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

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.


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]. Additionally, phosphorylated TH has biological characteristics to readily form intracellular aggregates [5]. Consistently, Lewy bodies are D 2L −/− [20], D2 null, and FABP3 −/− mice [21,22] were housed under climate-controlled conditions with a 12 h light and dark cycle. Primary cultured dopaminergic neurons were prepared from mice at the embryonic stage of 15.5th days. The experiments were conducted by employing two to three pregnant mice (4-12 embryos from one mother mouse) in each group and were performed in triplicates to test reproducibility. The number of animals used was the minimum number consistent with the aims of the experiment. Animal studies were conducted in accordance with the Tohoku University institutional guidelines. Ethical approval was obtained from the Institutional Animal Care and Use Committee of the Tohoku University Environmental and Safety Committee [2019PhLM0-021 (approved date: 1 December 2019) and 2019PhA-024 (approved date: 1 April 2019)].

Cell Culture
Primary cultures of mesencephalic neurons were prepared as previously described [4,9]. In brief, the mesencephalon was dissected from mouse embryos on the 15.5th day of gestation. The dissected tissues were then treated with papain (Sumitomo Bakelite, Tokyo, Japan) at 37 • C for 30 min and mechanically dissociated into single-cell suspensions. The cells were plated onto poly L-lysine-coated cover glass chambers at a density of 3 × 10 5 cells/cm 2 . The cultures were maintained in Eagle's minimum essential medium (Wako, Osaka, Japan) supplemented with 10% fetal calf serum for 1-4 days in vitro (DIV) or 10% horse serum for 5-12 DIV. The cells were cultured at 37 • C in an atmosphere with 5% CO 2 in air and 100% Biomedicines 2021, 9,49 3 of 14 relative humidity. All animal experiments were conducted in accordance with the general guidelines for animal experiments at Tohoku University.

Analyses of Fluorescence Intensity and Morphological Characteristics
Quantitative analysis of fluorescence intensity and Z-dimension analysis were performed using NIH ImageJ 1.53 software (National Institutes of Health, Bethesda, MD, USA) as previously described [5,9]. First, the background signal intensities were measured from regions without any cells and subtracted from all the images, and the remaining signals of the cells were used to define total cell areas.

Statistical Analyses
All values are expressed as mean ± standard error of the mean (SEM). Statistical significance was tested by one-way analysis of variance (ANOVA) with post-hoc Tukey's multiple comparison test or two-way ANOVA with post-hoc Bonferroni's multiple comparison test. A p-value < 0.05 was considered as statistically significant. All the statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA).

Generation of D 2L Specific Antibody and the Co-Localization Analysis of D 2L with FABP3 Distribution in Cultured Dopaminergic Neurons
To investigate the physiological significance of D 2L in α-synuclein uptake coupled with FABP3, we first generated an anti-D 2L antibody that specifically recognizes the D 2L specific sequence CTHPEDMKL, which does not exist in D2 short type receptors (D 2S ) ( Figure 1). Immunocytochemical analysis revealed that the immunoreactivities of the generated anti-D 2L antibody (red) were observed in WT-derived TH + neurons (green), whereas no immunoreactivities were observed in D 2L −/− TH + neurons, indicating that this anti-D 2L antibody specifically identifies D 2L receptors in the mesencephalic neurons ( Figure 2A). Importantly, D 2L (red) was well co-localized with FABP3 (green) in TH + neurons (blue) in WT, whereas no D 2L signal was observed in D 2L −/− TH + neurons ( Figure 2B).

D2L is Critical for α-Synuclein Uptake in Cultured Dopaminergic Neurons
We investigated the physiological significance of D2L in α-synuclein uptake in dopaminergic neurons. To analyze D2L dependency in the uptake of α-synuclein, we exposed cultured neurons derived from either C57BL6 wild type (WT) mice, D2L knockout mice

