Hyperphosphorylated Tau in an α-synuclein-overexpressing transgenic model of Parkinson's disease

Abstract

Although clinically distinct diseases, tauopathies and synucleinopathies share a common genesis and mechanisms, leading to overlapping degenerative changes within neurons. In human postmortem striatum of Parkinson's disease (PD) and PD with dementia, we have recently described elevated levels of tauopathy, indexed as increased hyperphosphorylated Tau (p-Tau). Here we assessed tauopathy in striatum of a transgenic animal model of PD, overexpressing human α-synuclein under the platelet-derived growth factor promoter. At 11 months of age, large and progressive increases in p-Tau in transgenic mice, hyperphosphorylated at sites reminiscent of Alzheimer's disease, were noted, along with elevated levels of α-synuclein and glycogen synthase kinase 3β phosphorylated at Tyr216 (p-GSK-3β), a major kinase involved in the hyperphosphorylation of Tau. Differential Triton X-100 extraction of striata showed the presence of aggregated α-synuclein in the transgenic mice, along with p-Tau and p-GSK-3β, which was also confirmed through immunohistochemistry. After p-Tau formation, both Tau and microtubule-associated protein 1 (MAP1) dissociated from the cytoskeleton, consistent with the diminished ability of these cytoskeleton-binding proteins to bind microtubules. Increases in free tubulin and actin were also noted, indicative of cytoskeleton remodeling and destabilization. In vivo magnetic resonance imaging of the transgenic animals showed a reduction in brain volume of transgenic mice, indicating substantial atrophy. From immunohistochemical studies, α-synuclein, p-Tau and p-GSK-3β were found to be overexpressed and co-localized in large inclusion bodies, reminiscent of Lewy bodies. The elevated state of tauopathy seen in these platelet-derived growth factor-α-synuclein mice provides further confirmation that PD may be a tauopathic disease.

Figure 1. Western blots of protein levels in striata of PDGF-α-Syn overexpressing transgenic mice

Striata from PDGF-α-Syn transgenic mice and litter-mate non-transgenic mice [WT] were solubilized in RIPA buffer and analyzed by Western blots for α-Syn [A], p-GSK- 3β [A], and p-Tau [B] levels. α-Syn, p-GSK-3β and p-Tau levels were all expressed relative to GAPDH. All values are expressed as percent change relative to changes observed in WT control animals. Results are from 3–4 animals per group; [*, P < 0.05] and [**, P < 0.01] compared to age-matched WT animals. All blots are representative of samples.

Figure 2. Neurochemical changes of proteins in striata of postmortem human striata from PD patients and age-matched controls

Human striata were solubilized in RIPA buffer. [A]. Levels of α-Syn were estimated by Western blots and expressed relative to β-actin. p-Tau [B] and p-GSK-3β levels [C] were measured as described in the legend to Figure 1. All values are expressed as percent change relative to the control, non-diseased group. Results are from 6 controls and 6 PD patients. [*, P < 0.01] compared to striata from the control group. All blots are representative of samples.

Figure 3. Triton X-100 extraction of striatal lysates from PDGF-α-Syn overexpressing transgenic mice

Striata from 11 month old transgenic mice and age-matched litter mates were extracted in Triton X-100 and soluble and insoluble fractions were isolated as described under Methods. [A] α-Syn, p-GSK-3β and [B] p-Tau levels were all expressed relative to GAPDH. All values are expressed as percent change relative to changes observed in WT control animals. Results are from 3–4 animals per group; [*, P < 0.05] and [**, P < 0.01] compared to age-matched WT animals. All blots are representative of samples.

Figure 4. Immunohistochemical co-localization and distribution

(Upper Panel) Striatum stained with α-Syn (Red) and PHF-1 Tau (Green) with DAPI as merged, higher magnification with single antibody(s) and higher magnification merged. (Middle Panel) Striatum stained with α-Syn (Red) and p-GSK-3β (Green) with DAPI as merged, higher magnification with single antibody(s) and higher magnification merged. (Lower Panel) Striatum stained with p-GSK-3β (Red) and PHF-1 (Green) with DAPI as merged, higher magnification with single antibody(s) and higher magnification merged. White boxes highlighted on left panel indicate areas shown at higher magnification in right panel(s). Scale bar: 50uM.

Figure 5. Association of striatal proteins to cytosketon-free and cytoskeleton-bound fractions

Striata from 11 month old animals and age-matched litter mates were extracted in PIPES buffer and centrifuged, as described under Methods. The supernatant obtained upon PIPES extraction represented the cytoskeleton-free fraction, while the PIPES-insoluble pellet was further solubilized in SDS buffer and represented the cytoskeleton-bound fractions. [A] α-Syn and p-GSK-3β, and [B] p-Tau proteins were analyzed by Western blots, as described in legend to Fig. 3. All values are expressed as percent change relative to changes observed in WT control animals. Results are from 5–6 animals per group; [*, P < 0.05] and [**, P < 0.01] compared to age-matched WT animals. All blots are representative of samples.

Figure 6. Cytoskeletal remodeling in PDGF-α-Syn mice

Striata from 11 month old animals and age-matched litter mates were extracted to obtain isolations of cytoskeletal-free and cytoskeleton-associated fractions, as described under Methods. Total Tau, α-tubulin, β-actin and MAP1 proteins were analyzed by Western Blots and expressed relative to GAPDH. All values are shown as a percent change relative to changes observed in WT control animals. Results are from 3–6 animals per group; [*, P < 0.05] and [**, P < 0.01] with Student’s t-tests comparing age-matched WT and Tg animals. All blots are representative of samples.

Figure 7. Immunostaining of TH neurons

Immunostaining of transgenic [Tg] and wild type [Wt] mice was conducted as described in Methods. [A] Staining for TH (Green), DAPI (Blue) at lowest magnification (left) to highest magnification (right). White boxes highlighted on left panel indicate areas shown at higher magnification in right panel(s). Scale bar: 10uM. [B] TH-positive neurons in coronal sections of transgenic and wild type mice were analyzed. The data are from 6 mice each [12 months of age]. The counts in the SN were done by sterology, and no significant differences were found between transgenic and wild type mice.

Figure 8. Immunostaining demonstrating conformational changes in p-Tau in transgenic mice

Staining of p-Tau using the conformational-sensitive MC1 antibody (Red) was conducted as described in Methods, with DAPI (Blue) at lowest magnification (left) to highest magnification (right). White boxes highlighted on left panel indicate areas shown at higher magnification in right panel(s). Scale bar: 70uM.

Figure 9. Co-Immunoprecipitation studies of human samples using α-Syn antibodies

Lysates from control and PD striatum were prepared and subject to co-IP studies using α-Syn antibodies as described under Materials and Methods. Proteins present in the washed immunoprecipitates were analyzed by Western blots. Higher levels of α-Syn, p-Tau [pSer396/404] and p-GSK-3β were found present in the lysates from PD compared to control brains, consistent with increased expression of these proteins in the diseased brains.

Figure 10. In vivo molecular resonance imaging [MRI] of WT and Tg mice

Volumetric measurements after in vivo MRI were performed as described in Methods with the use of Paravision 4.0 software by defining threshold values that segmented brain mass from structures such as the ventricles and determining the pixels and area corresponding to brain on each slice. Data are the averages from two mice in each group [18–22 months of age], while the image is representative from each animal species. [*, P < 0.05] compared to age-matched WT animals.