To test the impact of 14-3-3 proteins on αsyn pathogenesis in vivo, we examined the effect of 14-3-3θ overexpression or 14-3-3 inhibition on behavioral deficits, αsyn inclusion formation, and neuronal counts in the in vivo fibril model (Fig. 1a, b). We previously created a transgenic mouse that overexpresses 14-3-3θ tagged with the HA tag under the Thy1.2 promoter [4, 6]. This transgenic mouse expresses HA-tagged 14-3-3θ in neurons located in the cortex, hippocampus, amygdala, and other areas, but no HA-tagged 14-3-3θ is detected in dopaminergic neurons in the SN (Additional file 2: Figure S3b). This mouse was used to examine the impact of 14-3-3θ manipulation in the cortex and amygdala (Fig. 1a). We also examined the impact of 14-3-3θ overexpression in the SN by using an adeno-associated virus (AAV) expressing 14-3-3θ-GFP that was injected into the SN by stereotactic means (Fig. 1b). For testing the impact of 14-3-3 inhibition with the pan-14-3-3 peptide inhibitor difopein, we used 2 different lines expressing difopein-eYFP under the Thy1.2 promoter [20]: (1) line 132, which primarily expresses difopein-eYFP in neurons in the cortex and amygdala but not within the SN, and (2) line 166, which expresses difopein-eYFP in TH-positive neurons in the SN but does not have expression in the cortex (Fig. 4a, d; Additional file 3: Figure S4a).
14-3-3θ overexpression reduces social dominance behavioral deficits induced by αsyn fibrils
Previous studies have shown motor and/or cognitive behavioral effects in response to αsyn PFF injection into the dorsolateral striatum [24, 28, 29]. WT and 14-3-3θ transgenic littermates were injected unilaterally with monomeric or fibrillary αsyn (5 µg) into the dorsolateral striatum at 8 to 12 weeks of age. Six months after PFF injection, we examined motor and non-motor behaviors in these mice. In the open field test, we observed no difference in velocity (2-way ANOVA: genotype F (1, 45) = 0.2994, p = 0.5870; PFF treatment F (1, 45) = 1.079, p = 0.3044; interaction F (1, 45) = 0.008179, p = 0.9283) or distance traveled (2-way ANOVA: genotype F (1, 45) = 0.2940, p = 0.5903; PFF treatment F (1, 45) = 1.088, p = 0.3024; interaction F (1, 45) = 0.007373, p = 0.9320) between WT and 14-3-3θ transgenic mice, whether injected with monomeric or fibrillar αsyn (Additional file 4: Figure S2a, b). We did observe an increase in the time 14-3-3θ mice spent in the periphery of the open field arena compared to WT mice, which may suggest an increase in anxiety with 14-3-3θ overexpression; however, PFF injection did not impact time spent in the periphery (2-way ANOVA: genotype F (1, 45) = 9.556, p = 0.0034; PFF treatment F (1, 45) = 0.5662, p = 0.4557; interaction F (1, 45) = 0.6048, p = 0.4408; Additional file 4: Figure S2c). While some studies have shown a motor deficit in mice injected with PFFs at 6 mpi on the pole test or rotarod test [24], we did not observe a consistent deficit in WT animals injected with PFFs on either test at 6 mpi (Additional file 4: Figure S2d, e). We also did not observe any differences in monomeric or fibrillar αsyn-injected 14-3-3θ mice with regard to motor function on the pole test (2-way ANOVA: genotype F (1, 44) = 2.217e−005, p = 0.9963; PFF treatment F (1, 44) = 0.01661, p = 0.8980; interaction F (1, 44) = 0.08812, p = 0.7680; Additional file 4 Figure S2d) or rotarod test (2-way ANOVA: genotype F (1, 45) = 0.08034, p = 0.7781; PFF treatment F (1, 45) = 2.507, p = 0.1203; interaction F (1, 45) = 0.02684; p = 0.8706; Additional file 4: Figure S2e).
We did observe a strong effect of PFF injections in WT animals in the social dominance test, which is thought to be a measure of prefrontal cortical and amygdala function [27, 30,31,32,33], at 6 mpi (Fig. 1c). PFF-injected transgenic 14-3-3θ mice with 14-3-3θ overexpression primarily in the cortex showed a rescue of the social dominance deficit observed in WT littermates injected with PFFs (1-way ANOVA: F (2, 59) = 5.581; p = 0.0060; Fig. 1c).
