Tau knockout exacerbates degeneration of parvalbumin-positive neurons in substantia nigra pars reticulata in Parkinson's disease-related α-synuclein A53T mice

Abstract

α-Synuclein (α-syn)-induced neurotoxicity has been generally accepted as a key step in the pathogenesis of Parkinson's disease (PD). Microtubule-associated protein tau, which is considered second only to α-syn, has been repeatedly linked with PD in association studies. However, the underlying interaction between these two PD-related proteins in vivo remains unclear. To investigate how the expression of tau affects α-syn-induced neurodegeneration in vivo, we generated triple transgenic mice that overexpressed α-syn A53T mutation in the midbrain dopaminergic neurons (mDANs) with different expression levels of tau. Here, we found that tau had no significant effect on the A53T α-syn-mediated mDANs degeneration. However, tau knockout could modestly promote the formation of α-syn aggregates, accelerate the severe and progressive degeneration of parvalbumin-positive (PV⁺) neurons in substantia nigra pars reticulata (SNR), accompanied with anxiety-like behavior in aged PD-related α-syn A53T mice. The mechanisms may be associated with A53T α-syn-mediated specifically successive impairment of N-methyl-d-aspartate receptor subunit 2B (NR2B), postsynaptic density-95 (PSD-95) and microtubule-associated protein 1A (MAP1A) in PV⁺ neurons. Our study indicates that MAP1A may play a beneficial role in preserving the survival of PV⁺ neurons, and that inhibition of the impairment of NR2B/PSD-95/MAP1A pathway, may be a novel and preferential option to ameliorate α-syn-induced neurodegeneration.

Keywords: SNR; neurodegeneration; parvalbumin; synuclein.

Figure. 1

Pitx3-A53T α-Syn × Tau^−/− mice showed anxious behavior at 12-month-old. (A) All the mice were weighed monthly for 18 months (M). (B) The latency to fall was quantified by the rotarod test. The horizontal movement (C) and vertical movement (D) were measured using the open-field test. (E) The ratio of time in central zone over total time with any events of mice in the open-field test was measured. (F) The pathways traced by 12- and 18-month-old mice in the open-field test. n ≥ 10 male mice per genotype per time point. Values are mean±SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (Triple transgenic versus age-matched nTg); ^$P < 0.05 (Pitx3-A53T α-Syn × Tau^+/− versus age-matched Pitx3-A53T α-Syn × hTau, Pitx3-A53T α-Syn × Tau^+/+ and Pitx3-A53T α-Syn × Tau^−/−); ^#P < 0.05, ^###P < 0.001 (Pitx3-A53T α-Syn × hTau, Pitx3-A53T α-Syn × Tau^+/+ and Pitx3-A53T α-Syn × Tau^+/− versus age-matched Pitx3-A53T α-Syn × Tau^−/−).

Figure. 2

The effects of tau on A53T α-syn-mediated mDANs degeneration and α-syn aggregation. (A) TH immunohistochemistry staining of the midbrain coronal sections of mice. Scale bar: 100 μm. (B-D) The numbers of TH⁺ neurons in the SNC (B), VTA (C), and RRF (D) of mice. (E) Western blot showed the expression levels of tau and TH in midbrains of 2-month-old mice. (F,G) Bar graphs quantified the levels of tau and TH in midbrain homogenates, respectively. (H) Immunofluorescent images showed α-syn staining (green) in the mDANs (red) at SNC regions of 2-month-old mice. Scale bar: 10 μm. (I) Western blot showed the expression levels of α-syn-positive HMW aggregates in midbrains of 12-month-old mice. (J) Bar graph quantified the levels of total α-syn in midbrain homogenates. (K) Bar graph quantified the levels of α-syn-positive HMW aggregates in midbrain homogenates. n = 5 per genotype per time point. Values are mean±SEM. ***P < 0.001 (Triple transgenic versus age-matched nTg); ^###P < 0.001 (Triple transgenic versus age-matched Pitx3 × hTau, Tau^+/− and Tau^−/−).

