Depletion of microglia with CSF-1R inhibitor prevented motor deficits and neurodegeneration in mouse model of PD
The lack of predictive power related to neuroprotection in acute neurotoxin-induced PD models has led to the development of progressive models of PD, e.g., the α-synuclein-induced PD model. We found that delivery of the rAAV-hSYN vector induced the progressive loss of TH+ neurons in SN and striatal TH+ fibers, which was significant from 4 weeks post-injection and maintained at 8 weeks (Supplementary Fig. 1a-c). There was already a significant change in microglia morphology between ramified and amoeboid cells (with cell body enlargement and process retraction) at 2 weeks, which became more pronounced at 4 weeks and was maintained until 8 weeks (Supplementary Fig. 1d-h). The density of astrocytes significantly increased from 2 weeks, and the cells were highly prevalent at 8 weeks post-injection, as indicated by the positive staining pattern in the SNpc for the pan-astrocyte marker GFAP (Supplementary Fig. 1i-j). These results indicated there was chronic degeneration, as well as a chronic inflammatory state, after rAAV-hSYN vector injection.
To investigate the effect of long-term microglia depletion on the chronic pathogenesis of PD, we used the CSF-1R inhibitor PLX5622 in the PD model (Fig. 1a). To demonstrate its effect in our study, PLX5622 was formulated for addition to rodent chow and administered to adult male C57BL/6J mice for 7, 14, and 21 d. When compared with the control Veh-treated mice, mice subjected to oral PLX5622 treatment showed almost complete microglial elimination (92.5% reduction) within 21 d, detected as a loss of immunofluorescent staining for the pan-microglial marker IBA1 (7 d = 48.9 ± 5.4; 14 d = 39.5 ± 1.8; 21 d = 12.4 ± 1.8 vs. sham = 164.4 ± 12.6; PLX5622 effect, F (3, 12) = 88.36, p < 0.0001, ANOVA; p < 0.0001 vs. sham; Supplementary Fig. 2a-c).
After rAAV-hSYN injection, PLX5622 treatment significantly prevented postinjury motor performance (Fig. 1b-d). We assessed forelimb akinesia by conducting the cylinder test, and mice injected with human α-synuclein showed a preference for ipsilateral paw touches at 8 weeks post-injection, as expected (hSYN + vehicle = 25.1 ± 10.8% s vs. sham + vehicle = 57.6 ± 2.2 s; p = 0.0108 vs. sham + vehicle). Notably, in PD mice, PLX5562 showed tended to decrease forepaw asymmetry compared with vehicle treatment (hSYN + PLX5562 = 57.4 ± 5.3% vs. hSYN + vehicle = 25.1 ± 10.8%; F (3, 17) = 8.465, p = 0.0012; p = 0.0081 vs. hSYN + vehicle; Fig. 1b). Motor balance was also evaluated using an accelerating rotarod task. Vehicle-treated PD mice spent less time on the accelerating rotarod than the vehicle-treated sham mice (hSYN + vehicle = 101.3 ± 5.8 s vs. sham + vehicle = 293.2 ± 2.1 s; p < 0.0001 vs. sham + vehicle). In contrast, PLX5622-treated PD mice spent more time on the rotarod than vehicle-treated PD mice (hSYN + PLX5622 = 180.9 ± 14.6 s vs. hSYN + vehicle = 101.3 ± 5.8 s; F (3, 17) = 88.61, p < 0.0001; p < 0.0001; Fig. 1c). Open-field locomotor activity was examined using an infrared activity monitor. Intranigral injection of rAAV-hSYN significantly reduced the total distance traveled (hSYN + vehicle = 8441.0 ± 1974.0 mm vs. sham + vehicle = 14482.0 ± 1017.0 mm; F (3, 17) = 4.964, p = 0.0118; p = 0.0553 vs. sham + vehicle) and average movement speed (hSYN + vehicle = 28.6 ± 6.5 mm/s vs. sham + vehicle = 49.3 ± 3.3 mm/s; F (3, 17) = 6.153, p = 0.005; p = 0.0206 vs. sham + vehicle). Mice receiving PLX5622 tended to show improved locomotor activity (total distance traveled: hSYN + PLX5622 = 13263.0 ± 1852.0 mm, p = 0.05; average movement speed: hSYN + PLX5622 = 46.2 ± 4.4 mm/s, p = 0.0444; Fig. 1d).
