| Background: Parkinson’s disease(PD) is the most common central nervous system degenerative motor disease. The pathological characteristics showed that the relatively selective loss of midbrain dopamine neurons, and the residual dopaminergic neurons were characterized by the formation of Lewy bodies(LBs) and Lewy neurites(LNs). The toxicity of the oligomeric α-Synuclein(α-Syn) and the aggregated α-Syn will eventually cause the apoptosis of midbrain dopaminergic neurons(m DANs). The microtubule associated protein Tau is mainly distributed in the axons of neurons, and its normal physiological function is stabilizing microtubule structure and regulating microtubule assembly, which is essential for axonal growth and transport in the cell. An increasing number of studies have indicated that Tau protein is also involved in the pathogenesis of PD. Tau is also a component of the aggregated fibers in LBs, where Tau co-localized with α-Syn, and they also interacted and promoted aggregation synergistically. However, it still remains unknown the certain interaction between them, and their co-effect on the pathogenesis and pathologic process of PD. Therefore, we generated four lines of triple transgenic mice with different Tau expression(high expression, normal expression, half-dose expression and no expression), which also co-expressing A53 T mutant α-Syn especially in the m DANs, so as to a valuable mouse genetic model to investigate how the different expression of Tau affect the α-Syn-induced degeneration in PD.Objective:1. To study the effects of different expression levels of Tau on the aggregation of A53 T α-Syn.2. To study the effects of different expression levels of Tau on the neurotoxicity mediated by A53 T α-Syn.3. To further explore the possible mechanism of the interaction between α-Syn and Tau in vivo.Methods: 1. Generation of triple transgenic mice: the lines of pituitary homeobox 3(Pitx3) promoter-controlled tetracycline transactivator(t TA)(Pitx3-t TA, Pitx3) mice were crossbred with the lines of human willd-type Tau inducible transgenic(tet O-h Tau, h Tau) mice in which the expression of human wild-type Tau were under transcriptional control of tetracycline operator(tet O) and Tau-/- mice, and obtaining F1 offsprings, which only leaving Pitx3/h Tau and Pitx3/0/Tau+/- mice. Meanwhile, we also crossed the lines of human α-Syn A53 T inducible transgenic(tet O-A53 T, A53T) mice in which the expression of human α-Syn A53 T were under transcriptional control of tetracycline operator(tet O), with h Tau and Tau-/- mice to obtain A53T/h Tau and A53T/0/Tau+/- mice. Then, Pitx3/h Tau were crossbred with A53 T mice, A53T/h Tau were crossbred with Pitx3 mice, and Pitx3/0/Tau+/- were crossbred with A53T/0/Tau+/- mice. Finally, we use genotyping to obtain Pitx3/A53T/h Tau mice, Pitx3/A53T/0/Tau+/+, Pitx3/A53T/0/Tau+/-, Pitx3/A53T/0/Tau-/-, and non-transgenic(n Tg) mice.2. Behavioral experiment: using rotarod, grip strength test, and open field to detect the behavior of triple transgenic male mice and non-transgenic(n Tg) mice at 2-, 6-,12- and 18-month-old.3. Immunohistochemistry for detecting the expression of the DA neurons marker—tyrosine hydroxylase(TH) in the substantia nigra pars compacta(SNC), ventral tegmental area(VTA), and retro-rubral field(RRF), counting and analyzing the numbers of TH+ neurons.4. Immunofluorescence staining assay for detecting α-Syn, ionized calcium-binding adaptor molecule-1(Iba1), glial fibrillary acidic protein(GFAP), parvalbumin(PV), microtubule-associated protein 1A(MAP1A) and postsynaptic density-95(PSD-95) in the substantia nigra pars reticulata(SNR) of triple transgenic mice and n Tg mice..5. Jade-C and Tunel staining were used to detect the cell death and apoptosis in midbrain of triple transgenic mice and n Tg mice. And using Nissl staining to label