogether, these results show that promoting G3BP1 expression in the cerebellum of a polyQ SCA disease model significantly reduced motor behavior impairments and neuropathological abnormalities.