To investigate the effect of microglia depletion on neuropathology, animals were sacrificed at 8 weeks post-injection. Brains were removed and midbrain sections were cut and stained for TH. As expected, rAAV-hSYN caused a significant loss of nigral dopaminergic neurons. We accounted the number of TH+ nigral neurons and compared it to that recorded for the contralateral intact side. Consistent with the development of motor impairment, a progressive loss of TH+ neurons were seen in the SNpc (hSYN + vehicle = 37.6 ± 2.6 vs. sham + vehicle = 109.5 ± 4.1; F (3, 17) = 59.14, p < 0.0001; p < 0.0001 vs. sham + vehicle). Contrastingly, PLX5622 treatment reduced TH+ neuron loss (hSYN + PLX5622 = 66.1 ± 5.7; p = 0.0021 vs. hSYN + vehicle; Fig. 1e, f). In parallel, striatal TH+ fibers were also preserved by PLX5622 in PD mice (hSYN + PLX5622 = 34.6 ± 3.3 vs. hSYN + vehicle = 14.5 ± 0.85; F (3, 17) = 170.7, p < 0.0001; p = 0.0023 vs. hSYN + vehicle; Fig. 1e, g). Moreover, the abnormal phosphorylated-α-synuclein (p-α-syn) level in the SNpc was significantly reduced by PLX5622 at 8 weeks post-injection (hSYN + PLX5622 = 0.20 ± 0.053 vs. hSYN + vehicle = 0.63 ± 0.054; F (3, 17) = 51.77, p < 0.0001; p < 0.0001 vs. hSYN + vehicle). However, PLX5622 alone had no effect on α-synuclein phosphorylation in the brain (Fig. 1h, i).
In summary, long-term administration of PLX5622 for 11 weeks led to improved motor function and neuropathology in rAAV-hSYN-injected mice.
Long-term PLX5622 administration depletes microglia and modifies astrocyte reactivity state in rAAV-hSYN-injected PD mice
We quantified the number of IBA1+ cells to confirm the efficacy of microglia depletion after long-term PLX5622 administration. This revealed the widespread depletion of microglia: 80% in rAAV-hSYN (hSYN + PLX5622 = 29.06 ± 2.93 vs. hSYN + vehicle = 145.7 ± 9.18; F (3, 16) = 116.2, p < 0.0001; p < 0.0001 vs. hSYN + vehicle; Fig. 2a, b) and 82% in control mice (sham + PLX5622 = 16.10 ± 3.89 vs. sham + vehicle = 89.16 ± 3.81, p < 0.0001). To investigate whether the few remaining cells in PLX5622-fed mice treated with rAAV-hSYN were residual microglia or macrophages, we utilized an Ms4a3Cre-RosaTdT model, in which all granulocyte-monocyte progenitor cells and their lineages, but not microglia, irreversibly and persistently express tdTomato red fluorescent protein35. At 8 weeks post-injection, few tdTomato+ macrophages were detected in the SNpc of rAAV-hSYN mice, but tdTomato+ macrophage numbers were increased in the SNpc in rAAV-hSYN mice after microglia depletion (Fig. 2c). Most of the IBA1+ cells were residual microglia. These results indicated that the contribution of monocyte-derived macrophages to the chronic stage of the PD model was limited. Moreover, microglia depletion with PLX5622 enabled the incorporation of cells from the circulation. Together, the data show that microglia were efficiently depleted by long-term PLX5622 administration in control animals and to a lesser degree in rAAV-hSYN-injected mice.
Astrocytes and microglia communicate via cytokines and cell-surface markers during aging, neurodegeneration, and CNS injury36. In situ analysis of GFAP+ astrocytes in the SNpc at 8 weeks post-injection showed that α-synuclein overexpression increased astrocyte GFAP expression (hSYN + vehicle = 0.06 ± 0.0013 vs. sham + vehicle = 0.008 ± 0.0016; p < 0.0001 vs. sham + vehicle). Notably, PLX5622 reduced GFAP expression in PD mice compared with that in vehicle-treated PD mice (hSYN + PLX5622 = 0.019 ± 0.0052 vs. hSYN + vehicle = 0.06 ± 0.0013; F (3, 17) = 51.11, p < 0.0001; p < 0.0001 vs. hSYN + vehicle; Fig. 2d, e). A1 astrocytes with upregulated C3 expression, a neurotoxic astrocyte subtype induced by activated microglia, have been found in the diseased tissues of patients with PD and other neurodegenerative diseases37. The expression level of astrocyte C3 was significantly increased 8 weeks after rAAV-hSYN injection (hSYN + vehicle = 0.53 ± 0.06 vs. sham + vehicle = 0.19 ± 0.03; p = 0.0002 vs. sham + vehicle), while microglial depletion significantly decreased the expression level of C3 (hSYN + PLX5622 = 0.30 ± 0.04 vs. hSYN + vehicle = 0.53 ± 0.06; F (3, 17) = 11.6, p = 0.0002; p = 0.0053 vs. hSYN + vehicle; Fig. 2f, g). These results suggest that, upon microglia depletion, there was an efficient blockade of astrocyte activation and the emergence of an A1 neurotoxic astrocyte subtype.
