Parkinson’s disease (PD) is a debilitating neurodegenerative disorder affecting millions worldwide. Characterized by motor deficits, including tremors, rigidity, and impaired movement, PD stems from the progressive loss of dopaminergic neurons in the substantia nigra, a critical brain region for motor control. Hallmarks of PD also include excessive microglial activation and synaptic loss, particularly in the striatal region, which contributes to the early decline in motor function. However, exercise trainings have been proven to improve PD symptoms, delay the disease progression as well as affect excessive microglial synaptic phagocytosis. Emerging research highlights the potential of exercise as a therapeutic intervention, capable of not only alleviating symptoms but also modifying the underlying disease processes. This article delves into the intricate mechanisms through which exercise exerts its protective effects, focusing on the interplay between microglia, the complement system, and synaptic health.
The Pathological Landscape of Parkinson's Disease
The progressive degeneration of dopaminergic neurons in the substantia nigra is the primary pathological change in Parkinson’s disease (PD). Excessive microglial activation and synaptic loss are also typical features observed in PD samples. In the early stages of PD, there is already a loss of synapse in the striatal region, accompanied by a reduction of dopamine transporter protein.
Exercise: A Promising Intervention for Parkinson's Disease
Exercise trainings have been proven to improve symptoms and delay the progression of PD. Previous studies have shown that exercise trainings can enhance the balance, gait, and overall functional capacity, and reduce the incidence of falls in PD patients. After exercise training, the number of microglia in the striatum tends to decrease, with more of those shifting into the resting state compared to the MPTP-PD mice. Concurrently, exercise trainings rescue the down-regulation of PSD95 and synaptophysin in the striatum of MPTP-treated mice. That indicated synaptic function in the striatal region is enhanced. Experiment with electron microscopy also revealed significant neurogenesis of dendrites and axons in the striatal region following exercise.
Microglia: Guardians of the Brain and Key Players in PD
Microglia, the brain's resident immune cells, play a dual role in PD. While they normally act as protectors, clearing debris and pathogens, in PD, they become excessively activated, contributing to neuronal damage and synaptic loss. Microglia are known to participate in synaptic phagocytosis in various diseases, and synaptic alterations are early events in many neurodegenerative diseases. Increased microglia-mediated synaptic loss through phagocytosis exacerbates cognitive impairment. Also, persistent microglial phagocytic activity results in decreased synaptic density and cognitive decline after stroke and in multiple sclerosis. Similarly, excessive microglial synaptic phagocytosis has been observed in various PD animals.
Exercise Restricts Microglia-Synapse Engulfment
Exercise training was reported to effectively restrict microglia-synapse engulfment. For example, long-term voluntary exercise can protect hippocampal synapses in APP/PS1 mice, reduce microglial activity and dendritic spine loss. In a TDP-43-induced model of ALS, treadmill exercise was found to prevent the worsening of motor dysfunction and further inhibit microglial synaptic phagocytosis.
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Complement System: An Unforeseen Contributor to Synaptic Loss
The complement system, traditionally known for its role in immune defense, has emerged as a significant player in neurodegenerative diseases, including PD. In PD, the complement cascade becomes abnormally activated, leading to the tagging of synapses for removal by microglia, exacerbating synaptic loss.
CD55: A Key Molecule in Attenuating Complement-Mediated Synaptic Phagocytosis
CD55 probably accounts for the attenuation of complement-mediated synaptic phagocytosis in the striatum after treadmill training.
Study Design: Investigating the Impact of Exercise on Microglia and Synapses in a PD Mouse Model
To elucidate the mechanisms underlying exercise-induced neuroprotection, researchers employed a mouse model of PD, induced by injecting mouse-derived α-synuclein preformed fibrils (M-α-syn PFFs) into the substantia nigra. This model mimics key pathological features of PD, including dopaminergic neuron loss and microglial activation. The mice were then subjected to treadmill exercise training, and their brains were analyzed using a combination of techniques, including immunocytochemistry, RNA-seq, and proteomics.
Methods of Studying the Impact of Exercise on Microglia and Synapses in a PD Mouse Model
- Animals and Housing: 5 mice per cage were housed in a SPF environment equipped with a ventilation system. The ambient temperature was maintained at 22 °C ± 2 °C, and relative humidity ranged from 40 to 70%. The mice were kept in a 12-h light/12-h dark cycle (7:00-19:00) with access to ample food and water.
