Synaptic connector, nucleic acid, dopamine synapse formation promoter, pharmaceutical composition, method for promoting formation of dopamine synapse, method for treating parkinson's disease, use of synaptic connector, functional variant of cbln4, complex, and method for producing artificial synaptic connector

Abstract

A synaptic connector that promotes the formation of dopamine synapse, the synaptic connector being a molecule capable of connecting a deleted in colorectal cancer (DCC) protein present on the surface of the front part of dopamine synapse and a delta-type glutamate receptor (GluD) protein present on the surface of the rear part of dopamine synapse.

Description

Synaptic connectors, nucleic acids, dopamine synapse formation promoters, pharmaceutical compositions, methods for promoting dopamine synapse formation, methods for treating Parkinson's disease, uses of synaptic connectors, functional variants of CBLN4, complexes, and methods for producing artificial synaptic connectors.

[0001] The present invention relates to synaptic connectors, nucleic acids, dopamine synapse formation promoters, pharmaceutical compositions, methods for promoting dopamine synapse formation, methods for treating Parkinson's disease, the use of synaptic connectors, functional variants and complexes of CBLN4, and methods for producing artificial synaptic connectors.

[0002] Rapid information transmission via glutamate and GABA is achieved by the release and reception of neurotransmitters at synapses in milliseconds. In addition to cell adhesion molecules such as neuroxin and neuroligin, secretory factors such as Wnt and FGF are known to play important roles in synapse formation, maturation, and maintenance. Recently, it has been reported that C1q family molecules, which share structural characteristics with the innate immune molecule complement C1q, can simultaneously bind to presynaptic and postsynaptic receptors after secretion, thereby regulating synapse formation, maintenance, and maturation. Cerebellin (Cbln1-Cbln4) is a representative example; for instance, Cbln1 has been shown to strongly regulate synapse formation in the cerebellum by cross-linking presynaptic neuroxin with postsynaptic glutamate receptor δ2 (GluD2) to form a complex.

[0003] On the other hand, neuromodulatory transmission of dopamine, serotonin, acetylcholine, and other neurotransmitters has traditionally been described as "volume transmission," where neurotransmitters released from the axonal ampulla diffuse and exert their effects. However, in recent years, it has been suggested that, at least in dopaminergic axons, molecules common to glutamatergic and GABAergic transmission pathways may be involved in the dopamine release process, and that discrete adhesion and contact sites with postsynaptic neurons may exist. However, the molecules and mechanisms that form synapses in dopaminergic axons were completely unknown. Therefore, there is a strong need to identify the molecules responsible for dopamine neurotransmission and to elucidate the mechanisms of formation and maintenance of transmission sites based on them. Since dopamine-dependent circuits are involved in motor function, motivation, and reinforcement learning, and are also associated with pathological conditions such as Parkinson's disease, addiction, and depression, establishing a technological foundation that can selectively construct or enhance dopamine transmission sites in the mature brain is extremely important.

[0004] To date, it has been reported, for example, that Cbln1 is a synaptic connector that forms synapses, and that synaptic-like structures are observed in dopamine neurotransmission.

[0005] Dual character, asynaptic and synaptic, of the dopamine inervation in adult rat neostrium: a quantitative autoradiographic and immunocytochemical analysis. Liu C, Kershberg L, Wang J, Schneeberger S, Kaeser PS. Dopamine Secretion Is Mediated by Sparse Active Zone-like Release Sites. Cell. 2018 Feb 8;172(4):706-718. e15.

[0006] The present invention aims to solve the aforementioned conventional problems and achieve the following objectives. Specifically, the present invention aims to provide synaptic connectors, nucleic acids, dopamine synapse formation promoters, pharmaceutical compositions, methods for promoting dopamine synapse formation, methods for treating Parkinson's disease, the use of synaptic connectors, functional variants and complexes of CBLN4, and methods for manufacturing artificial synaptic connectors, all of which can promote the formation of dopamine synapses.

[0007] The means for solving the aforementioned problems are as follows: <1> A synaptic connector that promotes the formation of dopamine synapses, wherein the synaptic connector is a molecule that has the ability to connect the DCC (Deleted in Colorectal Cancer) protein present on the surface of the dopamine presynaptic site with the GluD (Delta-type Glutamate receptor) protein present on the surface of the dopamine postsynaptic site. <2> The synaptic connector according to <1>, wherein the GluD is GluD1. <3> The synaptic connector according to <1> or <2>, wherein the synaptic connector is a protein, nucleic acid, or small molecule compound. <4> The synaptic connector according to any one of <1> to <3>, wherein the synaptic connector is a protein. <5> The synaptic connector according to any one of <1> to <4>, wherein the synaptic connector is celeveline 4 (Cbln4) or a functional variant thereof. <6> The synaptic connector according to <5>, wherein the Cbln4 or its functional variant is human Cbln4 or its functional variant. <7> The synaptic connector according to <6>, wherein the Cbln4 or its functional variant is a protein consisting of an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. <8> The synaptic connector according to any one of <1> to <4>, wherein the synaptic connector is an artificial complex of a binding domain that binds to DCC and a binding domain that binds to GluD. <9> The synaptic connector according to <8>, wherein the synaptic connector is a complex of a nanobody that binds to DCC containing an amino acid sequence represented by SEQ ID NO: 3 and a nanobody that binds to GluD containing an amino acid sequence represented by SEQ ID NO: 4. <10> The synaptic connector according to <9>, wherein the synaptic connector is a polypeptide containing an amino acid sequence represented by SEQ ID NO: 5. <11> A nucleic acid comprising a nucleotide sequence encoding the synaptic connector according to any one of <4> to <10>. <12> A dopamine synapse formation promoter comprising the synaptic connector according to any one of <1> to <10>.<13> The dopamine synapse formation promoter according to <12>, wherein the synaptic connector is Cbln4 or a functional variant thereof. <14> The dopamine synapse formation promoter according to <13>, wherein Cbln4 or a functional variant thereof is translated from a polynucleotide containing an encoded base sequence. <15> The dopamine synapse formation promoter according to <14>, wherein the polynucleotide is mounted on an expression vector. <16> The dopamine synapse formation promoter according to <15>, wherein the expression vector is a plasmid vector or a viral vector. <17> The dopamine synapse formation promoter according to <16>, wherein the viral vector is one of an adeno-associated virus (AAV) vector, an adenovirus vector, or a lentiviral vector. <18> The dopamine synapse formation promoter according to <12>, wherein the synaptic connector is a complex of a binding domain that binds to DCC and a binding domain that binds to GluD. <19> The dopamine synapse formation promoter according to <18>, wherein the complex is translated from a polynucleotide comprising a nucleotide sequence encoding a binding domain that binds to DCC and a binding domain that binds to GluD. <20> The dopamine synapse formation promoter according to <19>, wherein the polynucleotide is mounted on an expression vector. <21> The dopamine synapse formation promoter according to <20>, wherein the expression vector is a plasmid vector or a viral vector. <22> The dopamine synapse formation promoter according to <21>, wherein the viral vector is one of an adeno-associated virus (AAV) vector, an adenovirus vector, or a lentiviral vector. <23> A pharmaceutical composition comprising the dopamine synapse formation promoter according to any one of <12> to <22>. <24> The pharmaceutical composition according to <23> for the treatment of Parkinson's disease. <25> A method for promoting dopamine synapse formation, comprising the step of administering the synapse connector according to any one of <1> to <10> to a target. <26> A method for treating Parkinson's disease, comprising the step of administering a therapeutically effective amount of the synaptic connector described in any of <1> to <10> above.<27> A synaptic connector according to any one of <1> to <10> above for use in promoting the formation of dopamine synapses. <28> A synaptic connector according to any one of <1> to <10> above for use in the treatment of Parkinson's disease. <29> Use of a synaptic connector according to any one of <1> to <10> above in the manufacture of a dopamine synapse formation promoter. <30> Use of a synaptic connector according to any one of <1> to <10> above in the manufacture of a pharmaceutical composition for the treatment of Parkinson's disease. <31> A functional variant of Cbln4 comprising the amino acid sequence represented by SEQ ID NO: 2. <32> A nucleic acid encoding the functional variant of Cbln4 according to <31> above. <33> A complex comprising a binding domain that binds to DCC comprising the amino acid sequence of SEQ ID NO: 3 and a GluD binding domain that binds to the ATD region of GluD1 shown in SEQ ID NO: 6. <34> The complex according to <33> above, comprising a binding domain that binds to DCC comprising the amino acid sequence represented by SEQ ID NO: 3 and a binding domain that binds to GluD comprising the amino acid sequence of SEQ ID NO: 4. <35> The complex according to <33> or <34>, comprising a binding domain that binds to DCC, a binding domain that binds to GluD, an immunoglobulin Fc, and a linker sequence. <36> The complex according to <35>, comprising the amino acid sequence represented by Sequence ID No. 5. <37> A nucleic acid encoding the complex according to any one of <33> to <36>. <38> A method for producing an artificial synaptic connector that promotes the formation of dopamine synapses, characterized by binding a molecule that binds to DCC protein and a molecule that binds to GluD protein. <39> The method for producing an artificial synaptic connector according to <38>, wherein each of the molecule that binds to DCC protein and the molecule that binds to GluD protein is a binding domain that binds to DCC protein and a binding domain that binds to GluD protein. <40> A DCC binding domain comprising the amino acid sequence represented by Sequence ID No. 3. <41> A nucleic acid encoding the binding domain according to <40>. <42> A GluD binding domain comprising the amino acid sequence represented by Sequence ID No. 4. <43> A nucleic acid encoding the binding domain according to <42>.