Remodeling of extracellular matrix following long-term PLX5622 administration
To explore the possible mechanisms by which the elimination of microglia promotes recovery, bulk RNA-seq was performed using substantia nigra tissues carefully dissected from mouse brains (Supplementary Fig. 3) from the four groups: Group 1 (Sham + Veh), Group 2 (hSYN + Veh), Group 3 (Sham + PLX), and Group 4 (hSYN + PLX; Fig. 3a), RNA-seq differential gene expression were verified by qPCR (Supplementary Fig. 4). RNA-seq expression data were analyzed by principal component analysis (PCA) (Fig. 3b). In our model, principal component 1 (PC1) captured the variation caused by hSYN, while PC2 captured the variation resulting from PLX5622 treatment. Gene expression signatures from PC 1–2 clustered mice from each of the four treatment groups were as expected. Pathways related to immune and inflammatory responses, such as leukocyte activation and cytokine production, were dominant in the gene ontology biological process (GO–BP) analysis, and these were upregulated by rAAV-hSYN injection and downregulated by microglia depletion (Fig. 3c). The MGnD (microglial neurodegenerative phenotype)38 was defined by alterations in the expression of 96 genes in total, with the upregulation in 28 inflammatory genes and the downregulation of 68 homeostatic microglial genes38. An assessment of gene expression in the four groups indicated most inflammatory genes associated with MGnD increased after rAAV-hSYN injection and decreased following microglia depletion. Of the homeostatic microglial genes that were reduced in MGnD microglia, most were also reduced following PLX5622 administration, confirming that microglia depletion was highly efficient upon PLX5622 administration (Fig. 3d).
Analysis of differentially expressed genes (DEGs) in substantia nigra tissues from hSYN + PLX and hSYN mice and their associated biological processes by GO demonstrated a strong induction of pathways related to extracellular matrix organization (Fig. 4a, b). To identify molecular functions that could be involved in dopaminergic neuron survival following PLX5622 administration, the top 32 genes statistically increased by > 2-fold in hSYN + PLX mice were analyzed using the STRING network database (Fig. 4c). This revealed the involvement of a prominent group of ECM genes. We analyzed the ECM-related gene expression and found two transcripts in the CCN gene family to be upregulated, CCN2 (Ctgf) and CCN3 (Nov) (Fig. 4d).
Collectively, the evidence indicates that, after microglia depletion, there was an increase in ECM-related gene expression in the rAAV-hSYN-injected mouse brain.
P2RY12 inhibition halts disease progression in PD mice
Microglia express a core gene profile that includes P2RY12. The P2RY12 receptor is necessary for microglial-directed motility in response to CNS injury39. To confirm the effect of microglia depletion by PLX5622 in rAAV-hSYN mice, we used PSB-0739, a potent and competitive antagonist of P2RY12, to inhibit microglia activation in response to α-synuclein overexpression (Fig. 5a). Mice treated with PSB-0739 showed a significantly reduced microglia density in the SNpc after rAAV-hSYN injection (hSYN + PSB-0739 = 113.1 ± 45.18 vs. hSYN + vehicle = 319.3 ± 45.18, p = 0.0021; Fig. 5b, c). Moreover, P2RY12 inhibition prevented the deterioration in motor performance of AAV-hSYN mice, as assessed by cylinder, rotarod, and locomotion tests, as well as reducing TH+ neuron loss and striatal TH+ fiber loss (cylinder test: hSYN + PSB-0739 = 52.63 ± 1.32% vs. hSYN + vehicle = 45.08 ± 0.16%, p = 0.0005; rotarod test: hSYN + PSB-0739 = 286.3 ± 8.42 s vs. hSYN + vehicle = 187.4 ± 6.69 s, p < 0.0001; locomotion test: hSYN + PSB-0739 = 15110 ± 1037 mm vs. hSYN + vehicle = 11690 ± 946.4 mm; p = 0.0408; hSYN + PSB-0739 = 50.38 ± 3.46 mm/s vs. hSYN + vehicle = 38.96 ± 3.16 mm/s; p = 0.0407; TH+ neuron: hSYN + PSB-0739 = 85.81 ± 3.32 vs. hSYN + vehicle = 54.75 ± 2.59; p < 0.0001; TH+ fiber: hSYN + PSB-0739 = 64.58 ± 2.58% vs. hSYN + vehicle = 21.32 ± 2.84%; p < 0.0001 vs. hYSN + vehicle; Fig. 5d-i). These findings implicate the necessity of functional P2RY12 for microglia activation in the progression of the disease. P2RY12 inhibition improved functional recovery and reduced dopaminergic cell death in PD mice.