A method for constructing an alpha-synuclein pre-fibril induced brain-derived parkinson's disease mouse model and application thereof

Abstract

The application belongs to the technical field of experimental animal model construction, and particularly relates to a construction method and application of an alpha-synuclein preformed fibril induced brain-derived Parkinson's disease mouse model, which comprises the following steps: injecting an effective amount of alpha-synuclein preformed fibril into the bilateral substantia nigra regions of A53T alpha-synuclein transgenic mice. The model constructed by the application rapidly appears motor dysfunction, non-motor symptoms, loss of substantia nigra dopaminergic neurons and alpha-synuclein pathological aggregation within 3 months after injection, significantly shortens the disease cycle of a traditional A53T transgenic mouse model, and for the first time, inhibition of subventricular zone adult neurogenesis and peripheral colon tissue barrier damage are observed in the model, which provides an important tool for studying the mechanism of PD non-motor symptoms.

Description

Technical Field

[0001] This invention relates to a method for constructing and applying an α-synuclein preformed fibrous mouse model of brain-derived Parkinson's disease, belonging to the field of experimental animal model construction technology. Background Technology

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. Its core pathological features include the progressive loss of dopaminergic neurons in the substantia nigra of the midbrain and the abnormal aggregation of α-synuclein (α-syn) to form Lewy bodies. Current clinical treatments (such as levodopa and deep brain stimulation) can only alleviate motor symptoms and cannot stop or reverse the neurodegenerative changes. The pathogenesis of PD is intricate and complex, and the current consensus is that it is not driven by a single factor. Therefore, constructing animal models that can rapidly and accurately mimic the core pathological features of PD is crucial for studying the disease's mechanisms and developing new therapies.

[0003] In existing research, the A53T transgenic mouse is one of the more commonly used animal models of Parkinson's disease (PD). However, this model has inherent limitations: its heterozygous onset period is relatively long, usually requiring more than 22 months for obvious pathological changes and behavioral phenotypes to appear, and the symptoms are usually mild. In addition, some studies failed to reproduce the dopaminergic neuron death and motor behavioral disorders reported in earlier literature during independent validation, and the reproducibility of the model needs to be improved. Other studies have constructed animal models by injecting α-synuclein preformed fibers (PFFs) into the amygdala, striatum, or substantia nigra of transgenic mice, but these models have limitations in functional assessment. For example, the basolateral amygdala injection model is mainly suitable for studying non-motor symptoms (such as constipation), but it is difficult to effectively assess motor function, while motor symptoms (resting tremor, rigidity, bradykinesia, postural and gait abnormalities) remain the core of human PD diagnosis and disease progression assessment.

[0004] In summary, in order to study more deeply the molecular mechanisms by which α-synuclein pathology leads to adult neurogenesis disorders (especially hippocampal neurogenesis disorders), there is an urgent need in this field for an animal model with a shorter onset period and more significant Parkinson's disease-related motor and non-motor symptoms (such as constipation) so that mechanism exploration and intervention studies can be carried out within a controllable experimental time window. Summary of the Invention

[0005] In view of the above-mentioned technical problems in the prior art, the present invention provides a method for constructing and applying a mouse model of brain-derived Parkinson's disease induced by α-synuclein prefibrillation.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: In a first aspect, the present invention provides a method for constructing a mouse model of brain-derived Parkinson's disease induced by α-synuclein preformed fibrosis, comprising the following steps: An effective amount of α-synuclein pre-fibrils was injected into the bilateral substantia nigra regions of A53T α-synuclein transgenic mice.

[0007] Based on the above technical solution, the present invention can be further improved as follows: Furthermore, the A53T α-synuclein transgenic mice are heterozygous mice aged 2-4 months.

[0008] Furthermore, the injection site was the bilateral substantia nigra region of the A53T α-synuclein transgenic mouse. The injection coordinates of the bilateral substantia nigra region were: with the anterior fontanelle as the origin, AP: -3.10 mm, ML: ±1.30 mm, DV: -4.65 mm.

[0009] Furthermore, the effective injection amount of the α-synuclein pre-formed filament is 8μg-15μg, preferably 10μg.

[0010] Furthermore, the injection concentration of the α-synuclein pre-formed filament is 4 mg / mL-6 mg / mL, and the injection volume is 0.5 μL-2 μL per side of the substantia nigra. Preferably, the injection concentration is 5 mg / mL, and the injection volume is 1 μL per side of the substantia nigra.

[0011] Furthermore, the preparation of the α-synuclein pre-fibrillated injection solution includes: dissolving monomeric α-synuclein in PBS, shaking and incubating for 6-8 days to obtain the α-synuclein pre-fibrillated stock solution, and then performing ultrasonic treatment.