[0008] According to the present invention, it is possible to solve the aforementioned problems in the conventional era, achieve the aforementioned objectives, and promote the formation of dopamine synapses, and to provide synaptic connectors, nucleic acids, dopamine synapse formation promoters, pharmaceutical compositions, methods for promoting the formation of dopamine synapses, methods for treating Parkinson's disease, uses of synaptic connectors, functional variants and complexes of CBLN4, and methods for manufacturing artificial synaptic connectors.

[0009] Figure 1A shows the staining images of HA-Cbln1 and TH in HA-Cbln1 knock-in mice. Scale bar: 1 mm. Figure 1B is a high-magnification image of the frontal linear nucleus (RLi), substantia nigra compacta (SNc), and lateral substantia nigra (SNL) regions of Figure 1A. Scale bar: 10 μm. Figure 1C shows the staining images of HA-Cbln2 and TH in HA-Cbln2 knock-in mice. Scale bar: 1 mm. Figure 1D is a high-magnification image of the interfascicular fossa (IF), parabrainstem pigment zone (PBP), substantia nigra compacta (SNc), and lateral substantia nigra (SNL) regions of Figure 1C. Scale bar: 10 μm. Figure 1E shows the staining images of HA-Cbln4 and TH in HA-Cbln4 knock-in mice. Scale bar: 1 mm. Figure 1F is a high-magnification image of the interfascicular zone (IF) and parabrainstem pigment zone (PBP) of the ventral tegmental area (VTA) and the substantia nigra pars compacta (SNc) portion of Figure 1E. Scale bar: 10 μm. Figure 1G is a stained image showing the distribution of HA-Cbln4 (green) in the dorsal striatum (caudate nucleus-putamen) and nucleus accumbens (NAc) together with tyrosine hydroxylase (TH). Scale bar: 1 mm. Figure 1H is a stained image showing the expression of HA-Cbln4 in the nucleus accumbens (NAc). Scale bar: 5 μm. Figure 2A is a stained image showing the distribution of HA-Cbln4 and VMAT2 along TH-positive axons in the nucleus accumbens (NAc). Scale bar: 5 μm. Figure 2B shows a stained image of the distribution of HA-Cbln4 and VGluT2 along TH-positive axons in the nucleus accumbens (NAc). Scale bar: 5 μm. Figure 2C shows a stained image of the distribution of HA-Cbln4 and VGluT3 along TH-positive axons in the nucleus accumbens (NAc). Scale bar: 5 μm. Figure 2D shows a stained image of the distribution of HA-Cbln4 and RIM1 / 2 along TH-positive axons in the nucleus accumbens (NAc). Scale bar: 5 μm. Figure 2E shows a stained image of the morphology of TH-positive axons in wild-type (WT) mice and Cbln4 knockout (Cbln4 KO) mice. For both WT mice and Cbln4 KO mice, enlarged views of the regions enclosed by A-C in the leftmost figure (left figure) are shown to the right. Scale bars: 10 μm (left figure) and 1 μm (enlarged figures A-C). Figure 2F shows stained images of the distribution of VMAT2 along TH-positive axons in WT mice and Cbln4 KO mice. Scale bar: 5 μm.Figure 2G shows a stained image of the distribution of VGluT2 along TH-positive axons in WT mice and Cbln4 KO mice. Scale bar: 5 μm. Figure 2H shows a stained image of the distribution of VGluT3 along TH-positive axons in WT mice and Cbln4 KO mice. Scale bar: 5 μm. Figure 2I shows a stained image of the distribution of RIM1 / 2 along TH-positive axons in WT mice and Cbln4 KO mice. Scale bar: 5 μm. Figure 3A shows a stained image showing the co-localization of HA-Cbln4 and GluD1 in the dorsal striatum and nucleus accumbens (NAc). Scale bar: 1 mm. Figure 3B shows a high-magnification stained image showing the co-localization of HA-Cbln4 and GluD1 along TH-positive dopaminergic axons within the nucleus accumbens (NAc) shell. Scale bar: 5 μm. Figure 3C is a schematic diagram and stained image of an experiment showing reduced distribution of GluD1 in the nucleus accumbens (NAc) of Cbln4 conditionally knockout (Cbln4 cKO) mice. Scale bar: 5 μm. Figure 3D is a diagram showing attenuation of dopaminergic neural pathways in GluD1 knockout (GluD1 KO) mice. Scale bar: 5 μm. Figure 3E is a schematic diagram and stained image of an artificial synapse assay showing that Cbln4 can induce dopaminergic synapses from primary cultured dopaminergic mice in a GluD1-dependent manner in vitro. Scale bar: 5 μm. Figure 3F shows a schematic diagram and stained image of a synaptic assay demonstrating that Cbln4 can induce dopaminergic synapses from human iPS cell-derived dopaminergic neurons in a GluD1-dependent manner in vitro. Scale bar: 5 μm. Figure 4A shows a stained image showing DCC expression in the ventral tegmental area (VTA) near the striatum. Scale bar: 10 μm. Figure 4B shows a stained image showing DCC expression in the nucleus accumbens (NAc). Scale bar: 5 μm. Figure 4C shows a stained image showing a decrease in DCC signaling in the nucleus accumbens (NAc) of Cbln4 conditionally knockout (Cbln4 cKO) mice. Scale bar: 5 μm. Figure 4D shows a stained image showing a decrease in dopaminergic axons in the nucleus accumbens (NAc) of DCC conditionally knockout (DCC cKO) mice. Scale bar: 5 μm.Figure 4E is a schematic diagram of a clustering assay using Cbln4-binding beads. Figure 4F is a staining image showing that Cbln4 recruits endogenous DCC and VMAT2 in vitro in dopaminergic neurons. Scale bar: 5 μm. Figure 5A is an experimental design to investigate the involvement of Cbln4 in social interaction, social cognition, and reward sensitivity. Figure 5B is a graph showing the results of a contact test (social approach) between male Cbln4 cKO mice and wild-type female mice (mean ± standard error; P < 0.05 vs. control group). Figure 5C is a graph showing the results of a social cognition test (mean ± standard error; P < 0.05 vs. control group). Figure 5D is a graph showing the results of a sucrose preference (reward sensitivity) test (mean ± standard error; P < 0.05 vs. control group). Figure 6A shows a schematic diagram and stained image of an experiment demonstrating that injection of Cbln4 into the putamen can induce dopaminergic synapses in a GluD1 and DCC-dependent manner. Scale bar: 10 μm. Figure 6B shows a schematic diagram and graph of experimental results demonstrating that Cbln4 enhances apomorphine-induced rotational motility. Figure 6C shows a schematic diagram and experimental results demonstrating the binding activity of wild-type and mutant Cbln4 proteins to GluD1 ATD. Figure 6D shows a schematic diagram and experimental results demonstrating the binding activity of wild-type and mutant Cbln4 proteins to DCC FN4-6. Figure 6E is a schematic diagram showing the method for creating a unilateral Parkinson's disease model. Figure 6F is a graph showing the effect of Cbln4 protein administration on a unilateral Parkinson's disease model. Figure 6G shows a staining image in the substantia nigra pars compacta (SNc) administered with 6-OHDA, illustrating the effect of Cbln4 administration in preventing the loss of dopaminergic neurons (TH-positive neurons). Scale bar: 100 μm. Figure 7 is a schematic diagram showing the designs of two types of dopaminergic (DA) synaptic connectors. Figure 8 is a schematic diagram showing an implementation example of an antibody-based dopaminergic (DA) synaptic connector (antibody-type DA synaptic connector). Figure 9 shows the structure and amino acid sequence of the antibody-type DA synaptic connector, DA1-GD51-Fc. Figure 10 shows the structural analysis results of the GD51 and GluD1 ATD complex using cryo-electron microscopy.Figure 11 is a schematic diagram and stained image of an experiment showing that the antibody-type DA synapse connector (DA1-GD51-Fc) induces DA synapse formation in vitro. Figure 12 is a schematic diagram and stained image of an experiment showing that the antibody-type DA synapse connector (DA1-GD51-Fc) induces DA synapse formation in vivo, and the results of the behavioral experiment. Figure 13 is the high-affinity DACbln4 mutant (Cbln4 ｈｉｇｈ ). The figure shows the amino acid sequence (SEQ ID NO: 2). Figure 14 is a stained image showing that the high-affinity DA synapse connector, Cbln4 ｈｉｇｈ induces more dopamine synapse formation in vitro compared to wild-type Cbln4. Scale bar: 50 μm. Figure 15 is a schematic diagram and stained image of an experiment showing that the high-affinity DA synapse connector (Cbln4 ｈｉｇｈ ) induces DA synapse formation in vivo, and the results of the behavioral experiment.