Since our transgenic 14-3-3θ line does not demonstrate 14-3-3θ overexpression in the nigra (Additional file 2: Figure S3b), we tested the impact of 14-3-3θ overexpression in the SN using an adeno-associated virus (AAV) expressing 14-3-3θ-GFP [18]. WT mice were stereotactically injected with AAV-GFP or AAV-14-3-3θ/GFP into the SN at 8 weeks of age, and then monomeric or fibrillar αsyn was injected into the ipsilateral dorsolateral striatum four weeks later. While there was a slight PFF effect on the open field test (2 way ANOVA: genotype F (1, 52) = 0.04865, p = 0.8263; PFF treatment F (1, 52) = 10.01, p = 0.0026, interaction (1, 52) = 0.2352, p = 0.6297) and on the pole test (2 way ANOVA: genotype F (1, 52) = 0.02336, p = 0.8791; PFF treatment F (1, 52) = 4.251, p = 0.0442; interaction F (1, 52) = 0.7770, p = 0.3821), no significant differences were observed between GFP mice injected with monomer vs. GFP mice injected with PFFs or between GFP mice injected with PFFs and 14-3-3θ mice injected with PFFs on either pole test or open field testing at 6 mpi (Additional file 4: Figure S2i–k). No significant motor deficit was observed in mice injected with AAV-GFP or AAV-14-3-3θ/GFP with or without PFFs in the wire hang test at 6 mpi (2-way ANOVA: genotype F (1, 52) = 1.554, p = 0.2181; PFF treatment F (1, 52) = 2.342, p = 0.1320; interaction F (1, 52) = 0.2655, p = 0.6086; Additional file 4: Figure S2l).
14-3-3 inhibition exacerbates social dominance behavioral deficits induced by αsyn fibrils
We next examined whether inhibition of 14-3-3s with the pan-14-3-3 peptide inhibitor difopein affects behavioral deficits in the in vivo PFF model. Transgenic mice expressing difopein in the cortex showed a deficit in social dominance after PFF injection that was not observed in WT mice at 3 mpi (1-way ANOVA: F (2, 42) = 3.139, p = 0.05; Fig. 1d). At 6 mpi, the win rate on the social dominance test was lower in difopein mice injected with PFFs compared to WT mice injected with PFFs (1-way ANOVA: F (2, 41) = 7.359, p = 0.0019; Fig. 1e). PFF treatment showed an overall slight increase in velocity (2-way ANOVA: genotype F (1, 56) = 0.4789 p = 0.4918; PFF effect F (1, 56) = 10.57, p = 0.0020; interaction F (1, 56) = 0.2741, p = 0.6027) and distance traveled (2-way ANOVA: genotype F (1, 56) = 0.1289, p = 0.7209; PFF effect F (1, 56) = 11.94, p = 0.0011; interaction F (1, 56) = 0.6675, p = 0.4174) in mice on the open field test, but no dramatic differences were noted between individual experimental groups at 6 mpi (Additional file 4: Figure S2f, g). No differences were noted between genotype or PFF treatment in the percent time spent in the periphery of the open field arena (2-way ANOVA: genotype F (1, 56) = 0.3703, p = 0.5453; PFF treatment F (1, 56) = 0.6636; p = 0.4187; interaction F (1, 56) = 0.9522, p = 0.3333; Additional file 4: Figure S2h).
Similar to that observed with the AAV-14-3-3θ injections into the SN, expression of difopein in the SN did not impact motor function significantly. PFF or genotype did not impact distance traveled (2-way ANOVA: genotype effect F (1, 60) = 0.6861, p = 0.4108; PFF treatment F (1, 60) = 0.2927, p = 0.5905; interaction F (1, 60) = 5.109; p = 0.0274) or velocity (2-way ANOVA: genotype effect F (1, 60) = 0.2982, p = 0.5870; PFF treatment F (1, 60) = 0.03472, p = 0.8528; interaction F (1, 60) = 13.13, p = 0.0006) on the open field test at 6 mpi, although a slight interaction effect was noted (Additional file 4: Figure S2m, n). WT and nigral difopein mice did not demonstrate any motor deficit on the rotarod test at 6 mpi (2-way ANOVA: genotype F (1, 56) = 0.6888, p = 0.4101; PFF treatment F (1, 56) = 2.386, p = 0.1281; interaction F (1, 56) = 0.09658, p = 0.7571; Additional file 4: Figure S2o, p).