neurons.6. Western blot analysis was used to detect the expression of Tau, TH, high molecule weight-α-Syn(HMW-α-Syn), phospho-Ser129-α-Syn(p Ser129), microtubule-associated protein 1A(MAP1A), and postsynaptic density-95(PSD-95) in the midbrain of triple transgenic mice and n Tg mice at 2-,6-,12- and 18-month-old mice.7. Using of co-immunoprecipitation(co-IP) experiments to explore whether the α-Syn and MAP1 A have a direct interaction.Results:1. Triple transgenic mice(Pitx3/A53T/0/h Tau, Pitx3/A53T/0/Tau+/+, Pitx3/A53T/0/Tau+/- and Pitx3/A53T/0/Tau-/-) displayed motor abnormalities resembling PD symptoms, including resting tremor, postural instability after brith, and started to gain less body weight at 1 months of age compared to n Tg mice(p < 0.001). Grip strength test showed taht both forelimb and hindlimb grip strength of triple transgenic mice decreased obviously than that of n Tg mice(p < 0.001), while there were no differences among the triple transgenic mice. In the rotarod test, triple transgenic mice displayed significant impairment in motor coordination and balance, and Pitx3/A53T/0/Tau+/- mice showed much weaker than the other triple transgenic mice from 6-month-old(p < 0.05). The results of open field test reflected that Pitx3/A53T/0/Tau-/- mice displayed an anxiety-like increased horizontal behavior at 12-month-old.2. Compared to n Tg mice, a robust and progressive loss of m DA neurons was observed in the SNC and VTA of triple transgenic mice, but there were no significant differences among themselves. Approximately 20% m DANs were lost in the SNC and 30% were lost in the VTA of all the triple transgenic mice at 2 months of age, and by the time the mice were 18 months of age, approximately 60% and 50% m DANs were lost in the SNC and VTA, respectively(P < 0.001). Whereas the numbers of m DANs in RRF of the triple transgenic mice developed a decrease from 6 months of age(P < 0.05), and decreased more obviously at 12-month-old(P < 0.001). Western blot analysis showed that the expression levels of TH in the triple transgenic mice were much lower than those in n Tg mice(P < 0.001), while there were no obvious differences among hemselves.3. α-Syn signals increased dramatically in the cytosol, nucleus, and processes of m DA neurons in the triple transgenic mice at each time point(2,6,12 and 18 months of age) as compared with n Tg mice. HMW-α-Syn aggregates in the midbrain homogenates of the triple transgenic mice were formed from 6 months of age and significantly increased at 12 and 18 months of age as compared with those of n Tg mice(P < 0.001), while Pitx3/A53T/0/Tau-/- mice exhibited more HMW-α-Syn expression than the other triple transgenic mice. However, there was no significant difference in the expression levels of p Ser129 α-Syn in the midbrain among all the triple transgenic mice and n Tg mice.4. Jade-C and Tunel staining showed that from 12 months of age, the triple transgenic mice developed cell apoptosis in substantia nigra pars reticulata(SNR), and Pitx3/A53T/0/Tau-/- mice displayed more apoptotic cells. In addition, GFAP and Iba1 immunofluorescence staining revealed that the activated astrocytosis and microgliosis in the SNR of Pitx3/A53T/0/Tau-/- mice obviously increased at 2 months of age as compared with the other triple transgenic mice.5. All the triple transgenic mice have developed less number of PV+ neurons in the SNR as compared with those in n Tg mice. There were no differences in thenumber of PV+ neurons in the SNR of Pitx3/A53T/h Tau, Pitx3/A53T/0/Tau+/+ and Pitx3/A53T/0/Tau+/- mice, which PV+ neurons could maintain survial until 18 months of age. However, the numbers of PV+ neurons in the SNR of Pitx3/A53T/0/Tau-/- mice decreased significantly at 12- and 18-month-old compared with those of n Tg mice(P < 0.001), and even compared with those of the other