[0010] (Synapse Connector) The synapse connector of this embodiment is a synapse connector that promotes the formation of dopamine synapses. The synapse connector is a molecule having the ability to connect a DCC (Deleted in Colorectal Cancer) protein present on the surface of the presynaptic part of the dopamine synapse and a GluD (Delta-type Glutamate receptor) protein present on the surface of the postsynaptic part of the dopamine synapse.

[0011] (Dopamine Synapse Formation Promoter, and Pharmaceutical Composition) The dopamine synapse formation promoter of this embodiment is a drug for promoting the formation of dopamine synapses, includes the synapse connector of this embodiment, and further includes other components as required. The pharmaceutical composition of this embodiment includes the dopamine synapse formation promoter of this embodiment, and further includes other components as required.

[0012] (Method for Promoting the Formation of Dopamine Synapses) The method for promoting the formation of dopamine synapses of this embodiment is a method for promoting the formation of dopamine synapses, and includes the step of administering the synapse connector of this embodiment to a subject.

[0013] (Method for treating Parkinson's disease) The method for treating Parkinson's disease according to this embodiment is a method for treating Parkinson's disease, and includes a step of administering a therapeutically effective amount of the synaptic connector according to this embodiment to a subject.

[0014] (Use) In one embodiment, there is provided the use of the synaptic connector for use in the treatment of Parkinson's disease. Also, in one embodiment, there is provided the use of the synaptic connector in the production of a dopamine synapse formation promoter. Also, in one embodiment, there is provided the use of the synaptic connector in the production of a pharmaceutical composition for treating Parkinson's disease.

[0015] (Functional variant of Cbln4) In one embodiment, there is provided a functional variant of Cbln4 containing the amino acid sequence represented by SEQ ID NO: 2, which has an improved function of promoting the formation of dopamine synapses as compared with wild-type Cbln4.

[0016] (Complex) In one embodiment, there is provided a complex containing a DCC binding domain containing the amino acid sequence of SEQ ID NO: 3 and a GluD binding domain that binds to the ATD region of GluD1 shown in SEQ ID NO: 6. More preferably, the complex is a complex containing a DCC binding domain containing the amino acid sequence of SEQ ID NO: 3 and a GluD binding domain containing the amino acid sequence of SEQ ID NO: 4. The complex may further contain an FC fragment of an immunoglobulin or a linker.

[0017] (Method for producing an artificial synaptic connector) The method for producing an artificial synaptic connector according to this embodiment is a method for producing an artificial synaptic connector that promotes the formation of dopamine synapses, and is characterized by binding a molecule that binds to a DCC protein and a molecule that binds to a GluD protein. More preferably, it is a method for producing an artificial synaptic connector characterized by binding a DCC binding domain that binds to a DCC protein and a GluD binding domain that binds to a GluD protein.

[0018] [Parkinson's Disease] As of 2023, 2.64 million people in Japan, the United States, and five European countries are affected by Parkinson's disease, and the number continues to increase along with the aging population. Current treatment for Parkinson's disease is symptomatic, mainly centered on levodopa, and there is no fundamental treatment that can suppress the progression of neurodegeneration. Although symptomatic drugs are effective in the early stages, their effectiveness decreases as the disease progresses, and because dopamine levels are not properly regulated in response to diurnal fluctuations and environmental changes, side effects (motor complications) due to dopamine deficiency or excess occur, significantly reducing quality of life.

[0019] This invention aims to achieve a groundbreaking therapeutic effect by restoring natural dopamine secretion by developing a method for repairing synapses between endogenous dopamine neurons and the striatum, using endogenous Cbln4 or its functional variant, or an artificially created synaptic connector that promotes dopamine synapse formation by substituting the function of Cbln4. Furthermore, by repairing synapses, this method can be developed into a fundamental treatment that can suppress the progression of neurodegeneration.

[0020] [Dopamine Synapses] Dopamine neurotransmission regulates movement, reward, motivation, and learning through circuits such as the substantia nigra-striatal pathway and the ventral tegmental area-nucleus accumbens pathway. Dopaminergic axons form numerous varicosities in the striatum and other areas, from which they release dopamine. Traditionally, volume transmission, in which released dopamine diffuses into the extracellular space and acts on receptors, has been emphasized. However, in recent years, it has been suggested that molecular-specific localization and discrete contact with target cells may also exist at dopamine release sites.

[0021] The inventors of this invention have revealed for the first time in the world that Cbln4 binds to DCC present on the surface of dopaminergic axons (presynaptic terminal) and GluD present on the surface of the striatum (postsynaptic terminal) where dopamine receptors are present, thereby forming a structure similar to a synapse in high-speed nerve information transmission mediated by glutamate and GABA, known as a "dopamine synapse."

[0022] In order to solve the above-mentioned objectives, the inventors diligently conducted research and, as demonstrated in the examples described later, obtained the following findings, and based on these findings, completed the present invention. Specifically, they found that Cbln4, expressed in dopamine-producing neurons of the ventral tegmental area (VTA), binds to DCC and GluD1 between dopaminergic axons and the striatum. It was revealed that Cbln4 is a component derived from the ventral tegmental area (VTA) located in a position that cooperates with presynaptic DCC and postsynaptic GluD1, and that Cbln4 induces presynaptic site assembly and promotes dopamine synapse formation in a GluD1-dependent manner. Subsequently, they found that in the substantia nigra-striatal pathway, administration of Cbln4 to the striatum induces functional dopamine synapses. Therefore, in Parkinson's disease model mice exhibiting motor impairment due to a decrease in dopamine synapses in the substantia nigra striatum, administration of Cbln4 to the striatum induced functional dopamine synapses and rescued the motor impairment. Furthermore, it suppressed the progression of substantia nigra dopamine neuronal cell death. Thus, we found that the Cbln4 protein, a molecule capable of connecting DCC and GluD, functions as a synaptic connector that promotes dopamine synapse formation.