[0012] Furthermore, the injection site is the striatum or medial forebrain tract of A53T α-synuclein transgenic mice.

[0013] Furthermore, the model exhibits a Parkinson's disease-like phenotype within 3 months after injection, the phenotype including one or more of the following: (a) Motor dysfunction, preferably a decrease in distance traveled in the open field test, a shortened fall time in the swivel test, or a decrease in maximum grip force in the grip test; (b) Non-motor symptoms, preferably olfactory dysfunction or slowed intestinal motility; (c) The number of tyrosine hydroxylase-positive dopaminergic neurons in the substantia nigra is reduced; (d) Increased pathological aggregation of α-synuclein in the substantia nigra region; (e) A decrease in the number of Ki67-positive proliferating cells in the subventricular region indicates inhibition of adult neurogenesis; (f) Pathological changes in colon tissue, preferably decreased expression of Occludin protein, reduced goblet cells, or infiltration of inflammatory cells.

[0014] Secondly, the present invention provides an application of the construction method described above for preparing animal models of Parkinson's disease for screening or evaluating drugs for the prevention, relief or treatment of Parkinson's disease.

[0015] Thirdly, the present invention provides an application of the construction method described above for preparing an animal model of Parkinson's disease for studying the association between α-synuclein pathology and adult neurogenesis disorders.

[0016] Fourthly, the present invention provides an application of the construction method described above for preparing an animal model of Parkinson's disease for studying the correlation between central and peripheral intestinal pathology.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The mouse model obtained by the construction method of the present invention can stably induce motor disorders, α-synuclein aggregation and dopaminergic neuron loss within 3 months after injection, which significantly shortens the disease cycle of the traditional A53T transgenic mouse model.

[0018] This invention is the first to demonstrate significant inhibition of adult neurogenesis (Ki67-positive cells) in the subventricular region of the brain in this model, providing an important tool for studying the mechanisms of non-motor symptoms of PD (such as constipation and cognitive impairment).

[0019] This invention is the first to discover that central injection of PFF can lead to decreased expression of the barrier protein Occludin, reduced goblet cells, and inflammatory infiltration in the peripheral colon, providing new experimental evidence for the study of bidirectional regulation of the "brain-gut axis".

[0020] This invention verifies that the model can stably induce phenotypes in both female and male mice, thus expanding the applicability of the model. Attached Figure Description

[0021] Figure 1 This is a representative schematic diagram of the movement trajectory of the mouse in the open field test during the kinematic test in Embodiment 1 of the present invention; Figure 2 This refers to the distance the mouse moved in the open field test during the kinematic test in Embodiment 1 of the present invention; Figure 3 The fall time in the mouse rotarod experiment during the kinematic test of Embodiment 1 of the present invention; Figure 4 This represents the maximum gripping force of the mouse in the gripping force experiment during the kinematic test of Embodiment 1 of the present invention; Figure 5 This refers to the intestinal peristalsis time of mice in the intestinal peristalsis detection in Example 1 of the present invention; Figure 6 The time it takes for the mice in the olfactory experiment of Example 1 of this invention to find food; Figure 7 This refers to the resting time of the mice in the tail suspension experiment in Example 1 of this invention; Figure 8 The spontaneous alternation rate of mice in the Y-maze spontaneous alternation rate experiment of Example 1 of this invention; Figure 9 The images show the immunohistochemical images of representative dopaminergic neurons and α-synuclein in the substantia nigra of mice during the brain pathology verification in Example 1 of this invention. Figure 10 This is a statistical analysis of the number of dopaminergic neurons in the substantia nigra of mice during the pathological verification of the brain in Example 1 of the present invention. Figure 11 This is a statistical analysis of the average optical density value of α-synuclein in the substantia nigra of mice during the pathological verification of the brain in Example 1 of the present invention. Figure 12 This refers to the distance the female mouse moved in the open field test during the kinematic test in Embodiment 2 of the present invention. Figure 13 This represents the maximum gripping force of the female rat in the gripping force experiment during the kinematic test of Embodiment 2 of the present invention; Figure 14 This refers to the time it took for the female mouse to find food in the olfactory experiment of Example 2 of this invention; Figure 15 These are images of immunohistochemical staining of typical substantia nigra dopaminergic neurons and substantia nigra α-synuclein in a female mouse during brain pathology verification in Example 2 of this invention. Figure 16 This is a statistical analysis of the number of dopaminergic neurons in the substantia nigra of female mice during the pathological verification of the brain in Example 2 of the present invention. Figure 17 This is a statistical analysis of the average optical density value of α-synuclein in the substantia nigra of female mice during the pathological verification of the brain in Example 2 of the present invention; Figure 18 This is a hematoxylin-eosin staining image of mouse colon during peripheral intestinal pathological verification in Example 1 of the present invention (arrows indicate goblet cells); Figure 19 This refers to the colonic pathology score of mice in the peripheral intestinal pathology verification of Example 1 of the present invention. Figure 20 This is an image of the Occludin protein immunoblot from mouse colon tissue in Example 1 of the present invention; Figure 21 The statistical chart of the mouse Occludin / GAPDH ratio in Example 1 of this invention, normalized to the PBS group; Figure 22The image shows a representative Occludin immunofluorescence staining of a mouse from Example 1 of this invention; Figure 23 This is a statistical graph of the average optical density values ​​of mouse Occludin immunofluorescence staining in Example 1 of the present invention; Figure 24 This is a representative Ki67 fluorescent staining image of the subventricular region of a mouse in Example 1 of this invention; Figure 25 This is a statistical chart of the number of Ki67+ mice in Example 1 of the present invention, where each point represents a brain slice image of each mouse. Detailed Implementation