triple transgenic mice(P < 0.05).6. In the SNR of all the triple transgenic mice, Tunel+ cells also expresssed Nissl+, which suggested that the apoptosis cells were neurons. However, Neu N+ neurons were only expressed in the SNR of Pitx3/A53T/0/Tau-/- mice. And in the SNR of Pitx3/A53T/0/Tau-/- mice, before the loss of PV+ neurons(6 months of age), Neu N+ was highly coexpressed with PV+; after the loss of PV+ neurons(12 and 18 months of age), Neu N+ was highly coexpressed with Tunel+. All these findings suggested that in the SNR of Pitx3/A53T/0/Tau-/- mice, the loss of PV+ neurons may undergo a transitional stage of neuronal degeneration, and Neu N expression may be a marker labeling the degeneration of PV+ neurons in the SNR of the Pitx3/A53T/0/Tau-/- mice.7. In the SNR of triple transgenic mice, α-Syn expressed mainly arounding the PV+ neurons, and there was the most aggregated α-Syn in the SNR of Pitx3/A53T/0/Tau-/- mice. Besides the abundant loss of PV+ neurons in the SNR, we found the PV projection fibers from SNR to the striatum decreased either in the Pitx3/A53T/0/Tau-/- mice.8. The expression levels of MAP1 A in the midbrain homogenates of Pitx3/A53T/0/Tau-/- mice were increased at 2- and 6-month-old, while those were decreased significantly to a minimum at 12- and 18-month-old. However, the MAP1 A expression levels were constant in the midbrain homogenates of the other triple transgenic mice and n Tg mice until 18 months of age, and there were no differences in the expression levels of MAP1 A among the other triple transgenic mice and n Tg mice. Immunofluorescence assay revealed that MAP1 Adegraded in the SNR of Pitx3/A53T/0/Tau-/- mice from 12-month-old. However, the morphological structure and expression of MAP1 A in the SNR of the other transgenic mice and n Tg mice still did not change obviously at 18-month-old.9. The expression levels of PSD-95 in the midbrain homogenates of Pitx3/A53T/0/Tau-/- mice were decreased significantly from 2 months of age(P < 0.001), while these of the other triple transgenic mice were decreased significantly from 6 months of age(p < 0.001) as compared with those of n Tg mice. Immunofluorescence staining revealed that PSD-95 had been degraded in the SNR of Pitx3/A53T/0/Tau-/- mice from 2-month-old. However, PSD-95 degradation occurred late in the SNR of the other triple transgenic mice until they were 12-month-old.Conclusions:1. Tau reduction worsens the motor disabilities of Pitx3/A53 T mice, whereas Tau knockout further causes anxiety-like behavior in Pitx3/A53 T mice.2. Tau reduction promotes the aggregation of α-Syn and exacerbates A53 T α-Syn-mediated neurodegeneration in the SNR.3. PV+ neurons are selectively and progressively lost in the SNR of Pitx3/A53T/0/Tau-/- mice, and may undergo the processes from PV+ normal to Neu N+ neurodegeneration and then to Tunel+ apoptosis; whereas the apoptotic cells in the SNR of the other triple transgenic mice are PV- neurons.4. In the SNR, PV colocalizes with MAP1 A. With increased α-Syn aggregation, MAP1 A, compensating for Tau deficiency in Pitx3/A53T/0/Tau-/- mice at the early stage, is decreased significantly from 12 months of age. The degeneration of MAP1 A could induce the loss of PV+ neurons, which should be the main reason causing the anxiety-like behavior in Pitx3/A53T/0/Tau-/- mice. These indicate that MAP1 A may play a "protective" role in promoting the survival of PV+ neurons.5. Aggregation of α-Syn induces the expression of PSD-95 significantly decreased. The degradation of PSD-95 in the SNR of Pitx3/A53T/0/Tau-/- mice occurs earlier than that of the other triple transgenic mice and precedes the degradation of MAP1 A. These suggest that the degradation of PSD-95 may be the upstream mechanism leading to the degeneration of MAP1 A. |
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