[0023] Based on these findings, the inventors further conceived and fabricated an artificially created dopamine (DA) synapse connector (also referred to as an artificial synapse connector, synapse connector, or "Bispecific Neuron Engineer (BiNE)") capable of connecting DCC and GluD, and demonstrated that this artificial synapse connector functions as an artificial synapse connector that promotes the formation of dopamine synapses.

[0024] Based on the above findings, the inventors discovered that a molecule capable of connecting the DCC protein present on the surface of the presynaptic dopamine site to the GluD protein present on the postsynaptic site can be used as an artificial synaptic connector to promote dopamine synapse formation, thus completing the present invention. According to the present invention, an artificial synaptic connector that can promote dopamine synapse formation can be provided. The artificial synaptic connector can contribute as a fundamental treatment that can suppress the progression of neurodegeneration in Parkinson's disease.

[0025] The following describes the synaptic connector of this embodiment, a dopamine synapse formation promoter, a pharmaceutical composition, a method for promoting dopamine synapse formation, a method for treating Parkinson's disease, the synaptic connector in use, a functional variant of Cbln4 or nucleic acid encoding said functional variant, a complex or nucleic acid encoding said complex, and a method for manufacturing an artificial synaptic connector.

[0026] <Synaptic Connector> The synaptic connector is not particularly limited and can be appropriately selected depending on the purpose, as long as it is a molecule that has the ability to connect the DCC protein present on the surface of the dopamine presynaptic site with the GluD protein present on the surface of the dopamine postsynaptic site. For example, it may be a protein, nucleic acid, or small molecule compound.

[0027] [DCC Protein] The DCC (Deleted in Colorectal Cancer) protein is a single-pass transmembrane receptor expressed on the cell surface and is primarily known as a receptor for the axon guidance factor netrin-1. In the developing nervous system, DCC is involved in axon extension and guidance, neural circuit formation, and the regulation of cell adhesion signaling. It induces cytoskeletal rearrangement through ligand binding via its extracellular domain and downstream signal transduction via its intracellular domain. In the adult brain, it has also been suggested to be involved in synapse formation, plasticity, and the maintenance of circuit function, making it an important molecular basis for the construction and maintenance of neural circuits.

[0028] [GluD Proteins] GluD (δ-type glutamate receptors; GluD1 / GRID1 and GluD2 / GRID2) proteins are transmembrane receptor proteins classified as part of the ionotropic glutamate receptor family. Generally, their properties as classical ligand-gated channels are limited or atypical, and they are known to function as "synaptic organizer receptors" that regulate synapse formation and maintenance through intermolecular interactions via their extracellular domains. In particular, GluD2 is highly expressed in cerebellar Purkinje cells, binds to Neuroxin via Cbln1, and is essential for parallel fiber-Purkinje cell synapse formation. On the other hand, GluD1 is expressed in a wide range of brain regions and is suggested to be involved in circuit-specific cell adhesion and organization mechanisms. GluD1 protein is preferred as the GluD protein to which dopamine synaptic connectors bind.

[0029] [Cbln4] Cbln4 (cerebellin 4) is a secreted glycoprotein belonging to the C1q family, possessing a C1q-like domain common to complement C1q, and is a member of the cerebellin group (Cbln1-Cbln4) which is expressed selectively in neural circuits and cell types. Cbln4 can form multimers extracellularly and function as a secreted organizer that binds to cell surface molecules presynaptic and synaptic connections to organize intercellular interactions. Human Cbln4 protein may have the amino acid sequence represented by Sequence ID No. 1.

[0030] Throughout this specification, gene names and protein names, whether uppercase or lowercase, are used to include orthologs of all mammalian species. In this specification, the animal species from which a gene name or protein name is derived is distinguished by prefixing the gene name or protein name with the animal species name.

[0031] There are no particular limitations on the form in which the synaptic connector is a protein, and it can be appropriately selected depending on the purpose. However, in one embodiment, it is preferably Cbln4 or a functional variant thereof, and more preferably human Cbln4 or a functional variant thereof. Specifically, it is preferably a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.

[0032] Here, "functional mutant" refers to a variant of Cbln4 that has the ability to link DCC protein and GluD protein. It may retain functions equivalent to Cbln4, or it may be a variant that has improved function in promoting dopamine synapse formation compared to Cbln4. The amino acid sequence of the functional mutant is preferably 90% or more identical to each amino acid sequence represented by SEQ ID NO: 1, and more preferably 95% or more identical. Alternatively, it may be a functional mutant of CBLN4 that includes the amino acid sequence represented by SEQ ID NO: 2.

[0033] The synaptic connector of the present invention, which is a molecule having the ability to connect the DCC protein present on the surface of the dopamine presynaptic site to the GluD protein present on the surface of the dopamine postsynaptic site, may be a complex, and more preferably a complex of a binding domain that binds to DCC and a binding domain that binds to GluD. In one embodiment, the synaptic connector is preferably a complex of a binding domain that binds to DCC, which includes the amino acid sequence represented by SEQ ID NO: 3, and a binding domain that binds to the amino-terminal domain (ATD) of GluD1, which includes the amino acid sequence represented by SEQ ID NO: 6 (preferably a binding domain that binds to GluD, which includes the amino acid sequence represented by SEQ ID NO: 4). Furthermore, it is more preferable that the synaptic connector is a complex of a binding domain that binds to DCC, which includes the amino acid sequence represented by SEQ ID NO: 5, and a binding domain that binds to GluD.

[0034] <Binding Domain> There are no particular restrictions on the binding domain that constitutes the synaptic connector and binds to DCC or GluD, and can be appropriately selected depending on the purpose, but it is preferably one of the following: antibody, nanobody, aptamer, special peptide, or small molecule compound. Among these, nanobody is more preferred because it has a molecular size of about 1 / 10 that of a typical antibody.

[0035] <Nanobodies> Nanobodies are the smallest protein fragments (single-chain antibodies) that recognize antigens, obtained by genetically modifying antibodies. They are antibody fragments VHH (variable domain of the HCAb heavy chain) derived from camelid animal species such as alpacas and camels.

[0036] Furthermore, the synaptic connector may include multiple domains of Cbln4 or its functional variant, as long as it is a molecule capable of linking the DCC protein and the GluD protein. It is preferable that the nanobody forms a trimer, as the binding domain of the Cbln4 protein to GluD is a trimer, and it can efficiently interact with the binding domain of the trimer, thereby enabling interaction between the Cbln4 protein and GluD.

[0037] The aforementioned synaptic connector may be an artificial synaptic connector consisting of a fusion protein formed by further fusing peptides such as tags, linkers, and protein domains, as long as it is a molecule capable of linking the DCC protein and the GluD protein. Examples of tags to be fused include HA tags and His tags. Examples of protein domains to be fused include immunoglobulin Fc.

[0038] As the Fc, human IgG Fc is preferred. As the human IgG, those lacking antibody-dependent cell-mediated cytotoxicity (ADCC) activity and complement-dependent cell-mediated cytotoxicity (CDC) activity are preferred. Examples of human IgG lacking ADCC / CDC activity include wild-type or S228P-mutated IgG4, IgG1 LALA (L234A / L235A), IgG1 aglycosylated (N297A), IgG4 FALA (F234A / L235A), and IgG4 SPLE (S228P / L235E).

[0039] Examples of the artificial synaptic connector being a low-molecular-weight compound include molecular glue.

[0040] Examples of the artificial synaptic connector being nucleic acid include aptamers (nucleic acid antibodies).