[0022] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0023] Example 1 (1) Preparation of α-synuclein prefibrils The concentration of α-synuclein monomeric protein was adjusted to 5 mg / mL with PBS buffer. The solution was then incubated at 1000 rpm for 7 days at 37°C in a constant-temperature shaker until the solution became turbid, thus obtaining mature α-synuclein preformed filaments (α-synuclein PFF). Before use, the α-synuclein PFF stock solution was sonicated to reduce aggregate size and enhance its seeding activity.

[0024] (2) Stereoscopic injection into the mouse brain Experimental animal selection: 3-month-old A53T α-synuclein transgenic heterozygous mice (male, M83 strain) were selected.

[0025] PFF group: Mice were anesthetized and fixed on a stereotaxic apparatus. Using Bregma's point (anterior fontanelle) as the origin, the coordinates of the bilateral substantia nigra were determined: AP: -3.10 mm, ML: ±1.30 mm, DV: -4.65 mm. Using a microsyringe, 1 μL of α-synuclein preformed filaments (concentration 5 mg / mL) were slowly injected into each bilateral substantia nigra at a rate of 200 nL / min. The needles were left in place for 5 minutes after injection to allow for full absorption, then the needles were slowly withdrawn, and the wounds were sutured.

[0026] Control group (PBS group): Mice were injected with an equal volume of PBS.

[0027] (3) Model behavioral validation Behavioral tests were conducted monthly after the injection, and the results showed: Open field test (see results) Figures 1-2 In the first and second months after injection, no obvious motor impairment was observed in the PFF group. However, three months after injection, the distance traveled by mice in the PFF group in the open field test was significantly reduced. The spontaneous movement distance in the PBS group was 2889.0 ± 188.3 cm, while that in the PFF group decreased to 2003.3 ± 1086.8 cm (P < 0.05). The PFF group showed severe spontaneous movement impairment.

[0028] Rotating bar experiment (see results) Figure 3 ): No obvious abnormalities were observed in the PFF group in the first and second months after injection. In the third month after injection, the mice in the PFF group showed obvious motor disorders. The fall time of the transgenic mice in the PBS group was 158.3 ± 40.4 s, while that in the PFF group it was reduced to 67.1 ± 63.3 s. Compared with the PBS group, the fall time in the PFF group was significantly shortened (P < 0.05).

[0029] Grasp force experiment (see results) Figure 4 The PBS and PFF groups also showed results consistent with the above-mentioned motor tests. No obvious abnormalities were observed in the PFF group in the first and second months after injection. In the third month after injection, the grip strength of the PBS group was 218.6 ± 16.2 gf, while that of the PFF group decreased to 107.3 ± 58.0 g. The maximum grip strength of the PFF group decreased significantly (P < 0.0001), indicating that the PFF group model mice had typical motor disorders.

[0030] Although motor dysfunction is the most prominent feature of Parkinson's disease, many patients also experience a variety of non-motor symptoms such as constipation, depression, and loss of smell. Therefore, in addition to testing the motor function of transgenic mice, changes in non-motor function were also examined. The results showed: Intestinal peristalsis test (results can be found in...) Figure 5 ): PFF mice showed delayed intestinal emptying in the third month after injection. The intestinal motility time in the PFF group was 165.0 ± 75.17 min, while the intestinal motility time in the PBS group was only 63.5 ± 7.3 min, which was significantly prolonged (P < 0.05).