D 2L Is Critical for α-Synuclein Uptake in Cultured Dopaminergic Neurons
We investigated the physiological significance of D 2L in α-synuclein uptake in dopaminergic neurons. To analyze D 2L dependency in the uptake of α-synuclein, we exposed cultured neurons derived from either C57BL6 wild type (WT) mice, D 2L knockout mice (D 2L −/− ), or D2null mice (D2 null) to 1 µM ATTO-550-labeled α-synuclein monomer at DIV 10. We discovered that the uptake ability of ATTO-550-labeled α-synuclein monomers was drastically abolished in D 2L −/− neurons, whereas WT TH + neurons took them up ( Figure 3A, **** p < 0.0001 vs. WT). The uptake of the α-synuclein monomer significantly increased at 6 h after the exposure to 1 µM α-synuclein monomer ATTO-550 in the NeuO + and FFN206 + neurons, markers for neuronal and VMAT2 positive cells, respectively (Supplementary Materials Figure S1). Additionally, we also investigated the ability of α-synuclein uptake in D2 null mice-derived mesencephalic neurons and revealed that the D2 receptor is critical for the uptake of ATTO-550-labeled α-synuclein monomers in TH + neurons ( Figure 3B, **** p < 0.0001 vs. WT). These data indicate that the dopamine D2 receptor, especially the D 2L type, is important for α-synuclein uptake into dopaminergic neurons. creased at 6 h after the exposure to 1 μM α-synuclein monomer ATTO-550 in the NeuO and FFN206 + neurons, markers for neuronal and VMAT2 positive cells, respectively (Supplementary Materials Figure S1). Additionally, we also investigated the ability of α-synuclein uptake in D2 null mice-derived mesencephalic neurons and revealed that the D2 receptor is critical for the uptake of ATTO-550-labeled α-synuclein monomers in TH + neurons ( Figure 3B, **** p < 0.0001 vs. WT). These data indicate that the dopamine D2 receptor, especially the D2L type, is important for α-synuclein uptake into dopaminergic neurons.

The C-Terminal of α-Synuclein Is Essential for the Uptake into Dopaminergic Neurons
We further investigated the molecular mechanism of α-synuclein uptake in dopaminergic neurons. Since the C-terminal of α-synuclein is assumed to perform an extensive role in neurodegeneration [25][26][27][28], we analyzed the importance of α-synuclein C-terminal region for its uptake in dopaminergic neurons in our culture system. First, we generated the C-terminal-truncated α-synuclein ∆130-140 ( Figure 4A). In the uptake analysis at 48 h after the addition of fluorescence-labeled WT or ∆130-140 α-synuclein, the truncated form showed a reduction in its uptake into TH + neurons ( Figure 4B, **** p < 0.0001). These data suggest that the C-terminal 131-140 amino acids of α-synuclein is important for its uptake by dopaminergic neurons.
ATTO-550-labeled α-synuclein monomer fluorescence intensity of individual TH + neurons. **** p < 0.0001 in wild type (WT) versus D2L −/− , n = 34 in three independent experiments. (B) Representative images of TH + mesencephalic neurons derived from wild type or D2 null knockout mice. Neurons were treated with ATTO-550-labeled α-synuclein monomer in the same condition as in (A) and stained with anti-TH antibody (TH, green) and dopamine D2 receptor (DRD2, blue). The magnified images were enlarged by three times. Scale bar 10 μm. The quantitative analysis of ATTO-550-labeled αsynuclein monomer fluorescence intensity of individual TH + neurons on the right. **** p < 0.0001 in WT versus D2 null knockout (D2 null), n = 28 in three independent experiments.

The C-Terminal of α-Synuclein is Essential for the Uptake into Dopaminergic Neurons
We further investigated the molecular mechanism of α-synuclein uptake in dopaminergic neurons. Since the C-terminal of α-synuclein is assumed to perform an extensive role in neurodegeneration [25][26][27][28], we analyzed the importance of α-synuclein C-terminal region for its uptake in dopaminergic neurons in our culture system. First, we generated the C-terminal-truncated α-synuclein Δ130-140 ( Figure 4A). In the uptake analysis at 48 h after the addition of fluorescence-labeled WT or Δ130-140 α-synuclein, the truncated form showed a reduction in its uptake into TH + neurons ( Figure 4B, **** p < 0.0001). These data suggest that the C-terminal 131-140 amino acids of α-synuclein is important for its uptake by dopaminergic neurons.

D2L Receptors and FABP3 are Critical for the Uptake of α-Synuclein Fibrils and Monomers in Dopaminergic Neurons
The fibril form of α-synuclein shows a higher interaction with the membrane and exacerbates cytotoxicity in cultured cell lines [33]. Thus, we examined whether D2L is crit-