[0041] <Nucleic Acid> The nucleic acid (polynucleotide) containing the base sequence encoding the synaptic connector is not particularly limited as long as it contains the base sequence encoding the amino acid sequence of the synaptic connector described above, and can be appropriately selected depending on the purpose. For example, it may be in the form of mRNA or DNA, or it may be in the form of being incorporated into an expression vector, and any of these can be appropriately selected depending on the purpose.

[0042] The nucleic acid having a base sequence encoding a synaptic connector that induces the formation of dopamine synapses in the present invention is not particularly limited as long as it is a nucleic acid having a triple base sequence encoding each amino acid of the synaptic connector, and can be appropriately selected depending on the purpose. Examples of the base sequence of the nucleic acid encoding the synaptic connector include a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, and a base sequence encoding a functional variant of Cbln4. The base sequence encoding the functional variant is preferably 90% or more identical to the base sequence encoding the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, and more preferably 95% or more identical. It may also be a base sequence encoding the amino acid sequence represented by SEQ ID NO: 2, or a base sequence encoding each of the synaptic connectors described above.

[0043] Furthermore, the nucleic acid having a base sequence encoding an artificial synaptic connector may be any nucleic acid encoding a fusion protein in which a binding domain that binds to a DCC protein and a binding domain that binds to a GluD protein are bound, or it may be a nucleic acid encoding a fusion protein that further contains peptides such as a tag, linker, or protein domain. Examples of the protein domain include the Fc fragment of an immunoglobulin.

[0044] <Dopamine synapse formation promoter and pharmaceutical composition> The dopamine synapse formation promoter containing a synapse connector may be the synapse connector itself or a pharmaceutical composition. The pharmaceutical composition may be a protein preparation containing a synapse connector, a composition containing nucleic acid encoding a synapse connector, may contain other drugs, or may contain a pharmaceutically acceptable carrier.

[0045] "Pharmacologically acceptable" means a non-toxic component or composition that is physiologically acceptable and, when administered to humans, does not typically cause gastrointestinal disorders, dizziness, or other allergic reactions, or similar reactions. Examples of such carriers include solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads, microsomes, and biodegradable nanoparticles.

[0046] The dopamine synapse formation promoter and / or pharmaceutical composition may be formulated with a suitable carrier depending on the route of administration. There are no particular restrictions on the route of administration, and it can be appropriately selected depending on the purpose; for example, it may be administered parenterally or orally. Examples of parenteral administration routes include the striatum, midbrain, cerebrospinal fluid (CSF), nasal mucosa, veins, subcutaneous tissue, and muscles.

[0047] The dopamine synapse-promoting agent and / or pharmaceutical composition may be formulated using methods known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to a subject.

[0048] The dopamine synapse-forming agent and / or pharmaceutical composition, formulated in the manner described above, may be administered in an effective dose via various routes, including parenteral, oral, nasal, subcutaneous, intravenous, or intramuscular. In the foregoing, “effective dose” means the amount of substance administered to a patient that allows for the monitoring of diagnostic or therapeutic effects.

[0049] The dosage of the dopamine synapse formation promoter and / or pharmaceutical composition can be appropriately selected according to the route of administration, target recipient, target disease and its severity, age, sex, weight, individual differences, and disease state. The dopamine synapse formation promoter and / or pharmaceutical composition can be appropriately selected in terms of the content of the active ingredient, synaptic connector, according to the degree of the disease, but generally, when using adults as a reference, the active ingredient may be administered in amounts of 1 μg to 10 μg (for example, 5 μg) per dose, repeated once every few days.

[0050] A dopamine synapse formation promoter and / or pharmaceutical composition containing Cbln4 or its functional variant, or nucleic acids encoding synaptic connectors such as antibodies or nanobody complexes, may contain mRNA encoding synaptic connectors, or in another embodiment, nucleic acids encoding synaptic connectors mounted on an expression vector, or other drugs, or a pharmaceutically acceptable carrier.

[0051] The dopamine synapse formation promoter and / or pharmaceutical composition preferably contains mRNA encoding a synaptic connector, and more preferably has a 5' cap structure and a 3' tail structure from the viewpoint of mRNA stabilization. Examples of the 5' cap structure include, but are not limited to, m7GpppNp-RNA, m7GpppNmp-RNA, and m7GpppNmpNmp-RNA (wherein m represents a 2'-O-methyl residue). Examples of the 3' tail structure include a 3' poly(A) tail (e.g., 10 to 200 adenosine nucleotides), a 3' poly(C) tail (e.g., 10 to 200 cytosine nucleotides), and combinations thereof.

[0052] The mRNA encoding the synaptic connector may include a 5' untranslated region and / or a 3' untranslated region. The 5' untranslated region and / or 3' untranslated region is, for example, 50 to 500 nucleotides and preferably contains elements that affect mRNA stabilization, translation, etc.

[0053] From the viewpoint of mRNA stabilization, the mRNA encoding the synaptic connector is preferably encapsulated in lipid nanoparticles. Examples of lipid nanoparticles include cKK-E12 (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione), OF-02, Target 23, Target 24, ICE, HGT5000, HGT5001, HGT4003, DOTAP (1,2-dioleyl-3-trimethylammoniumpropane), DODAP (1,2-dioleyl-3-dimethylammoniumpropane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammoniumpropane), DLinDMA, and DODAC. It is preferable to include cationic lipids selected from the group consisting of DDAB, DMRIE, DOSPA, DOGS, DODMA, DMDMA, DODAC, DLenDMA, DMRIE, CLINDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, K-K-DMA, DLin-K-XTC2-DMA, DLin-KC2-DMA, dialkylamino-based, imidazole-based, and guanidine-based cationic lipids, and may further contain non-cationic lipids, PEG-modified lipids, etc.

[0054] mRNA encoding synaptic connectors may consist of unmodified nucleotides or may contain one or more modified nucleotides. Examples of modified nucleotides include pseudouridine, N-1-methylpseudridine, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 4'-thiouridine, 4'-thiocytidine, and 2-thiocytidine.

[0055] A dopamine synapse formation promoter and / or pharmaceutical composition containing an expression vector encoding a synaptic connector such as an antibody, nanobody, or aptamer may contain an expression vector encoding a synaptic connector, other drugs, and a pharmaceutically acceptable carrier.

[0056] There are no particular restrictions on the expression vector encoding the synaptic connector, and any known expression vector can be appropriately selected depending on the purpose. Examples include plasmid vectors and viral vectors. Examples of viral vectors include adeno-associated virus (AAV), adenovirus, lentiviral vector, retrovirus, poxvirus, baculovirus, vaccinia virus, herpes simplex virus, Epstein-Barr virus, adenovirus, geminivirus, and karimovirus. Among these, adeno-associated virus (AAV) vectors, adenovirus vectors, and lentiviral vectors are preferred, with adeno-associated virus vectors preferred because they are derived from non-pathogenic viruses, are highly safe, and can efficiently introduce genes into non-dividing cells. The expression vector preferably comprises a promoter, a nucleic acid sequence encoding the synaptic connector functionally linked to the promoter, and a 5' untranslated region (UTR). The promoter may be a constitutive expression promoter or a conditionally regulated expression promoter, and any known promoter can be appropriately selected depending on the purpose.

[0057] The mRNA and / or expression vector encoding the synaptic connector may be in liquid form or lyophilized powder form. The dopamine synapse formation promoter and / or pharmaceutical composition and syringe may be pre-filled for single-dose administration. The vial may contain the composition in lyophilized powder or liquid form.

[0058] In one embodiment, the dose of mRNA and / or expression vector encoding the synaptic connector is preferably at least 0.5 mg / kg based on the subject's body weight. Examples of such doses include 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, and 50 mg / kg.

[0059] In one embodiment, the expression of synaptic connectors encoded by mRNA and / or expression vectors can be detected at least 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or 1 month after administration.

[0060] When the dopamine synapse formation promoter and / or pharmaceutical composition is used in combination with other drugs, the dopamine synapse formation promoter and / or pharmaceutical composition and the other drugs may be contained in one formulation, or they may be contained in separate formulations. If they are separate formulations, each formulation may be administered simultaneously, or they may be administered separately at different times.