14-3-3θ overexpression delays αsyn inclusion formation
Given the reversal of the social dominance deficit in 14-3-3θ mice, we next examined the impact of 14-3-3θ on αsyn inclusion formation. At 3 mpi, we observed a dramatic number of inclusions that stained positive for phosphorylated S129-αsyn (pS129-αsyn) in the sensorimotor cortex of WT mice injected with PFFs (Fig. 2a, c). By 6 mpi, inclusion numbers in WT mice were dramatically reduced (Fig. 2d). Other groups have also noticed a decline in αsyn inclusions over time in mice, presumably due to the loss of neurons that develop inclusions [34,35,36]. Monomer-injected mice failed to stain for pS129-αsyn (Additional file 2: Figure S3a). Inclusions in PFF-injected mice were primarily associated with neurons instead of glial cells, as demonstrated by co-staining for pS129-αsyn with the neuronal marker NeuN or the astrocytic marker GFAP (Fig. 2b; Additional file 2: Figure S3c). In 14-3-3θ transgenic mice injected with PFFs compared to WT mice injected with PFFs, we observed a 41% reduction in the number of inclusions that stained positive for pS129-αsyn in the cortex at 3 mpi (unpaired, two-tailed t-test: t(13) = 2.754, p = 0.0164; Fig. 2a, c). However, at 6 mpi, 14-3-3θ mice injected with PFFs showed a 2.4-fold increase in inclusion counts in the cortex compared to WT mice injected with PFFs (unpaired, two-tailed t-test: t(17) = 3.232, p = 0.0049; Fig. 2d). These inclusions in 14-3-3θ mice were also associated with neurons instead of glial cells (Fig. 2b; Additional file 2: Figure S3c). In the amygdala, we observed a 60% decrease in the number of pS129-αsyn positive inclusions at 3 mpi in 14-3-3θ transgenic mice injected with PFFs compared to WT mice injected with PFFs (unpaired, two-tailed t-test: t(15) = 2.757, p = 0.0147) but then a 60% increase in the number of inclusions in the amygdala at 6 mpi (unpaired, two-tailed t-test: t(13) = 2.193, p = 0.0471) in 14-3-3θ transgenic mice injected with PFFs compared to WT mice injected with PFFs (Fig. 2c, d). No differences in inclusion counts were noted between 14-3-3θ and WT mice at either 3 mpi (unpaired, two-tailed t-test: t(13) = 0.1161, p = 0.9094) or 6 mpi (unpaired, two-tailed t-test: t(10) = 0.5746, p = 0.5782) in the SN, in which 14-3-3θ overexpression is not seen in these transgenic mice (Fig. 2c, d). These findings suggest that inclusion formation in the cortex and amygdala is delayed by 14-3-3θ overexpression, and that higher levels of inclusions seen at 6 mpi in the 14-3-3θ mice compared to WT mice could be due to a reduction in neuronal loss.
Since our transgenic 14-3-3θ line does not demonstrate 14-3-3θ overexpression in the SN, we also examined nigral inclusion formation in the mice stereotactically injected with AAV-GFP or AAV-14-3-3θ/GFP in the SN (Fig. 3a). Consistent with our transgenic data, the number of inclusions positive for pS129-αsyn in the SN was decreased in mice injected with AAV-14-3-3θ/GFP compared to mice injected with AAV-GFP at 3 mpi (unpaired, two-tailed t-test: t(25) = 2.229, p = 0.0350 Fig. 3b, d). By 6 mpi, the number of inclusions positive for pS129-αsyn in the SN were increased in mice injected with AAV-14-3-3θ/GFP compared to mice injected with AAV-GFP (unpaired, two-tailed t-test: t(25) = 2.481, p = 0.0202; Fig. 3c, d). As observed in the 14-3-3θ transgenic mice, the pS129-αsyn inclusions were associated primarily with neurons instead of glial cells (Fig. 3e). These findings suggest that inclusion formation in the nigra is also delayed by 14-3-3θ overexpression in the SN. The subsequent increase in inclusion formation at 6 mpi could reflect a reduction in neuronal loss in mice overexpressing 14-3-3θ in the SN.
14-3-3 inhibition accelerates αsyn inclusion formation
We next examined whether inhibition of 14-3-3s with the pan-14-3-3 peptide inhibitor difopein affects αsyn inclusion formation in the in vivo PFF model. The difopein transgenic line that expresses difopein-eYFP in neurons in the cortex but not within the SN revealed increased inclusion counts in the cortex at 3 mpi compared to WT mice (unpaired, two-tailed t-test: t(29) = 2.441, p = 0.0210; Fig. 4a–c). However, at 6 mpi, inclusion counts were significantly lower by 43% in the cortical difopein mice compared to WT injected with PFFs (unpaired, two-tailed t-test: t(25) = 2.233, p = 0.0347; Fig. 4c). Similarly, in the amygdala, inclusion counts were increased by 31% at 3 mpi (unpaired, two-tailed t-test: t(21) = 2.070, p = 0.05) but showed a non-significant decrease (53%) at 6 mpi (unpaired, two-tailed t-test: t(15) = 2.021, p = 0.0616) in difopein mice compared to WT mice (Additional file 3: Figure S4).