D 2L Receptors and FABP3 Are Critical for the Uptake of α-Synuclein Fibrils and Monomers in Dopaminergic Neurons
The fibril form of α-synuclein shows a higher interaction with the membrane and exacerbates cytotoxicity in cultured cell lines [33]. Thus, we examined whether D 2L is critical for α-synuclein monomers as well as fibrils. We previously demonstrated that FABP3 is critical for the uptake of α-synuclein monomers in TH + cells [9]. Therefore, we first determined that FABP3 is also required for α-synuclein fibril uptake. Importantly, TH + dopaminergic neurons did not take up α-synuclein fibrils in the absence of FABP3 ( Figure 6A, FABP3 −/− ; Figure 6B, p < 0.0001 vs. WT). Moreover, D 2L knockout showed a prominent decrease in the ability of α-synuclein fibril uptake ( Figure 6A, D 2L −/− ; Figure 6B p < 0.0001 vs. WT). In the TH + neurons derived from WT mice, α-synuclein fibrils formed intracellular aggregations in the cell body ( Figure 6A, WT) and showed Lewy neurite-like morphologies in the neuronal processes ( Figure 6C). These data suggest that both FABP3 and D 2L receptors are critical for the α-synuclein fibrils as well as monomers. is critical for the uptake of α-synuclein monomers in TH + cells [9]. Therefore, we first determined that FABP3 is also required for α-synuclein fibril uptake. Importantly, TH + dopaminergic neurons did not take up α-synuclein fibrils in the absence of FABP3 ( Figure  6A, FABP3 −/− ; Figure 6B, p < 0.0001 vs. WT). Moreover, D2L knockout showed a prominent decrease in the ability of α-synuclein fibril uptake ( Figure 6A, D2L −/− ; Figure 6B p < 0.0001 vs. WT). In the TH + neurons derived from WT mice, α-synuclein fibrils formed intracellular aggregations in the cell body ( Figure 6A, WT) and showed Lewy neurite-like morphologies in the neuronal processes ( Figure 6C). These data suggest that both FABP3 and D2L receptors are critical for the α-synuclein fibrils as well as monomers.

D 2L Receptors Are Predominantly Co-Localized with α-Synuclein Fibril ATTO-550 in Dopaminergic Neurons
Finally, we investigated the spatial characteristics of D 2L receptors coupled with αsynuclein fibril uptake. After 48 h of the treatment with 1 µM α-synuclein fibril ATTO-550, cells were fixed and analyzed through immunocytochemistry. Importantly, we found that αsynuclein fibrils taken up by TH + neurons were eminently co-localized with D 2L immunoreactivities ( Figure 7A, WT). Z-dimension analysis revealed that D 2L tightly co-localized with α-synuclein fibrils ( Figure 7B). D 2L −/− TH + neurons showed no intracellular aggregation of α-synuclein fibrils ( Figure 7A, D 2L −/− ). These data indicate that D 2L receptors are involved in the uptake process of α-synuclein fibrils and their intracellular aggregation.

Discussion
In the current study, we demonstrated that dopamine D 2L receptor is critical for the uptake of α-synuclein fibrils and monomers in dopaminergic neurons using D 2L -deficient mice, specifically lacking dopamine D2 long type receptors, as well as D2 null mice. We also employed FABP3 −/− mice and found that FABP3 is critical for the uptake of α-synuclein fibrils in addition to monomers [9]. D 2L receptors and FABP3 (Figure 2) as well as D 2L and α-synuclein fibrils (Figure 7) were highly co-localized. Dopaminergic neurons required the C-terminal region of α-synuclein to take up the protein into the cells (Figure 4). Additionally, dopaminergic neurons took up α-synuclein in a caveola-dependent manner ( Figure 5). Our data indicate a novel mechanism by which D 2L receptors are required for the caveola-mediated α-synuclein uptake through its C-terminal region coupled with FABP3 ( Figure 8).

