Loss of tau delays onset of motor symptoms after PFF inoculation
We showed that tau is required for synaptic and memory deficits caused by A53T mutant human α-synuclein (HuαSA53T) expression in primary neurons and in the TgA53T model [4, 5]. While tau expression did not impact αS-dependent motor abnormalities in presymptomatic mice, studies have proposed that a pathological relationship exists between tau and αS. Interestingly, recent studies show that while αS promotes pathological spreading of tau in brain , tau did not promote spreading of αS pathology [6, 11]. Therefore, we aimed to further evaluate the role of tau in onset and progression of α-synucleinopathy derived from HuαS.
In order to induce αS pathology in a temporally regulated manner, we used intramuscular (i.m.) injections of wild-type (WT) HuαS PFF into our TgA53T mouse model , as well as TgA53T mice crossed to a background lacking endogenous mTau−/− expression (TgA53T/mTau−/−) and genotypic controls (mTau−/− and nTg; Fig. 1a). Following PFF inoculations, tissue samples for histology and biochemical analyses were collected at 40- and 70-days post inoculation (dpi), as well as when the mice reached disease end stage (classified as ataxic deterioration to the point of complete hindlimb paralysis preventing ambulation; Fig. 1a). In addition, prior to 70 dpi, mice underwent a battery of behavioral tests: pole, rotarod, and open field testing.
All PFF-inoculated TgA53T animals in wildtype background developed motor symptoms and reached end stage by ~100 dpi. Significantly, the average time to reach ataxic onset and end stage was significantly delayed in TgA53T/mTau−/− compared to TgA53T (p=0.0122 and p=0.0022 respectively; Fig. 1b, c). Moreover, the progression from initial onset of motor symptoms to end stage was also significantly prolonged in TgA53T/mTau−/− compared to TgA53T (p=0.0247; Fig. 1d), indicating that the loss of tau delayed the progression of α-synucleinopathy-associated neurodegeneration.
Consistent with the delay in disease progression associated with the loss of endogenous mTau expression, behavioral analysis at 70 dpi also showed loss of tau significantly attenuates HuαSA53T-mediated behavioral abnormalities in TgA53T mice. The rotarod test of motor coordination showed that while TgA53T mice had a significantly decreased latency to fall compared to nTg and mTau−/− controls, TgA53T/mTau−/− mice had a similar latency to fall as control mice (Fig. 1e). With pole test performance, an additional indicator of motor coordination, TgA53T mice required significantly more time to perform the test than the control mice, however TgA53T/mTau−/− mice were not significantly different from the controls (Fig. 1f, g). As previously documented , both TgA53T and TgA53T/mTau−/− groups exhibit similar hyperactivity in open field test (Additional file 1: Fig. S1a). Interestingly, while the TgA53T group presented increased anxiety-associated behavior as they spent less time in the center, such behavior was not observed in TgA53T/mTau−/− mice (Additional file 1: Fig. S1b, c). Collectively, these results show that the loss of tau expression delays onset and progression of overt disease as well as intermediate behavioral deficits in the TgA53T model of α-synucleinopathy.
Loss of tau expression does not impact pS129 αS pathology in end stage TgA53T mice but leads to modest reduction in pS129 αS accumulation in presymptomatic TgA53T mice
The delay in disease/ataxic onset, as well as behavioral deficits, in the TgA53T/mTau−/− mice compared with TgA53T mice seems to contradict prior studies using mouse αS PFF inoculation model [6, 11]. Thus, we asked whether lack of tau expression impacts subcortical α-synucleinopathy in TgA53T model.
To survey if the expression of tau was correlated with bulk changes in αS pathology, we performed biochemical analysis of lysates from spinal cord and brain stem regions that are prone to develop αS pathology in the TgA53T model [3, 15]. Immunoblot analysis of spinal cord from end stage mice show similar levels of full length αS compared to age-matched controls (Fig. 2a) and a prominent increase in the phospho-serine129 αS (pS129 αS), a marker of pathological αS, in both TgA53T and TgA53T/mTau−/− mice (Fig. 2b). Further, consistent with a prior study , no difference in the levels of pS129 αS/total αS was observed between TgA53T and TgA53T/mTau−/− mice (Fig. 2c, d). To determine if there was a difference in the aggregation of αS that is not fully reflected by total pS129 αS levels, we examined Triton X-100 detergent-soluble and insoluble fractions from the spinal cord. Our results show that while PFF inoculation increases the amount of insoluble αS, expression of tau does not significantly impact the levels of insoluble αS but there was slightly increased αS in the soluble fraction (Additional file 1: Fig. S2). Moreover, tau expression did not impact the levels of SDS-stable oligomers that resolve at ~25 kDa, ~37 KDa, and > 200 kDa (Additional file 1: Fig. S2). Analysis of brainstem region showed that PFF-inoculation leads to similar increases in pS129 αS levels in both TgA53T and TgA53T/mTau−/− mice, similar to the patterns observed in the spinal cord (Additional file 1: Fig. S3).
