α-synuclein accumulation into dopaminergic neurons is a pathological hallmark of Parkinson’s disease. We previously demonstrated that fatty acid-binding protein 3 (FABP3) is critical for α-synuclein uptake and propagation to accumulate in dopaminergic neurons. FABP3 is abundant in dopaminergic neurons and interacts with dopamine D2 receptors, specifically the long type (D2L). Here, we investigated the importance of dopamine D2L receptors in the uptake of α-synuclein monomers and their fibrils. We employed mesencephalic neurons derived from dopamine D2L−/−, dopamine D2 receptor null (D2 null), FABP3−/−, and wild type C57BL6 mice, and analyzed the uptake ability of fluorescence-conjugated α-synuclein monomers and fibrils. We found that D2L receptors are co-localized with FABP3. Immunocytochemistry revealed that TH+ D2L−/− or D2 null neurons do not take up α-synuclein monomers. The deletion of α-synuclein C-terminus completely abolished the uptake to dopamine neurons. Likewise, dynasore, a dynamin inhibitor, and caveolin-1 knockdown also abolished the uptake. D2L and FABP3 were also critical for α-synuclein fibrils uptake. D2L and accumulated α-synuclein fibrils were well co-localized. These data indicate that dopamine D2L with a caveola structure coupled with FABP3 is critical for α-synuclein uptake by dopaminergic neurons, suggesting a novel pathogenic mechanism of synucleinopathies, including Parkinson’s disease.
Multiple system atrophy (MSA) is a neurodegenerative disease characterised by the accumulation of misfolded α-synuclein (αSyn) and myelin disruption. However, the mechanism underlying αSyn accumulation in MSA brains remains unclear. Here, we aimed to identify epsin-2 as a potential regulator of αSyn propagation in MSA brains. In the MSA mouse model, PLP-hαSyn mice, and FABP7/αSyn hetero-aggregate-injected mice, we initially discovered that fatty acid-binding protein 7 (FABP7) is related to MSA development and forms hetero-aggregates with αSyn, which exhibit stronger toxicity than αSyn aggregates. Moreover, the injected FABP7/αSyn hetero-aggregates in mice selectively accumulated only in oligodendrocytes and Purkinje neurons, causing cerebellar dysfunction. Furthermore, bioinformatic analyses of whole blood from MSA patients and FABP7 knock-down mice revealed that epsin-2, a protein expressed in both oligodendrocytes and Purkinje cells, could potentially regulate FABP7/αSyn hetero-aggregate propagation via clathrin-dependent endocytosis. Lastly, AAV5-dependent epsin-2 knock-down mice exhibited decreased levels of αSyn aggregate accumulation in Purkinje neurons and oligodendrocytes, as well as improved myelin levels and Purkinje neuron function in the cerebellum and motor performance. These findings suggest that epsin-2 plays a significant role in αSyn accumulation in MSA, and we propose epsin-2 as a novel therapeutic target for MSA.
Accumulation of α-synuclein protein into dopaminergic neurons is a pathological hallmark of Parkinson's disease. We previously demonstrated that fatty acid-binding protein 3 (FABP3) is critical for α-synuclein uptake into dopaminergic neurons and its propagation. FABP3 is abundant in dopaminergic neurons and interacts with the dopamine D2 receptor largely distributed in caveolae. In this study, we newly investigated the significance of caveolae formation in the uptake process of α-synuclein, coupled with FABP3. To disclose this issue, we employed mesencephalic neurons derived from dopamine D2-null knockout (D2-null) and FABP3 knockout (FABP3 -/-) as well as wild type C57BL6 mice, and treated with dynasore, a dynamin inhibitor, or caveolin-1 siRNA, and analyzed the ability of the uptake of fluorescence-conjugated α-synuclein monomer and fibrils. Our immunocytochemistry revealed that D2 receptors are co-localized with FABP3. Importantly, TH + D2-null neurons did not take up αsynuclein monomers. Moreover, exposure to dynasore, caveolin-1 knockdown, and the deletion of α-synuclein Cterminus abolished the uptake. D2 receptors and FABP3 were also essential for the uptake of α-synuclein fibrils. Intriguingly, D2 receptors and accumulated α-synuclein fibrils were well co-localized. These data indicate that caveolae formation with the D2 receptors coupled with FABP3 is critical for the uptake of α-synuclein via its Cterminus in dopaminergic neurons, suggesting a novel pathogenic mechanism of synucleinopathies including Parkinson's disease.
Background: Multiple system atrophy (MSA) is an adult-onset, fatal neurodegenerative disease featured by propagation of misfolded α-synuclein and dysregulation of myelin integrity in central nervous systems (CNS). In our previous study, we identified that fatty acid binding protein 7 (FABP7), one glial protein regulated α-synuclein (α-syn) toxicity and closely related to oligodendrocyte loss. Here, we investigated how FABP7 works in MSA pathological process. Methods: The mouse model of MSA: PLP-hαSyn transgenic mice (PLP-hαSyn mice) were used for understanding the underling role of FABP7 in α-syn aggregation. In vitro, FABP7/α-syn hetero-aggregates were constructed by recombinant human-α-syn and 6X His-tag human-FABP7. By immunohistochemical analyze of brain tissue from PLP-hαSyn mice and aggregates injection mice, we identified the toxicity of aggregates and the region or cell type in brain which aggregates prefer to propagate. Furthermore, through the bioinformatics analysis of MSA patient blood, PLP mouse brain tissue we found candidate proteins related to α-syn propagation. Then, the hypothesis was verified by adeno-associated virus (AAV) knockdown in aggregates injection mice. Findings: In PLP-hαSyn mice, FABP7 also forms hetero-aggregates with α-syn and the FABP7/α-syn hetero-aggregates exhibited higher molecular weight and stronger toxicity than α-syn aggregates both in vitro and in vivo. The same with PLP-hαSyn mice, in the FABP7/α-syn hetero-aggregates injected mice, aggregates prefer to accumulate in oligodendrocyte and purkinje neurons. By bioinformatics analysis we found the protein epsin-2 as a potential receptor which regulate FABP7/α-syn hetero-aggregates propagate to oligodendrocytes and purkinje neurons. In AAV-dependent epsin-2 knock down mice we observed decreased levels of FABP7/α-syn hetero-aggregates in purkinje neurons and oligodendrocytes. Conclusions: These data suggest that epsin-2 is an important potential therapeutic target for MSA via regulating FABP7/α-syn hetero-aggregates propagation. It is very meaningful for further investigation and developing specific inhibitors. Funding: This work was supported by the Strategic Research Program for Brain Sciences from the Japan Agency for Medical Research and Development (JP17dm0107071, JP18dm0107071, JP19dm0107071, and JP20dm0107071).
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