Discussion
In the current study, we demonstrated that dopamine D2L receptor is critical for the uptake of α-synuclein fibrils and monomers in dopaminergic neurons using D2L-deficient mice, specifically lacking dopamine D2 long type receptors, as well as D2 null mice. We also employed FABP3 −/− mice and found that FABP3 is critical for the uptake of α-synuclein fibrils in addition to monomers [9]. D2L receptors and FABP3 (Figure 2) as well as D2L and α-synuclein fibrils (Figure 7) were highly co-localized. Dopaminergic neurons required the C-terminal region of α-synuclein to take up the protein into the cells (Figure 4). Additionally, dopaminergic neurons took up α-synuclein in a caveola-dependent manner ( Figure 5). Our data indicate a novel mechanism by which D2L receptors are required for the caveola-mediated α-synuclein uptake through its C-terminal region coupled with FABP3 ( Figure 8). We demonstrated that dopaminergic neurons require dopamine D2L receptors, FABP3, and dynamin/caveolin-1coupled caveolae formation to take up α-synuclein monomers as well as fibrils. In this context, FABP3 and caveolin can interact with α-synuclein, and dopamine D2L receptors bind to FABP3. Caveolin and dopamine D2L receptors are abundant in lipid raft. The caveolae-mediated endocytosis is coupled with dopamine D2L receptors and FABP3, and the structure is abundantly associated with α-synuclein. The structure of D2L/FABP3 in the endocytotic process holds α-synuclein and does not release until its recycling. We also suggest that α-synuclein uptake by different mechanisms in other cell types, such as glial cells, is also conceivable.
Caveola is a lipid raft invagination of the plasma membrane with a defined omegashaped structure [34][35][36]. Caveolae are principally composed of caveolin proteins ( Figure  5 and Figure 8). There are three caveolin proteins, caveolin-1, caveolin-2, and caveolin-3 [34]. In neuronal cells, caveolae formation is strictly dependent on caveolin-1 [34], which Figure 8. Schematic illustration of α-synuclein uptake in dopaminergic neurons. We demonstrated that dopaminergic neurons require dopamine D 2L receptors, FABP3, and dynamin/caveolin-1-coupled caveolae formation to take up αsynuclein monomers as well as fibrils. In this context, FABP3 and caveolin can interact with α-synuclein, and dopamine D2L receptors bind to FABP3. Caveolin and dopamine D 2L receptors are abundant in lipid raft. The caveolae-mediated endocytosis is coupled with dopamine D 2L receptors and FABP3, and the structure is abundantly associated with αsynuclein. The structure of D 2L /FABP3 in the endocytotic process holds α-synuclein and does not release until its recycling. We also suggest that α-synuclein uptake by different mechanisms in other cell types, such as glial cells, is also conceivable.
Caveola is a lipid raft invagination of the plasma membrane with a defined omega-shaped structure [34][35][36]. Caveolae are principally composed of caveolin proteins (Figures 5 and 8). There are three caveolin proteins, caveolin-1, caveolin-2, and caveolin-3 [34]. In neuronal cells, caveolae formation is strictly dependent on caveolin-1 [34], which was knocked down in this study to elucidate the caveola-dependent uptake of α-synuclein. Caveolin-1 interacts with α-synuclein [37], suggesting that the uptake of the protein might be mediated by caveolaemediated endocytosis. The internalization of caveolae is also mediated by dynamin in mammalian cells [38]. Consistent with these results, we demonstrated that caveolin-1 knockdown or treatment with dynasore, a cell-permeable inhibitor of dynamin [32], abolishes α-synuclein uptake into dopaminergic neurons ( Figure 5). Dynamin-dependent α-synuclein uptake is also modulated by clusterin in the case of astrocytes [39], indicating the possible mechanism in clusterin-mediated regulation of neuronal uptake inhibition of extracellular α-synuclein.
Dopamine D 2L receptors bind to FABP3 by the 29 amino acid domain of the third cytoplasmic loop [18,40,41]. Furthermore, FABP3 is localized in mature neurons and interacts with α-synuclein in dopaminergic neurons in the substantia nigra [13,17,42,43], and directly binds to α-synuclein in vitro (in submission). Additionally, α-synuclein itself has the ability to interact with the plasma membrane to pass through by the formation of short fibrils [33]. In this study, we provided evidence that D 2L receptors and FABP3 are critical for α-synuclein uptake coupled with caveolae formation in TH + neurons. These data suggest a possible mechanism by which dopaminergic neurons passively take up membrane-bound α-synuclein in a caveolae-mediated and D 2L -FABP3-associated manner (Figure 8).
Dopamine D 2S and D 2L receptors may differentially contribute to dopaminergic signaling [44][45][46]. Interestingly, deletion of D 2L diminishes the typical antipsychotic racloprideinduced parkinsonism [44]. These data suggest the potential of drug development targeting the D 2L receptors to sequester α-synuclein to prevent its uptake into dopaminergic neurons in Parkinson's disease therapy. D 2L receptors play a more prominent role than D 2S in mediating emotional response, such as behavioral reactions to novelty and inescapable stress [47], indicating the possibility that protection of D 2L from α-synuclein is important in the uptake and propagation process of the protein. In addition to the FABP ligands [48][49][50], we will try to develop the molecules targeting α-synuclein to prevent its uptake into dopaminergic cells.
In conclusion, the present study demonstrated the physiological significance of dopamine D 2L receptors in the uptake and accumulation process of α-synuclein in neuronal cells. D 2L was critical for α-synuclein uptake coupled with FABP3 in a caveola-dependent manner. In addition, D 2L and FABP3 are crucial for the uptake of α-synuclein fibrils as well as monomers. Our data suggest that D 2L is a potential target for the development of prophylactic medicine by preventing α-synuclein propagation in the neurodegenerative process in Parkinson's disease and other synucleinopathies. Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.