[0061] Examples of methods for evaluating the expression level of the synaptic connector include Western blotting, immunohistochemistry (IHC), immunofluorescence (IF), immunochromatography, enzyme immunoassay (ELISA), chemiluminescence enzyme immunoassay (CLEIA), chemiluminescence immunoassay (CLIA), fluorescence enzyme immunoassay, aptamer method, immunoturbidimetry, dye colorimetric method, immunowax method, latex agglutination method, Jaffe method, and gold colloid colorimetric method. Examples of methods for evaluating the expression level of mRNA encoding the synaptic connector include in situ hybridization, quantitative reverse transcription polymerase chain reaction (RT-qPCR), and spatial transcriptome analysis.

[0062] There are no particular restrictions on the antibodies that can detect the aforementioned synaptic connectors, and they can be appropriately selected depending on the purpose. Furthermore, synaptic connectors with tags such as HA tags attached can be expressed, and they can be detected using antibodies against the tags.

[0063] There are no particular restrictions on the PCR primers or hybridize probes capable of detecting the mRNA and / or expression vector encoding the synaptic connector, and they can be appropriately selected depending on the purpose.

[0064] [Subjects] The subjects are generally mammals, such as humans, non-human primates, dogs, cats, mice, rats, cattle, horses, and pigs. Among these, humans are preferred. The subjects may be adults, infants, children, or the elderly, and subjects suspected of having Parkinson's disease, or subjects diagnosed or confirmed to have Parkinson's disease are preferred.

[0065] There are no particular restrictions on the specimens used to evaluate the expression levels of synaptic connectors, mRNA encoding synaptic connectors, and / or expression vectors. They can be appropriately selected depending on the purpose, and examples include pathways such as the striatum, midbrain, cerebrospinal fluid (CSF), nasal mucosa, veins, subcutaneous tissue, and muscles.

[0066] <Complex> The complex is a synaptic connector that promotes the formation of dopamine synapses. The complex may, for example, include a binding domain that binds to DCC containing the amino acid sequence of SEQ ID NO: 3 and a GluD binding domain that binds to the N-terminal domain (amino-terminal domain (ATD)) of human GluD1 shown in SEQ ID NO: 6. ATD is the largest extracellular domain of GluD and is located in the synaptic cleft.

[0067] The GluD-binding domain is preferably one that includes the amino acid sequence of SEQ ID NO: 4.

[0068] <Method for Manufacturing an Artificial Synaptic Connector> The method for manufacturing an artificial synaptic connector is characterized by binding a molecule that binds to DCC protein and a molecule that binds to GluD protein, and is a method for manufacturing an artificial synaptic connector that promotes the formation of dopamine synapses. Preferably, the method involves binding a binding domain that binds to DCC protein and a binding domain that binds to GluD protein, and is a method for manufacturing an artificial synaptic connector that promotes the formation of dopamine synapses.

[0069] Examples of the aforementioned molecules include proteins, nucleic acids, or small molecule compounds. Examples of the aforementioned proteins include antibodies, nanobodies, and special peptides. Examples of the aforementioned nucleic acids include aptamers. Examples of the aforementioned small molecule compounds include molecular glues.

[0070] The present invention will be described more specifically below based on examples, but the present invention is not limited to the following examples.

[0071] <Materials and Methods> <<Animals>> All procedures regarding the rearing and use of animals were carried out in accordance with the facility's guidelines, with the approval of the Keio University Animal Resources Committee. The following mouse strains were used: HA-Cbln1 knock-in (KI) mice, HA-Cbln2 knock-in (KI) mice, HA-Cbln4 knock-in (KI) mice, Cbln4 knockout (KO) mice, GluD1 knockout (KO) mice, 2×ALFA-DCC knock-in (KI) mice, Cbln4 conditional knockout (cKO) mice, conditional DCC knockout (cKO) mice (created in this study; described below), C57BL / 6N, ICR (Nippon SLC Co., Ltd.). Mice were maintained in a 12:12 light-dark cycle, and food and water were freely available. The sex of the embryos used for primary culture was not determined. Adult male mice (8-9 weeks old) were used for protein injection, immunohistochemistry, and behavioral studies. In some cases, adult female mice (8-9 weeks old) were also used for behavioral studies.

[0072] <<Cell Line>> HEK293 cells (tSA strain; donated by Dr. R. Horn of Thomas Jefferson University, Pennsylvania) were cultured in Dulbecco's modified Eagle medium (DME; D5796, Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Hyclone), 50 U / mL penicillin / streptomycin (15140-122, Thermo Fisher Scientific), and 2 mM L-glutamine. The cells were cultured in 10% CO2. ２ The temperature was maintained at 37°C in the incubator.

[0073] <<Culture of Isolated Dopaminergic Neurons>> Primary dopaminergic neurons were prepared from the ventral midbrain of ICR mouse embryos on day 13 (E13) for culture. Specifically, the ventral midbrain of ICR mice was excised using ice-cold phosphate-buffered saline (PBS) containing glucose, incubated at 37°C for 5 minutes, enzymatically digested with 0.25% trypsin-EDTA, and then gently ground using a plastic pipette tip. The resulting cells were centrifuged at 1,000 rpm for 2 minutes and pelletized. After removing the supernatant, the cells were resuspended in DMEM / F12 (11330-032, Gibco) supplemented with 10% FBS and divided into 5 × 10⁶ cells. ５ The cells were plated into a poly-D-lysine / laminin-coated 24-well plate to a cell-per-well ratio. The plated cell cultures were then placed in a humidified 5% CO2 solution. ２The culture medium was maintained at 37°C in an atmosphere. After 30 to 60 minutes, the medium was changed to a 10% hormone mix (100 μM Putrescin, 30 nM Sodium selenium, 200-fold dilution N3 supplement (4 μM Progesterone, 2 mg / mL Bovine insulin (Sigma, I-1882), 20 mg / mL Transferrin (Sigma, T-7786)), 1000-fold dilution Reverse T3 (3,3',5-Triiodo-L-thyronine), 2% B-27 supplement (Gibco), 0.5 mM The cultures were replaced with a Neurobasal / F12-based medium containing L-glutamine, 2 mM glucose, and penicillin-streptomycin. The cultures were then heated to 37°C and 5% CO2. ２ The samples were maintained in atmosphere and used in vitro (Days in vitro, DIV12) on day 12 for bead binding assays or artificial synapse assays.

[0074] <Details of the method> <<HA-tagged Cbln knock-in mice>> HA-Cbln1, HA-Cbln2, and HA-Cbln4 knock-in (KI) mice were prepared by electroporation of the fused body with some modifications to the previously reported method. At that time, the B6D2F1 zygotes received in vitro were cultured at 37°C for 15 minutes with SpCas9 protein (250 ng / ml; PNA Bio), gene-specific single-stranded guide RNA (sgRNA; 200 ng / ml; synthesized using MEGAshortscript T7 Translation Kit (Thermo Fisher Scientific)), and a 132-base single-stranded oligodeoxynucleotide (ssODN; 500 ng / ml; Integrated DNA Technologies) encoding the HA epitope tag flanked by homology arms. The sequences of sgRNA and ssODN are as follows. (However, sgRNA is described in DNA type.)

[0075] For creating HA-Cbln1 knock-in mice For creating HA-Cbln2 knock-in mice For creating HA-Cbln4 knock-in mice

[0076] For Cbln1, Cbln2, and Cbln4, the HA tag was inserted directly below the first amino acid. Electroporation was performed using a platinum block electrode (GE-101, BEX) connected to a gene transfer device CUY21EDIT II (BEX) at 150 mA; 1 ms on / 100 ms off; 2 pulses. After culturing the fertilized eggs to the 2-cell stage, they were transplanted into the fallopian tubes of pseudo-pregnant ICR females. The results of genome editing were verified by PCR and direct sequencing analysis of the target site. Sequencing analysis was performed using the CRISP-ID method.