We performed histological analysis for αS pathology (pS129 αS) to determine if the pattern of αS pathology was influenced or mediated by tau expression. The immunostained spinal cord sections were used to determine the percent (%) area covered by immunoreactivity. Consistent with the bulk biochemical analysis, our results show that both TgA53T and TgA53T/mTau−/− mice develop similar levels of pS129 αS pathology at end stage while the control animals (nTg and mTau−/− alone) do not exhibit pS129 αS staining (Fig. 3a-e). In addition, we also examined the microglia and astrocyte activation via Iba1 and GFAP immunostaining, respectively. In end stage animals, quantitative analysis of microglial activation (Iba1) showed significant activation with αS pathology but did not reveal any differences as a function of tau expression (Fig. 3f-j). Similarly, while the increase in GFAP staining with αS pathology was also significant compared to nTg and mTau−/− controls, there were no differences as a function of tau expression (Fig. 3k-o; see Additional file 1: Fig. S4 for representative low magnification images). In addition, pS129 αS, Iba1, and GFAP were elevated in end stage brainstem, cerebellum, and motor cortex regions, but not hippocampus, yet were not qualitatively different between TgA53T and TgA53T/mTau−/− mice. (Additional file 1: Fig. S5).
Because end stage of disease is reached at a later dpi in TgA53T/mTau−/− mice, it is possible that αS pathology was normalized between groups at terminal stages. Thus, we evaluated tissues at the same intermediate stage following PFF inoculation (70 dpi and 40 dpi). Similar to end stage analysis, immunoblots of spinal cord and brain stem lysates failed to show any differences in pS129 αS levels (Fig. 4 and Additional file 1: Fig. S3) or insoluble αS as a function of tau expression (Additional file 1: Fig. S6). Moreover, tau expression did not impact the levels of SDS-stable αS oligomers (Additional file 1: Fig. S6). Biochemical analysis of 40 dpi mice show lack of pS129 αS accumulation (Additional file 1: Fig. S7a, b).
We also performed histological analysis of pS129 αS and neuroinflammation as we have done for the end stage subjects above. Significantly, despite the lack of differences in bulk immunoblot analysis of total pS129 αS levels (Fig. 4), quantitative analysis of the pS129 αS staining in the spinal cord reveals a modest decrease in pS129 αS pathology in TgA53T/mTau−/− compared to TgA53T mice (Fig. 5a-e). This suggests that while the overall abundance of pS129 αS along the spinal cord is unchanged, there is a specific delay in the accumulation of pS129 αS in the grey matter of the cord. However, immunohistochemistry may not be sensitive enough to identify smaller changes in aggregates and levels of pS129 αS in white matter and in non-neuronal cells . As such, it is possible that the loss of tau expression leads to smaller pS129 αS structures that are not readily visible by light microscopy or slow accumulation of pS129 αS in larger neurons.
The analysis of microglial (Fig. 5f-j) and astrocytic (Fig. 5k-o) activation in presymptomatic animals at 70 dpi show a significant increase in microglial activation in mice with αS pathology. Consistent with the reduced αS pathology in TgA53T/mTau−/− animals, the activation status of microglia and astrocytes is reduced in these mice compared to TgA53T mice (Fig. 5j and o; see Additional file 1: Fig. S8 for representative low magnification images). In the brainstem, pS129 αS, Iba1, and GFAP were modestly increased at 70 dpi in TgA53T and TgA53T/mTau−/− mice. The overall abundance was qualitatively similar to the quantitative results seen with spinal cord sections (Additional file 1: Fig. S9). In addition, histological analysis of 40 dpi mice shows initial onset of pS129 αS was not altered by tau loss, nor was there profound inflammatory activation (Additional file 1: Fig. S7c-i).
