As described previously (Jiao et al., 2020), the line of pituitary homeobox 3 (Pitx3) promoter-controlled tetracycline transactivator (tTA) (Pitx3-tTA) mice, the lines of human α-syn A53T inducible transgenic mice (tetO-A53T) and human wild-type Tau inducible transgenic mice (tetO-hTau), in which the expression of human α-syn A53T and human wild-type Tau were under the transcriptional control of tetracycline operator (tetO), and the line of Tau^–/– mice were used to generate triple transgenic mice and maintained on C57BL/6J background. The mice were reared under specific-pathogen-free (SPF) conditions, with 12-h light/12-h dark cycles and fed a regular diet ad libitum. All experimental procedures performed in this study were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University.

The genotype of the mice was determined by PCR analysis of genomic DNA extracted from tail biopsy and had been further validated histologically, as described in the previous study (Jiao et al., 2020).

The mice were perfused transcardially with phosphate-buffered saline (PBS). Brains were separated and soaked in 4% paraformaldehyde for 48 h. Then, 30% sucrose solution was used for dehydration treatment. A cryostat (Leica SM 2010R, Germany) was used to cut the brains into continuous slices with a thickness of 40 μm. The following primary and secondary antibodies were used as recommended by the manufacturers: rabbit anti-human/mouse α-synuclein (α-syn; Santa Cruz Biotechnology, United States, sc-7011-R, 1:1000), mouse anti-PV (Sigma-Aldrich, United States, P3088, 1:500), rabbit anti-PV (Abcam, United States, ab11427, 1:1000), rabbit anti-c-fos (Sigma-Aldrich, United States, F7799, 1:1000), mouse anti-neuronal nucleus (NeuN; Chemicon, United States, MAB 377, 1:500), and Alexa 488 or Alexa 555 conjugated secondary antibody (Invitrogen, United States, 1:500). For antibodies produced in mice, the mouse-on-mouse immunodetection kit (Vector Laboratories, United States, BMK-2202) was used following the manufacturer’s protocol. A laser scanning confocal microscope (LSM 710; Zeiss, Germany) was used for observing and photographing. The paired images in all the figures were acquired under the same conditions and processed uniformly after collection.

According to the manufacturer’s protocol, apoptotic cells were labeled with an apoptosis detection kit (Roche, United States, 11684795910). The frozen sections were adhered to the slides and air-dried at 50°C for 30 minutes. Slides were rinsed in PBS for 5 min × 4 times, and then immersed in the permeabilization solution (0.1% sodium citrate, containing0.1% TritonX-100) and incubated on ice (2–8°C) for 2 min. Slides were washed in PBS for 5 min × 4 times. Afterward, the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) reaction mixture was added to the slides and incubated at 37°C in the dark for 1 h. Slides were then rinsed in PBS for 5 min × 4 times and in ddH2O for 5 min × 3 times. After air-drying for 2 h at 37°C in the dark, the slides were mounted with ProLong^® Gold anti-fading reagent (Invitrogen, United States, 1724814) before analysis.

Cell counting of TUNEL⁺, NeuN⁺, and PV⁺ cells was performed in serial coronal sections across the SNR (every fourth from bregma, −3.28 to −4.04 mm). Fluorescence images of TUNEL, NeuN, and PV on each of the coronal sections in the SNR of 2-, 6-, and 12-month-old Pitx3-A53Tα-Syn × Tau^–/– mice were captured with a laser-scanning confocal microscope (LSM 710; Zeiss, Germany) at 20 × magnification. The numbers of TUNEL⁺, NeuN⁺, and PV⁺ cells, PV and NeuN double-positive cells, and TUNEL and NeuN double-positive cells were counted with NIS-Elements BR Imaging software (Nikon Instruments, Japan). The percentage of PV and NeuN double-positive cells to the total number of PV⁺ cells and the percentage of TUNEL and NeuN double-positive cells to the total number of TUNEL⁺ or NeuN⁺ cells in the SNR were determined.

To quantify the relative immunofluorescence intensity of α-syn, c-fos, and PV in the SNR of mice, images were taken using identical settings and then analyzed using ImageJ software (NIH, United States). To measure c-fos and PV fluorescent intensity, only fluorescence within the PV⁺/c-fos⁺ co-staining neurons in the SNR was analyzed. All the images were converted to an 8-bit color scale and the background was subtracted. Areas of interest were selected by the freehand selection tools and subjected to measurement by mean gray value to determine the average intensity.

Three mice were used per genotype and at each time point. Counters were blinded to the information of the samples.

GraphPad Prism 7 (GraphPad Software, United States) was used for statistical analysis. Data were expressed as mean ± SEM. The one-way analysis of variance was used to compare the means of different groups, followed by Tukey’s honestly significant difference post hoc test, and significance was set at P < 0.05.