To test the impact of 14-3-3 inhibition on aggregation in nigral neurons, we measured inclusions in the nigral difopein transgenic line. Inclusion counts in the difopein nigral mice showed a non-significant increase at 3 mpi (unpaired, two-tailed t-test: t(14) = 1.123, p = 0.2804) and decreased significantly at 6 mpi (unpaired, two-tailed t-test: t(21) = 2.355, p = 0.0283) after PFF injection compared to WT mice (Fig. 4d–f). We conclude that 14-3-3 inhibition accelerates inclusion formation in response to αsyn fibrils, and that the reduction in inclusion counts at 6 mpi could reflect an increase in neuronal loss.
14-3-3s regulate reduction in dopaminergic neuron counts induced by αsyn fibrils
As noted above, αsyn inclusion numbers are much higher at 3 months after PFF injection than at 6 months after injection in WT animals. This reduction in αsyn inclusion numbers over time is presumably secondary to the death of neurons that develop αsyn inclusions [35]. 14-3-3θ overexpression in either the nigra or cortex reduced αsyn inclusion numbers at 3 mpi, but we observed an increase in αsyn inclusions at 6 mpi in 14-3-3θ-overexpressing mice compared to control mice. We hypothesized that this delayed increase in inclusion formation with 14-3-3θ overexpression is due to the rescue of neurons that normally die in response to PFFs in control mice. To test the impact of 14-3-3θ overexpression on dopaminergic neurons in the SN, we performed stereological analysis of TH-positive neuronal counts in the nigra in mice injected with AAV-GFP or AAV-14-3-3θ/GFP. As expected, striatal PFFs induced a 25% reduction in ipsilateral dopaminergic neuron counts in control mice injected with AAV-GFP into the ipsilateral nigra at 6 mpi (2-way ANOVA: genotype F (1, 43) = 0.8197, p = 0.3703; PFF treatment F (1, 43) = 10.01, p = 0.0029; interaction F (1, 43) = 2.514, p = 0.1202; Fig. 5a, b). In contrast, AAV-14-3-3θ/GFP mice injected with PFFs did not demonstrate a significant reduction in TH-positive dopaminergic neurons compared to AAV-14-3-3θ/GFP mice injected with monomeric αsyn at 6 mpi (Fig. 5a, b). This finding suggests that the increase in αsyn inclusions in 14-3-3θ-overexpressing mice at 6 mpi may be due to a reduction in dopaminergic neuronal loss.
We next examined TH-positive neuronal counts in response to PFFs in difopein-expressing mice. Stereological analysis of TH-positive counts in the nigra revealed a greater reduction in ipsilateral dopaminergic neuronal counts in PFF-injected mice expressing difopein in the SN compared to WT mice after PFF injection at 6 mpi (2-way ANOVA: genotype F (1, 46) = 3.208, p = 0.0798; PFF treatment F (1, 46) = 58.27, p < 0.0001; interaction F (1, 46) = 4.309, p = 0.0435; Fig. 5c, e). Stereological analysis at 3 mpi showed a non-significant reduction of TH-positive dopaminergic neuron numbers in difopein nigral mice, but no reduction in TH-positive dopaminergic neurons in WT mice injected with PFFs at 3 mpi (2-way ANOVA: genotype F (1, 26) = 3.282, p = 0.0816; PFF treatment F (1, 26) = 0.3335, p = 0.5686; interaction F (1, 26) = 1.105, p = 0.3028; Fig. 5d), as previously described by others [24]. We also examined whether TH-positive cell numbers correlated with pS129-αsyn inclusion numbers, but the association varied between the different cohorts, with the only significant correlation in the difopein 6 mpi cohort (Additional file 5: Figure S5).
To assess potential neuronal loss in the cortex of WT and transgenic difopein mice, we measured counts of layer IV pyramidal neurons. Since inclusion formation primarily occurs in layer IV and V pyramidal neurons [28], we used NECAB1 as a marker for layer IV pyramidal neurons and measured counts of NECAB1-positive neurons per area (Additional file 6: Figure S6b). At 6 mpi, we observed a non-significant reduction in NECAB1-positive neuronal density in WT mice injected with PFFs compared to those injected with monomeric αsyn, while difopein mice injected with PFFs did show a significant reduction in NECAB1-positive neuronal density compared to difopein mice injected with monomeric αsyn (2-way ANOVA: genotype F (1, 27) = 1.426, p = 0.2428; PFF treatment F (1, 27) = 35.68, p < 0.0001; interaction F (1, 27) = 4.681, p = 0.0395; Additional file 6: Figure S6a, c). While not statistically significant, there was a slight trend towards decreased NECAB1 counts in difopein mice injected with PFFs compared to WT mice injected with PFFs ( Additional file 6: Figure S6a, c). These findings in the SN and cortex suggest that the reduction in αsyn inclusion counts in difopein-expressing mice at 6 mpi could be due to the increased loss of neurons in difopein mice compared to WT mice.