- PD Mouse Model: The mouse model of PD was established by injecting mouse-derived alpha-synuclein preformed fibrils (M-α-syn PFFs) (AnaSpec, AS-56082-100) into the substantia nigra. Eight-week-old male mice were divided into Sham and PFFs groups. Animals were assigned a number, then use a random number table to assign the animals into groups. PFFs group received bilateral injections of PFFs into the substantia nigra (P3.0 mm, L/R1.3 mm, V4.5 mm), with a dosage of 2 μg per side at a continuous flow rate of 0.2 μl/min, while Sham group received an equivalent volume of saline.
- Behavior Tests: Behavior tests included pole test and rotarod test. The rotarod test included three training sessions, with speeds of 10 rpm/min constant, 2-20 rpm/min constant acceleration, and 4-40 rpm/min constant acceleration. Mice were then tested on the rotarod at 4-40 rpm/min constant acceleration for 5 min. Each experiment was conducted in triplicate, and the intervals between experiments exceeded thirty minutes. In the pole test, mice were trained to climb a vertical pole (50 cm height, 0.5 cm diameter) with a textured surface.
- Tissue Collection and Preparation: After behavior tests, brain tissues of mice were immediately collected. Following sodium pentobarbital anesthesia, blood was removed from the body using 1 × PBS, and the tissues were fixed by perfusion with PFA. After sucrose gradient dehydration, the tissues were embedded in OCT and stored at − 80 °C. For another subset of mice, brain tissues were dissected under a stereomicroscope after cervical dislocation, and the substantia nigra and striatum were isolated. Brain tissues were lysed using RIPA lysis buffer (Beyotime,P0013B) supplemented with protease inhibitor mixture (Roche, 4693116001) and phosphatase inhibitor mixture (Roche, 4906845001). BCA assay kit (Beyotime, P0010) was used to measure the total protein concentration.
- Western Blotting: Proteins were separated by SDS-PAGE, and then transferred to a PVDF membrane. Membranes were blocked in 5% skimmed milk for 1 h, incubated overnight with specific primary antibodies at 4 °C, washed three times in 1 × TBST, incubated with secondary antibodies at room temperature for 1 h, and finally detected using the ECL protein blotting substrate kit.
- Immunofluorescence: Frozen brain section (30 μm thickness) were collected for immunofluorescence. The substantia nigra and striatum brain slices were washed three times for 10 min each in 1 × PBS and then blocked with immunofluorescence blocking solution (Beyotime, P0260) for 1 h. After blockage, the slices were incubated overnight with primary antibodies at 4 °C. After three washes with 1 × PBS, the slices were incubated with secondary antibodies at room temperature for 1 h. Following three washes with 1 × PBS, the slices were coverslipped using mounting medium and imaged using a Leica confocal laser scanning microscope. 63 × oil objective was utilized to collect microglia confocal images. The microglia were scanned from top to bottom with 0.3-µm steps in the z direction with 2048 × 2048-pixel resolution and all collected images were merged to one image.
- Exercise Paradigm: After three weeks of PFFs administration, PFFs group was divided into PFFs and Exer groups. Exer group participated in treadmill exercise training, consisting of 5 days per week, 40 min per day, with a speed ranging up to 15 m/min (6 m/min for 5 min, 9 m/min for 5 min, 12 m/min for 20 min, 15 m/min for 5 min, and 12 m/min for 5 min) for 4 weeks.
- RNA Sequencing (RNA-Seq): RNA-Seq was conducted by GENE DENOVO. Total RNA was extracted using Trizol reagent kit. After reverse transcription and RNA amplification, RNA libraries were constructed. Prior to information analysis, raw data underwent data filtering to reduce analysis interference caused by invalid data. Preliminary filtering was performed on the obtained valid data, removing genes with zero expression in over half of the samples, and data normalization was conducted to ensure greater consistency within samples. PCA analysis was performed on three groups to ensure good clustering within each group. Further, differential expression gene (DEGs) analysis was conducted pairwise among the three groups, and then KEGG and GO functional enrichment analysis using DEGs were conducted.
- Proteomics: 4D-DIA relative quantitative proteomics by GENE DENOVO were applied in our study. Proteins were extracted from the samples, and the total concentration was determined using BCA assay. Subsequently, peptide samples were prepared and subjected to mass spectrometry detection. This process involved comparing the obtained mass spectrometry data to the reference library to identify and quantify the proteins present in each sample. The obtained protein quantification results were statistically analyzed and subjected to bioinformatics analysis. After inter-sample correction using the Limma package, differential multiples were calculated using the Limma package, and Gene Set Enrichment Analysis (GSEA) was performed using the ClusterProfiler package.
