Dopamine D2 Long Receptors Are Critical for Caveolae-Mediated α-Synuclein Uptake in Cultured Dopaminergic Neurons

Ichiro Kawahata,Luc Bousset,Tomoki Sekimori,Yasushi Kawata,Toshikuni Sasaoka,Ronald Melki,Kohji Fukunaga,Yanyan Wang,Haoyang Wang,Tomohiro Mizobata

doi:10.3390/biomedicines9010049

Abstract

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.

Highlights

We previously demonstrated that fatty acid-binding protein 3 (FABP3) is critical for the uptake of α-synuclein into dopaminergic neurons [9], thereby its nigrostriatal propagation [10]. α-synuclein is associated with FABPs as well as fatty acids and promotes protein aggregation [11,12,13]
Immunocytochemical analysis revealed that the immunoreactivities of the generated anti-D2L antibody were observed in wild type (WT)-derived tyrosine hydroxylase (TH)+ neurons, whereas no immunoreactivities were observed in D2L−/− TH+ neurons, indicating that this anti-D2L antibody identifies D2L receptors in the mesencephalic neurons (FigD2L was well co-localized with FABP3 in TH+ neurons in WT, whereas no ure 2A)
D2L Receptors are2LPredominantly Co-Localized with α-Synuclein Fibril ATTO-550 in we investigated the spatial characteristics of D2L receptors coupled with αwith αFinally, we investigated the spatial characteristics of D2L receptors coupled synuclein fibril uptake

Summary

Introduction

The synaptic protein α-synuclein is the major component of Lewy bodies and Lewy neurites, which are the pathological hallmarks of Parkinson’s disease [1,2]. The intracellular accumulation of α-synuclein and its fibrillation increases the phosphorylation level of tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine [3]. Pathological elevation of TH phosphorylation leads to its degradation by the ubiquitin-proteasome system and loss of TH protein in dopaminergic neurons [3,4]. Phosphorylated TH has biological characteristics to readily form intracellular aggregates [5]. Lewy bodies are Biomedicines 2021, 9, 49.

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