As described in our previous study, we continued to use the triple transgenic mice that overexpressed PD-related α-syn A53T missense mutation in the midbrain dopaminergic neurons with different tau gene dosage from high to low, which were named as Pitx3-A53Tα-Syn × hTau, Pitx3-A53Tα-Syn × Tau⁺/⁺, Pitx3-A53Tα-Syn × Tau^+/–, and Pitx3-A53Tα-Syn × Tau^–/– mice, respectively (Jiao et al., 2020).

Considering that Pitx3-A53Tα-Syn × Tau^–/– mice specifically developed severe degeneration of PV⁺ neurons in the SNR at 18-month-old, we presumed that this may be related to the accumulation of α-syn. We stained α-syn in the SNR of 18-month-old mice. The results showed that compared with nTg mice, α-syn was significantly overexpressed in the SNC of all triple transgenic mice, as expected (Figure 1A). But more prominently, Pitx3-A53Tα-Syn × Tau^–/– mice had the highest level of α-syn (^**P < 0.01, vs. nTg), while other triple transgenic mice had almost the same α-syn content in the SNR with no statistical difference as compared to nTg mice (Figures 1A,B). These results indicated that tau knockout could modestly exacerbate the accumulation of α-syn in the SNR of α-syn A53T conditional transgenic mice at old age.

As the expression product of the Fos gene, c-fos is most commonly used to detect the activity of neurons in the central nervous system. The expression level of c-fos is directly proportional to the activity of neurons (Dragunow and Faull, 1989; VanElzakker et al., 2008). To determine whether different tau protein expression levels affect the changes in PV⁺ neuron activity mediated by A53T α-syn, we stained PV and c-fos in the SNR region of 6- and 18-month-old mice. The results indicated PV staining almost costained with c-fos, demonstrating that the remnant PV⁺ neurons were active (Figures 2A,B). It was worth noting that, accompanied by the significantly decreased PV⁺ neurons, c-fos expression was reduced accordingly in the SNR of Pitx3-A53Tα-Syn × Tau^–/– mice at 18-month-old (Figures 2B–D). These suggested that the number of PV⁺ neurons was proportional to the level of their activities, and Pitx3-A53Tα-Syn × Tau^–/– mice showed practically degeneration of PV⁺ and c-fos⁺ neurons in the SNR at old age.

To determine whether the decrease of PV⁺ neurons was due to cell apoptosis, we stained PV and TUNEL in the SNR of 18-month-old mice. The results revealed that the less PV expressed, the more TUNEL signals were detected, as shown in the Pitx3-A53Tα-Syn × Tau^–/– mice. However, TUNEL did not costain with PV in the SNR of all the triple transgenic mice (Figure 3). These suggested that the degenerating PV⁺ neurons could not be labeled by TUNEL, and they might undergo a transitional stage between the expression of PV and TUNEL.

NeuN is a neuron-specific marker, but its expression in the SNR is species-specific. For example, the neurons in the gerbil SNR do not express NeuN, whereas the neurons in the rats’ SNR strongly express NeuN (Mullen et al., 1992; Kumar and Buckmaster, 2007; Gusel’nikova and Korzhevskiy, 2015). To determine the type of cells which presented massive apoptosis in the SNR but did not co-stain with PV, we examined TUNEL and NeuN staining in the SNR of 18-month-old mice. The results showed that TUNEL was highly costained with NeuN in the SNR of Pitx3-A53Tα-Syn × Tau^–/– mice (Figure 4A). The percentage of TUNEL⁺NeuN⁺ co-staining cells relative to the total number of TUNEL⁺ or NeuN⁺ cells reached 94.47 ± 2.12% or 92.72 ± 1.1%, respectively (Figure 4B). However, in the SNR of the other triple transgenic mice and nTg mice, the neurons rarely expressed NeuN, which did not co-stain with TUNEL either (Figure 4A). Considering the previous experimental results (Figure 3), we preliminarily speculated that NeuN may be suited for an indicator of the transitional stage of degenerating neurons in SNR between expression of PV and TUNEL, especially, at the beginning of 6-month-old.

To further explore whether NeuN can specifically label the degenerating PV⁺ neurons, we stained PV and NeuN in the SNR of Pitx3-A53Tα-Syn × Tau^–/– mice at 2- and 6-months old. The results showed that PV⁺ neurons in the SNR did not begin to degenerate at 2-month-old because the expression level of PV was high while the expression level of NeuN was low (Figure 5A). At 6-month-old, NeuN was gradually increased and highly costained with PV (Figures 5A,C). Combining the previous results, while the loss of PV⁺ neurons (12- and 18-month-old) becoming significantly (Figures 2, 3), NeuN was highly costained with TUNEL (Figures 4, 5B,C). Therefore, we suspected that in the SNR of Pitx3-A53Tα-Syn × Tau^–/– mice, the rapidly progressed loss of PV⁺ neurons may undergo a transitional stage, i.e., from decreasing PV expression, to increasing NeuN expression, finally to TUNEL expression (Figure 6); NeuN may be a compatible marker labeling the degeneration of PV⁺ neurons at the beginning of 6-month-old.