[0077] <<2×ALFA-tagged DCC knock-in mouse>> To create a DCC knock-in (KI) mouse with a tandem ALFA epitope tag (2×ALFA tag), genome editing was performed using CRISPR / Cas9. A single-stranded guide RNA (sgRNA) targeting the DCC locus was designed, and the tag was introduced directly below the signal peptide region. The sequences of the sgRNA and the single-stranded oligodeoxynucleotide (ssODN) flanked by homology arms are as follows. (However, the sgRNA is described in DNA type.)

[0078]

[0079] Immediately following the signal peptide, and following the first amino acid of the mature DCC protein, a 2×ALFA tag configuration consisting of proline-ALFA-proline-linker sequence-proline-ALFA-proline was inserted into the DCC gene. Correct genome editing was confirmed by PCR and sequencing analysis, similar to the methods described above.

[0080] <<DCC Conditional Knockout Mice>> DCC conditional knockout (cKO) mice were created by Dr. Yoshihiko Kawasaki of the National Institute of Genetics (NIG). A targeting construct was designed to introduce the loxP region flanking the latter half of exon 1 of the mouse DCC gene (chromosome 18). Three alleles resulting in loss-of-function mutations were established: - A flexed allele that retains the neomycin resistance cassette (DCC flex (Neo)) - A flexed allele that lacks the neomycin resistance cassette (DCC flex) - A deletion allele resulting from Cre-mediated excision of the flexed region (DCC Δ)

[0081] Frozen embryos of the DCC flux (Neo) strain are preserved at RIKEN (The Institute of Physical and Chemical Research), and currently three alleles are maintained: DCC flux (Neo), DCC flux, and DCCΔ. Furthermore, the conventional DCC deletion allele (DCC), first reported by Tessier-Lavigne et al. − These are also preserved as frozen embryos.

[0082] Genotyping of these mice was performed by PCR using the following primers:

[0083] <<Cbln4 Conditional Knockout Mice and Cbln4 Knockout (KO) Mice>> Cbln4 conditional knockout (cKO) mice were created by Dr. Masahiko Sakimura of Niigata University. A targeting vector was constructed using a 0.64 kb genomic fragment containing exon 3 of the Cbln4 gene and inserted into the KpnI / SacI site of the intermediate entry clone (pDME-1). A phosphorylated kinase (pgk) promoter-driven neomycin resistance cassette was sandwiched between the FRT site and the loxP site and positioned 300 bp upstream of exon 3. Furthermore, a second loxP site was inserted 143 bp downstream of the stop codon. For homologous recombination, upstream 3.74 kb and downstream 5.09 kb genome fragments were recovered from the BAC clone and subcloned into a 5' entry clone (pD5UE-2).

[0084] For homologous recombination, 3.74 kb upstream genomic fragments and 5.09 kb downstream genomic fragments were recovered from BAC clones and subcloned into 5' entry clone (pD5UE-2) and 3' entry clone (pD3DE-2), respectively. The targeting vector was assembled into the destination vector pDEST-DT containing the CAG promoter-driven diphtheria toxin gene using the MultiSite Gateway Three-Fragment Vector Construction Kit (Invitrogen). ES cell culture, chimera generation, and screening were performed according to previously reported methods. Correct targeting was confirmed by Southern blot analysis (Cbln4-flox mice). Cbln4 knockout (KO) mice were generated by crossing Cbln4-flox mice with telencephalon-Cre transgenic mice.

[0085] <<GluD1 knockout (KO) mice>> GluD1 knockout mice were generated using a classical embryonic stem cell-based homologous recombination strategy developed prior to CRISPR / Cas9 genome editing technology. In this strain, a pgk promoter-driven neomycin resistance cassette was inserted into exon 11 of the Grid1 (GluD1) gene, resulting in inhibition of GluD1 expression. The neomycin cassette was introduced to enable positive selection of correctly targeted ES cell clones and remains in the mutant allele as part of the original gene targeting design.

[0086] <<cDNA construct>> To generate recombinant Cbln4 protein, cDNA encoding mouse Cbln4 was fused to a GSGS linker and a C-terminal 6×His tag and cloned into the pCAGG vector (gift from Dr. Junichi Miyazaki of Osaka University). Three-point mutations were introduced by overlap extension PCR to generate a mutant of Cbln, Cbln4, which does not bind to GluD1. ΔＧｌｕＤ１(Y127A, S129A, D152A) were created. In addition, by substituting the seven amino acid residues (103-FFTLESV-108) between β-sheet A' and B' of Cbln4 with the corresponding eight amino acid residues (NFDSERST) of Cbln1, a Cbln4 mutant that does not bind to DCC, Cbln4 ΔＤＣＣ We constructed it.

[0087] A construct was created to express the amino-terminal domain (ATD) of mouse GluD1 on HEK293 cells for an in vitro synapse formation assay. Myc-tagged GluD1-ATD was cloned into a pDisplay vector (Thermo Fisher Scientific). The ATD sequence of mouse GluD1 used in construct preparation is shown in SEQ ID NO: 7. Furthermore, four point mutations (D21A, I23A, E58A, R341A) were introduced into the nucleic acid of the cloned myc-tagged GluD1-ATD by overlap extension PCR, resulting in GluD1-ATD that does not bind to the Cbln protein. ΔＣｂｌｎ We constructed it.

[0088] <<Production of Adeno-Associated Virus (AAV)>> AAV293 cells (240073, Agilent Technologies) were transfected with pAAV (Addgene), pAAV-DJ Rep / Cap (VPK-420-DJ, Cell Biolabs, Inc.), and pHelper plasmid using the calcium phosphate method. Transfection was performed in a 5% CO2 atmosphere. ２ The transfection was carried out for 16 hours in IMDM (12440053, Gibco) medium containing 5% FBS and penicillin / streptomycin. After transfection, the medium was replaced with DMEM containing 10% FBS, non-essential amino acids (11140050, Gibco), 2 mM I-glutamic acid, and penicillin / streptomycin, and then 10% CO2. ２ The cells were cultured for two days under the specified conditions. Afterward, the cells were harvested, and AAV particles were purified using an AAV purification kit (6666, Takara Bio Inc.) according to the manufacturer's instructions. The titer of the purified AAV was 1-3 × 10⁻⁶. １２ The concentration was GC / μL.

[0089] <<Production and Purification of Recombinant Proteins>> HEK293 cells were purified using the calcium phosphate method to produce pCAGGS-Cbln4 ＷＴ -His, pCAGGS-Cbln4 ΔＤＣＣ -His, or pCAGGS-Cbln4 ΔＧｌｕＤ１ -His was transfected. 5% CO ２ After 6–12 hours, the culture medium was changed to CD293 medium (Thermo Fisher Scientific) containing 4 mM-glutamine. Five days after transfection, the medium was collected, centrifuged at 2,000 rpm for 5 minutes, and the supernatant was filtered through a 0.22 μm filter. The His-tagged Cbln4 protein was purified using TALON Metal Affinity Resin (Takara Bio) according to the manufacturer's instructions, eluted with 200 mM imidazole, filtered, and concentrated through an Amicon Ultra-0.5 10K centrifuge filter (Millipore). The protein concentration was measured by absorbance at 280 nm.

[0090] <<Stereotactic Injection>> AAV-EF1α-Cre-mCherry or AAV-EF1α-mCherry was stereotactically injected into the ventral midbrain of DCC-flux or Cbln4-flux mice. 500 nL was administered to each site. The relative coordinates to the lambdoid suture were AP -1.0 mm, ML ±0.50 mm, and DV -4.2 mm.

[0091] For intrastriatal protein delivery, wild-type mice (8 weeks old) were given a stereotactic injection into the lateral dorsal striatum two days before fixation. Recombinant Cbln4 ＷＴ , Cbln4 ΔＤＣＣ , and Cbln4 ΔＧｌｕＤ１ The protein (2.5 mg / ml, 2 μL) was injected at coordinates AP-1.0 mm, ML-2.2 mm, and DV-3.6 mm (from the brain surface) from the bregma.