Figure. 3

Tau knockout accelerated the progression of A53T α-syn-mediated neurodegeneration in SNR. (A) Representative images for Fluoro-Jade C and TUNEL staining in the SNR of 12- and 18-month-old mice. Scale bar: 100 μm. (B,C) The numbers of Fluoro-Jade C⁺ and TUNEL⁺ cells in SNR. (D) TUNEL (green) was highly costained with Nissl (red) in the SNR of the triple transgenic mice at 12-month-old. Scale bar: 100 μm. (E) Percentage of TUNEL and Nissl double-positive neurons to the total number of TUNEL⁺ cells in the SNR of the triple transgenic mice at 12- and 18-month-old. n=5 per genotype per time point. Values are mean±SEM. **P < 0.01, ***P < 0.001 (Triple transgenic versus age-matched nTg); ^#P < 0.05, ^##P < 0.01 (Pitx3-A53T α-Syn × hTau, Pitx3-A53T α-Syn × Tau^+/+ and Pitx3-A53T α-Syn × Tau^+/− versus age-matched Pitx3-A53T α-Syn × Tau^−/−).

Figure. 4

Pitx3-A53T α-Syn × Tau^−/− mice developed severe and progressive degeneration of PV⁺ neurons in the SNR. (A) PV staining in the SNR of mice. Representative coronal sections of midbrain had been shown with white dotted lines demarcating the boundary between SNC and SNR. The ventrolateral area was considered as SNR. Scale bar: 100 μm. (B) The numbers of PV⁺ neurons in the SNR of mice. (C) The soma size of PV⁺ neurons in the SNR of mice. Values are mean ±SEM. *P < 0.05, ***P < 0.001 (Triple transgenic versus age-matched nTg); ^#P < 0.05 (Pitx3-A53T α-Syn × hTau, Pitx3-A53T α-Syn × Tau^+/+ and Pitx3-A53T α-Syn × Tau^+/− versus age-matched Pitx3-A53T α-Syn × Tau^−/−); ^$$P < 0.01 (18-month-old versus 6-month-old Pitx3-A53T α-Syn × Tau^−/−). (D) α-syn (green, C20) and PV (red) costaining in the SNR of 18-month-old mice. The arrowheads pointed to α-syn-positive aggregates. Scale bar: 10 μm. n = 5 per genotype per time point.

Figure. 5

Pitx3-A53T α-Syn × Tau^−/− mice specifically developed severe impairment of MAP1A in the SNR. (A,B) Western blot showed the expression levels of MAP1A in the midbrain homogenates of 2- and 12-month-old mice, respectively. (C) Western blot showed the expression levels of MAP1A in the midbrain homogenates of 18-month-old nTg and Tau^−/− mice. (D) Bar graph represented the expression levels of MAP1A in the midbrains of 2-, 6-, 12- and 18-month-old mice. Values are mean ± SEM. *P < 0.05 (12- and 18-month-old versus 6-month-old Pitx3-A53T α-Syn × Tau^−/−). (E,F) PSD-95 (green), MAP1A (red) and PV (blue) costaining in the SNR of 2- and 12-month-old mice, respectively. Arrowheads marked PSD-95 staining; arrows pointed to MAP1A staining; asterisks indicated PV⁺ neurons. Scale bar: 10 μm. n = 5 per genotype per time point.

Figure. 6

A53T α-syn mediated MAP1A impairment through the NR2B/PSD-95 pathway. (A,B) Representative immunoblotting showed the presence of α-syn, NR2B, PSD-95 and MAP1A positive bands in NR2B-immunoprecipitated protein fractions from the midbrain homogenates of 2 month-old nTg and Pitx3-A53T α-Syn × Tau^+/+ mice, respectively. Mouse or rabbit IgG were used as negative controls. (C,D) Costaining images in the SNR of 2-month-old nTg and Pitx3-A53T α-Syn × Tau^+/+ mice showed the apparent co-localization of PSD-95 and NR2B (C), α-syn and NR2B (D). Arrows pointed to the costainings. Scale bar: 10 μm. (E,F) Western blot showed the expression levels of NR2B in the midbrains of 2- and 12-month-old mice, respectively. (G) Bar graph illustrated the representative levels of NR2B. (H,I) Western blot showed the expression levels of PSD-95 in the midbrain homogenates of 2- and 12-month-old mice, respectively. (J) Bar graph showed the representative levels of PSD-95. Values are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (Triple transgenic versus age-matched nTg). n = 5 per genotype per time point.