[0031] Olfactory tests are commonly used to assess the olfactory function of animals. Olfactory tests were performed on mice at 1 month and 2 months post-injection (results see [link to results]). Figure 6As can be seen, olfactory dysfunction occurred in mice before obvious motor impairment. In the first month, the time required to find food in the PBS group was 85.3 ± 26.6 s, while in the PFF group it was 163.7 ± 51.2 s (P < 0.05). In the second month, the time required in the PBS group was 68.2 ± 25.3 s, while in the PFF group it was 104.0 ± 24.8 s (P < 0.05). This result not only demonstrates that α-synuclein PFF causes olfactory abnormalities in A53T transgenic mice, but also indicates that olfactory abnormalities occur before motor dysfunction. Notably, in repeated olfactory tests, the time to find food in all groups showed a decreasing trend month by month, suggesting that the mice exhibited a certain learning and adaptation effect to the testing environment and task. Nevertheless, at each time point (month 1 and month 2), the search time in the PFF group was significantly longer than that in the PBS group (P < 0.05), indicating that α-synuclein PFF injection still stably induced olfactory dysfunction, and that this dysfunction could be detected before the onset of motor symptoms.

[0032] The tail suspension test was used to assess depressive-like behavior in A53T transgenic mice (see results). Figure 7 No significant differences in depressive-like behaviors were observed between the PBS group and the PFF group.

[0033] The Y-maze test is a classic behavioral experiment in neuroscience research used to assess spatial working memory and exploratory instincts in rodents. It can keenly reflect cognitive impairment, learning difficulties, and emotional stress levels in mice under pathological conditions such as Parkinson's disease. (See results for the Y-maze test.) Figure 8 The study found that the spontaneous alternation rate was not statistically different between the PFF group and the PBS group; however, the PFF group showed a decreasing trend month by month.

[0034] In summary, olfactory dysfunction (significantly prolonged time to find food) appeared in the PFF group mice starting one month after injection; and slowed intestinal motility (significantly prolonged intestinal motility time) appeared three months after injection. No depressive-like behavior was observed in the tail suspension test, and the spontaneous alternation rate in the Y-maze showed a decreasing trend but no statistically significant difference.

[0035] (4) Pathological verification of the model brain Three months after injection, the mice underwent brain harvesting via perfusion. Immunohistochemical results showed (see...). Figures 9-11 ): Compared with the PBS group, the number of tyrosine hydroxylase-positive dopaminergic neuronal markers (TH) in the substantia nigra region of mice in the PFF group was significantly reduced (P < 0.05).

[0036] Compared with the PBS group, the mean optical density of α-synuclein in the substantia nigra region of mice in the PFF group was significantly increased (P < 0.05).

[0037] α-synuclein aggregation is a core pathological feature of Parkinson's disease (PD). The above results show that the animal model obtained by the method of the present invention can simulate the core pathological feature of α-synuclein aggregation in PD, and the course of the disease is significantly shortened.

[0038] (5) Pathological verification of the model's peripheral intestine Three months after injection, colon tissue was taken from mice for testing: Histopathology: See Figure 18 HE staining (hematoxylin and eosin staining) showed that, compared with the PBS group, the columnar epithelial cells of the colon in the PFF group mice were damaged, goblet cells were reduced, and obvious dark purple punctate clusters were visible near the muscularis mucosa, suggesting that inflammatory cell infiltration may exist in the PFF group. See also Figure 19 The pathological scores were 1.8 ± 0.5 in the PBS group and 3.5 ± 1.0 in the PFF group, showing a significant increase in the PFF group (P < 0.05).

[0039] Barrier function: Western blot (see...) Figures 20-21 The results showed that the Occludin / GAPDH gray value in the PBS group was 1.1 ± 0.2, while the Occludin / GAPDH gray value in the PFF group was 0.4 ± 0.2, a significant decrease. Occludin (closure protein) represents the integrity of tight junctions, i.e., the quantification of intestinal barrier function, while GAPDH (glyceraldehyde-3-phosphate dehydrogenase) represents the internal control of the protein. This ratio represents the expression level of Occludin per unit of total protein. Immunofluorescence (see [link to immunofluorescence analysis]). Figure 22-23 The results showed that the average fluorescence intensity of Occludin in the PBS group was 817489 ± 327647 au, while the average fluorescence intensity of Occludin in the PFF group decreased to 302020 ± 154880 a.u., which was significantly lower than that in the PBS group, indicating that the intestinal barrier function was impaired.