Loss of tau expression leads to reduced neurodegeneration in presymptomatic TgA53T mice but not in end stage mice
Thus far, our results indicate that while tau is associated with modest increases in early αS pathology, tau does not seem to impact the extent of αS pathology at end stage. However, it is possible that tau might be acting downstream of αS abnormalities [4, 5]. Because loss of motor neurons is a robust neurodegenerative phenotype in the TgA53T model , we examined if tau expression affects the loss of motor neurons in the spinal cord of TgA53T mice.
Our analysis of end stage mice following PFF inoculation show that, as with the normal aging model, presence of αS pathology and limb paralysis in the TgA53T mice was accompanied by severe loss of motor neurons in the ventral horn (Fig. 6a-e). Further, the mean motor neurons per lumbar spinal cord section in TgA53T/mTau−/− mice were not different from TgA53T mice. This was further demonstrated with loss of total NeuN+ content of the ventral horn (Fig. 6f), while dorsal horn neurons were left intact (Fig. 6g). Significantly, analysis of presymptomatic animals at 70 dpi show that the early presence of αS pathology in TgA53T mice was already associated with a significant loss of ventral horn motor neurons in the lumbar spinal cord, albeit less severe than in the end stage animals (Fig. 6h-l). More important, the loss of motor neurons was significantly attenuated in the TgA53T/mTau−/− mice, despite the significant presence of αS pathology, compared to the TgA53T mice (Fig. 6l; see Additional file 1: Fig. S10 for representative low magnification images). In addition, this protection was also observed after quantification of ventral horn neurons via NeuN+ staining (Fig. 6m), while dorsal horn neurons were not affected (Fig. 6n). This suggests that while there may be a mild delay of αS pathological progression as a function of tau expression, a robust delay in neuronal toxicity may be a more likely explanation for the observed delay in disease onset and behavioral deficits observed in the TgA53T/mTau−/− mice.
Loss of tau does not impact soluble α-synuclein oligomer formation or GSK3β activity
Using a different HuαSA53T-expressing transgenic mouse line (M83), it has been previously demonstrated that TgA53T (M83) mice accumulated soluble tau oligomers with aging, and the mice treated with tau-oligomer specific antibody (TOMA) can subsequently reduce αS oligomers and aggregates . Therefore, while the loss of tau expression does not alter overt αS aggregation or SDS-stable oligomers (Fig. 2, Additional file 1: Fig. S2 & S6), we tested if the loss of tau expression reduces the levels of soluble αS oligomers in our TgA53T model using the Syn33 antibody [4, 10, 24] with non-denaturing dot blot analysis.
Dot blot analysis of buffer soluble fractions from spinal cords of both end stage and 70 dpi spinal cord lysates from TgA53T animals reveals higher levels of αS oligomers as a function of HuαSA53T expression compared to nTg and mTau−/− controls. Further, the accumulation of Syn33+ oligomeric species were comparable between TgA53T and TgA53T/mTau−/− groups (Fig. 7a, b). Consistent with specificity of Syn33 to soluble oligomers, Syn33 did not react to the detergent insoluble fractions (Fig. 7c, d). These results show that tau expression does not impact the levels of soluble oligomers recognized by Syn33 in our TgA53T model. Similar results were seen in our analysis of cortical and hippocampal tissues .
We also examined whether the αS pathology in the TgA53T mouse model is associated with obvious increases in pathological tau. However, our analysis for AT8-positive tau shows that, even at end stage, accumulation of hyperphosphorylated tau was not detected (Additional file 1: Fig. S11g). Increased activation of glycogen synthase kinase 3β (GSK3β) has been proposed as a mediator of αS-induced neuronal dysfunction [5, 7, 26, 27]. Thus, we also examined spinal cord lysates for the levels of active GSK3β, as measured by phosphorylated Tyr-216 (pY216) . Our results show that neither total GSK3β levels nor pY216-GSK3β activation are increased as a function of αS pathology, nor impacted by tau expression (Additional file 1: Fig. S11a-f).