- ELISA: After blood collection, samples were put on ice for 30 min. Subsequently, centrifugation was performed at 12,000 rpm for 15 min to obtain the serum. Striatal tissues were homogenized using lysis buffer, and the supernatant was collected after centrifugation at 12,000 rpm for 15 min. ELISA kits were used to quantify dopamine (DA) level in the striatum and complement levels (C3 and C1q) in the serum.
- Golgi Staining: FD Rapid Golgi Stain Kit (FDNeurotech, PK401) was used for Golgi staining according to the manufacturer’s instructions. After cervical dislocation, mouse brain tissues were separated and washed in PBS to remove blood. The brain tissues were immersed in a mixture of Solution A and Solution B (1:1) for 14 days and then transferred to Solution C for dehydration, with the entire process conducted in the dark. After dehydration, brain tissues were sliced into 200 μm sections using a vibratome, and the sections were collected on chrome-alum-gelatin-coated slides.
- AAV-CD55 Overexpression: An adeno-associated virus (AAV) expressing CD55 (pAAV-EF1A-mCD55-mCherry:WPRE) (the AAV was constructed and packaged by VectorBuilder) was constructed and injected into the striatum (A0.8 mm, L/R1.2 mm, V3.6 mm). Virus injection included treatment and pre-treatment experiments. In the treatment experiment, eight-week-old male C57BL/6 J mice were divided into Sham, PFFs, and CD55-OE (overexpression) groups. PFFs and CD55-OE groups underwent PD modeling by receiving PFFs in the substantia nigra. CD55-OE group received AAV injection in the striatum, while Sham group and PFFs group received equivalent volumes of saline. Behavior tests, including pole test and rotarod test, were performed weekly, and mice were sacrificed for brain retrieval after three weeks. In the pre-treatment experiment, six-week-old mice were divided into Sham, PFFs, and CD55-OE groups. CD55-OE group received AAV injection in the striatum, while the PFFs and Sham groups received equivalent volumes of negative virus in the striatum. After two weeks, PFFs and CD55-OE groups underwent PD modeling in the substantia nigra, while Sham group received saline.
- Statistical Analysis: GraphPad Prism V.9 was used for all statistical analyses. All data are presented as mean ± SD. Normality of data was assessed, and statistical analysis was performed using unpaired two-tailed t-tests or one-way ANOVA. Differences between two groups were determined by unpaired two-tailed Student’s t-test. When comparing more than two groups, one-way ANOVA analysis was used, followed by Tukey’s post hoc test.
Key Findings: Exercise Mitigates Motor Deficits and Protects Dopaminergic Neurons
Injection of pre-formed fibrils of α-synuclein (PFFs) has been an alternative method proposed for modeling PD by increasing the level of α-synuclein in the substantia nigra. This model can more closely replicate the physiological processes that may occur in the brain of PD patients. Given that the substantia nigra is the primary brain region affected in PD, injection into the substantia nigra allows for a more direct observation of the impact of α-syn on dopaminergic neurons. Therefore, the substantia nigra was chosen as the main injection site. Prior to PFFs injection, baseline behavior tests were conducted on mice to ensure no significant intergroup differences. The modeling lasted for three weeks, during which behavior tests including pole test and rotarod test were performed every week. The results showed that after PFFs injection three weeks, mice in PFFs group exhibited a decline in behavioral abilities, with reduced latency time on the rotarod and prolonged climbing time compared to Sham group, which showed statistical differences. The results showed a significant decrease in TH protein levels in both the substantia nigra and striatum of PFFs group compared to Sham group . The number of TH-positive neurons in the substantia nigra also declined in PFFs group, according to the results of immunofluorescence assay. That reflected the loss and death of nigral dopaminergic neurons in PFFs-treated mice. Additionally, microglia, the central immune cells involved in the development of Parkinson’s disease in various groups was assessed. CD68 was chosen as an indicator of microglial activation, and detected an upregulation of nigral/striatal microglia in PFFs group, along with increased number of microglia and an elevated proportion of CD68 expression per microglia, suggesting that microglial aggregation and over-activation occur in the substantia nigra and striatum in the PFFs-induced mouse model. Moreover, PFFs administration significantly suppressed the expression of postsynaptic density protein 95 (PSD95) in the striatum, indicating the synaptic loss under that condition.
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