[0040] The above results indicate that bilateral substantia nigra injection of A53T transgenic mice via PFF may cause mechanical damage to their colonic tissue, suggesting that the mouse model involves not only pathological damage to the brain but also pathological damage to the colon. Based on the brain-gut axis theory, pathological changes in α-synuclein in the central nervous system can be transmitted to the distal intestine via vagal nerve efferent fibers, autonomic nerves, or inflammatory signaling pathways, inducing α-synuclein deposition and intestinal barrier dysfunction in the enteric nervous system. Therefore, this invention, based on verifying pathological changes in the brain, further conducts pathological and barrier function tests on peripheral intestinal tissue to comprehensively evaluate the phenotypic characteristics of the PFF-induced Parkinson's disease model at both ends of the brain-gut axis.

[0041] (6) Detection of adult neurogenesis in the model Three months after injection, mouse brain tissue was collected for immunofluorescence staining to detect cell proliferation in the subventricular region. Results are shown below. Figures 24-25 Compared with the PBS group, the number of Ki67 positive proliferating cells in the subventricular region of mice in the PFF group decreased from 47.1±15.4 to 29.9±15.9, showing a significant reduction (P<0.01), indicating that α-synuclein pathology can inhibit adult neurogenesis.

[0042] Example 2 Validation of gender differences in the model.

[0043] The same modeling and testing methods were used on A53T transgenic female mice as in Example 1. The results showed: Open field test (see results) Figure 12 In the third month, female mice in the PFF group showed a decrease in spontaneous activity. Compared with female mice in the PBS group, the spontaneous activity of female mice in the PFF group decreased from 2279.5±48.7 cm to 474.8±154.3 cm (P<0.0001).

[0044] Grip force test: (See results) Figure 13 ): In the third month, the grip strength of female mice in the PFF group decreased to 30.67±11.93gf (P<0.01).

[0045] In non-motor behavioral tests, female mice exhibited significant olfactory dysfunction in both month 1 and month 2. Figure 14 In the first month, the time required for female mice in the PBS group to find food was 51.6±17.4 s, while that in the PFF group was 175.7±75.2 s, which was significantly longer (P<0.05). The same trend was observed in the second month, with the time required for female mice in the PBS group being 40.0±3.6 s, while that in the PFF group was 117.9±23.6 s (P<0.05).

[0046] Immunohistochemical experiments were used to detect pathological changes in the brains of female mice. (See also...) Figures 15-16 In the PFF group, the number of dopaminergic neurons in the substantia nigra region of female mice decreased from 124.83±21.68 to 74.55±14.96 (P<0.05), and the deposition of α-synuclein also increased significantly. Figure 15 See also Figure 17 The optical density value of the PFF group increased from 0.50±0.37au to 1.73±0.98au (P<0.05).

[0047] As can be seen, female mice also showed reduced spontaneous activity, decreased gripping strength, olfactory dysfunction, and a decrease in TH-positive neurons and an increase in α-synuclein deposition in the substantia nigra region 3 months after injection, consistent with the male mouse phenotype, indicating that there was no sex difference in this model.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing an animal model of brain-derived Parkinson's disease induced by α-synuclein preformation fibers, characterized in that, Includes the following steps: An effective amount of α-synuclein pre-fibrils was injected into the bilateral substantia nigra regions of A53T α-synuclein transgenic mice.

2. The construction method according to claim 1, characterized in that, The A53T α-synuclein transgenic mice were 2-4 month old heterozygous mice.

3. The construction method according to claim 1, characterized in that, The effective injection dose of the α-synuclein pre-formed filament is 8 μg-15 μg.

4. The construction method according to claim 3, characterized in that, The injection concentration of the α-synuclein pre-fibrils is 4 mg / mL-6 mg / mL, and the injection volume is 0.5 μL-2 μL per side of the substantia nigra.

5. The construction method according to claim 4, characterized in that, The preparation of the α-synuclein pre-fibrillated injection solution includes: dissolving monomeric α-synuclein in PBS, shaking and incubating for 6-8 days to obtain the α-synuclein pre-fibrillated stock solution, and then performing ultrasonic treatment.

6. The construction method according to claim 1, characterized in that, The injection site is the striatum or medial forebrain tract of A53T α-synuclein transgenic mice.

7. The application of the construction method according to any one of claims 1-6, characterized in that, Animal models of Parkinson's disease used to prepare for screening or evaluation of drugs for the prevention, relief or treatment of Parkinson's disease.

8. The application of the construction method according to any one of claims 1-6, characterized in that, This was used to prepare an animal model of Parkinson's disease for studying the association between α-synuclein pathology and adult neurogenesis disorders.

9. The application of the construction method according to any one of claims 1-6, characterized in that, This was used to prepare an animal model of Parkinson's disease for studying the correlation between central and peripheral intestinal pathology.

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