We previously showed that α-synucleinopathy in TgA53T model was associated with chronic endoplasmic reticulum stress (ERS)  and dysfunction in autophagy-lysosomal pathways (ALP) . Because both ERS and ALP deficits follow the onset of αS pathology, we examined whether ERS and ALP in TgA53T model are affected in a tau-dependent manner. We performed biochemical (Western blot) analysis in 70 dpi and end stage spinal cord lysates for markers of ERS and ALP (Additional file 1: Fig. S12). While there was no obvious indication of ALP abnormalities at 70 dpi (Additional file 1: Fig. S12a), analysis of spinal cord lysates from end stage TgA53T mice show expected ALP abnormalities (Additional file 1: Fig. S12b). ALP markers (LC3 II/I ratio, P62, and pAMPK/AMPK ratio) in TgA53T or TgA53T/mTau−/− were not different from each other.
Analysis of ERS markers, Grp78 and p-eIF2α/eIF2α ratio, showed expected signs of chronic ERS in end stage TgA53T and TgA53T/mTau−/− mice (Additional file 1: Fig. S12b). While the levels of Grp78 was similarly increased in both TgA53T and TgA53T/mTau−/− mice, TgA53T/mTau−/− had modest but significantly reduced p-eIF2α/eIF2α ratio compared to TgA53T. No signs of ERS were seen in lysates from 70 dpi subjects (Additional file 1: Fig. S12a). Similar results for ALP and ERS markers were observed in brainstem lysates from 70 dpi and end stage animals (Additional file 1: Fig. S13). There were no signs of changes in ERS or ALP in the brainstem of 70 dpi animals (Additional file 1: Fig. S13a). However, in the brainstem, there was elevated ALP markers at end stage, but no sign of ERS in end stage subjects (Additional file 1: Fig. S13b).
Collectively, our results show that both ERS and ALP deficits associated with α-synucleinopathy in the TgA53T mouse model occurs after the αS pathology is well developed. Further, unlike the loss of motor neurons (Fig. 6) both ERS and ALP deficits seem to be independent of tau expression.
To determine whether tau mediates the initiation and progression of intraneuronal αS pathology following PFF exposure, we examined the neuronal uptake of WT αS PFF in primary hippocampal neurons cultured from nTg and mTau−/− mice. Cultured neurons were exposed to αS PFF for 2 hours. After this 2-hour incubation, PFF-containing media was removed, neurons were washed with PFF-free media to remove any extracellular PFF, and then fresh PFF-free media was added. Neuronal lysates were then collected at 0, 3, 6, 16, 24 and 48 hours following the 2-hour incubation with αS PFFs. As expected, neurons rapidly internalize αS PFF and internalized αS accumulates as a truncated protein  (Additional file 1: Fig. S14a, b). Quantitative analysis of uptake, truncation, and clearance of αS PFF shows that both nTg and mTau−/− neurons metabolize exogenous PFF almost identically (Additional file 1: Fig. S14a, b).
We next investigated whether tau expression affects αS aggregation and neuronal survival following αS PFF exposure. Primary nTg mouse hippocampal neurons were exposed to αS PFF and evaluated for the presence of pathological pS129 αS and neurodegeneration 14 days post-PFF exposure in vitro. αS PFF applied to primary hippocampal neurons in vitro led to a dose-dependent increase in pS129 αS accumulation at 14 days post-PFF in the absence of significant NeuN+ neuronal loss (Additional file 1: Fig. S14c-e). To determine if tau expression modulates PFF-induced αS aggregation in neurons, we examined the levels of pS129 αS in cultured nTg and mTau−/− cultures exposed to PFF treatment. To account for possible differences in the density of neurons and/or neurites between the cultures, the area of pS129 αS staining was normalized to the MAP2 stained area. These results show there was no difference in pS129 αS accumulation between nTg and mTau−/− neurons at 14 days post-PFF (Fig. 8a-c). The addition of αS PFF did not induce neurodegeneration in culture but did induce progressive loss of dendritic arborization (Fig. 8d, f; Additional file 1: Fig. S15). Specifically, αS PFF-induced αS aggregation in nTg neurons leads to simplification of dendritic arborization, as indicated by reduced MAP2 stained area per NeuN+ cells at 14 days post-PFF (Fig. 8d, f; Additional file 1: Fig. S15). Significantly, in mTau−/− neurons, while αS PFF induced comparable levels of pS129 αS staining at 14 days post-PFF (Fig. 8a-c), the loss of MAP2 was prevented (Fig. 8e, f). These results show that the loss of mTau expression attenuates PFF-induced neuronal toxicity without affecting the neuronal accumulation of pathological αS.