[0092] <<6-OHDA-induced Parkinson's disease (PD) model>> To selectively degenerate and remove dopaminergic neurons while protecting noradrenergic terminals, desipramine hydrochloride (25 mg / kg, i.p.) was administered 30 minutes prior to infusion of 6-hydroxydopamine (6-OHDA) or the solvent alone (control). Eight-week-old wild-type mice were anesthetized by intraperitoneal administration of a mixed anesthetic (MMB) containing medetomidine (0.3 mg / kg), midazolam (4 mg / kg), and butorphanol (5 mg / kg). Using a glass capillary tube, 6-OHDA (2 μg / μL dissolved in sterile saline with 0.02% ascorbic acid) or a solvent (sterile saline with 0.02% ascorbic acid) was injected into the right striatum of the brain at stereotactic coordinates of AP-1.0 mm, ML+2.2 mm from the lambdoid suture, and DV-2.4 mm from the brain surface. Two days after injection, 0.5 mg / kg of apomorphine was administered subcutaneously, and apomorphine-induced rotational movement was recorded for 50 minutes. Mice that rotated clockwise at a constant rotation speed were selected.

[0093] For rescue experiments, wild-type Cbln4 (Cbln4 ＷＴ ) Protein (2.5 mg / mL) 2 μL was injected into the striatum at AP-1.10 mm, ML+1.75 mm, and DV-3.5 mm through the lambda suture. In addition, to investigate the function of Cbln4 in vivo, Cbln4 ＷＴ Protein, Cbln4 ΔＤＣＣ Mutant proteins and Cbln4 ΔＧｌｕＤ１ The mutant protein (2 μL, 2.5 mg / mL) was injected at the same coordinates.

[0094] <> The TH Reporter Human iPS Cell line WD39 was established by Dr. Hideyuki Okano (Keio University) and provided by Dr. Masato Koike (Juntendo University). This TH Reporter iPSC line was differentiated into dopamine neurons using an established protocol for direct conversion to neurospheres, in accordance with previously reported methods.

[0095] <<Immunohistochemistry>> Mice were deeply anesthetized with 2.5% aveltin (tribromoethanol in 2.5% tert-amyl alcohol; 0.02 mL / g body weight, i.p.) and transcardiac perfused with 4% paraformaldehyde (PFA) in phosphate buffer. Brains were collected and fixed overnight at 4°C in 4% PFA fixative. Coronal fragments (50 μm) were prepared using a microslicer (DTK-1000N). These fragments were blocked at room temperature (RT) for 30 minutes (PBS, 10% normal donkey serum, 0.1% Triton X-100), and then incubated overnight at RT with primary antibodies diluted in buffer (PBS, 0.1% Triton X-100).Primary antibodies: Anti-Tyrosine Hydroxyrase (TH) antibody (1:1,000, mouse IgG1, 16570-10, Calbiochem), anti-TH antibody (1:1,000, sheep, AB1542, CHEMICON), anti-HA tag antibody (1:500, rabbit, 3724S, Cell Signaling), anti-HA antibody (1:500, rat, 11867423001, Roche), anti-His tag antibody (1:500, mouse IgG2a, D291-3, MBL), anti-His antibody (1:500, rabbit, 12698S, Cell Signaling Inc.), anti-GluD1 antibody (1:250, guinea pig, MSFR102510, Frontier Institute), anti-vesicular monoamine transporter 2 (VMAT2) antibody (1:250, rabbit, MSFR106410, Frontier Institute), anti-vesicular glutamate transporter 2 (VGluT2) antibody (1:400, goat, MSFR106270, Frontier Institute), anti-vesicular glutamate transporter 3 (VGluT3) antibody (1:250, goat, MSFR106340) Frontier Institute, anti-DCC antibody (1:500, mouse, ab16793, Abcam), anti-DCC antibody (10% normal donkey serum, 0.1% Triton X-100 in PBS), anti-Dopamine Transporter (DAT) antibody (1:250, rat, MAB369, Millipore), and anti-ALFA tag antibody (1:500, mouse IgG1 Fc fusion single-domain antibody), anti-Rab3-interacting molecule 1 and 2 (RIM1 / 2) antibody (1:250, rat, 140217, Synaptic Systems).

[0096] After three PBS washes, sections were incubated with secondary antibody (1:500 in blocking buffer) in RT for 2 hours. For confocal imaging, DyLight 405-labeled secondary antibody, Alexa Fluor 488 (Plus)-labeled secondary antibody, Alexa Fluor 555 (Plus)-labeled secondary antibody, Alexa Fluor 647 (Plus)-labeled secondary antibody, and Cy3-labeled secondary antibody were used. NeuroTrace® 435 / 455 blue fluorescent Nissl stain (Molecular Probes) was used in conjunction with the secondary antibody to label the cell bodies of nerve cells.

[0097] <<In vitro synapse formation assay>> For HEK293-induced presynaptic differentiation, DIV12 primary dopaminergic neurons are subjected to GluD1-ATD or GluD1-ATD in the presence of a mock or recombinant Cbln4-His (50 nM, calculated as a hexamer). ΔＣｂｌｎ 5 × 10 ４ HEK293 cells were co-cultured for 24 hours.

[0098] To induce bead-induced synapse formation, DIV12 neurons were incubated for 24 hours with Cbln4-His bound to streptavidin-coated Dynabeads M-280 (Thermo Fisher Scientific) via a biotinylated anti-His monoclonal antibody (MBL).

[0099] After incubation, cells were fixed in PBS containing 4% PFA at room temperature (RT) for 10 minutes, washed three times with PBS (5 minutes each), and then permeabilized / blocked for 30 minutes with PBS containing 10% normal donkey serum and 0.1% Triton X-100 (RT). Cells were incubated with each primary antibody (2 hours, RT), then washed, and then incubated with fluorophore-conjugated secondary antibodies (30 minutes, RT). For observation, coverslips were mounted using Fluoromount-G.

[0100] <<Image Analysis>> Images of the ventral tegmental area (VTA) and substantia nigra compacta (SNc) were acquired using a Ti-E inverted microscope (Nikon) equipped with a DS-Qi2 CMOS camera (Nikon) and 4× (NA 0.13) and 20× (NA 0.75) objective lenses. Images of the nucleus accumbens (NAc) and dorsal striatum (DS) were acquired using an SD-OSR system (Olympus) equipped with a CSU-W1 scan unit (Yokogawa Electric), an ORCA-Flash 4.0 CMOS camera (Hamamatsu Photonics), and a 100× objective lens (NA 1.45).

[0101] <<Confocal Image Analysis>> The analysis was performed using a custom script in Fiji:ImageJ. After subtracting the background using the Rolling Ball algorithm (radius = 30 pixels), Otsu thresholding was performed to segment the positive pixels of each channel. Bead-related regions, GluD1-positive regions, or TH-positive regions were identified, and pixels positive for the corresponding signals within these regions were extracted. For quantitative comparison, the average fluorescence intensity of the extracted pixels was calculated.

[0102] For intracellular co-localization, fluorescence images were binarized using a U-Net-based segmentation model and analyzed in three dimensions using the Fiji DiAna plugin. U-Net was trained with over 10,000 iterations on approximately 1,600 neuron images to detect intracellular fluorescence. Binary masks were generated for NeuroTrace (cell body marker) and region of interest (ROI) channels (e.g., HA and TH). Cell bodies positive for both NeuroTrace and ROI channels were identified by comparison with the corresponding target channels, and the number of co-localized cells was quantified based on three-dimensional reconstruction in DiAna.

[0103] <<Apomorphine-Induced Rotation Test>> Mice were accustomed to a transparent cylinder measuring 14 cm in height and 15 cm in diameter for 5 minutes, and spontaneous rotation during this time was recorded to evaluate baseline asymmetry. Apomorphine (0.5 mg / kg in 0.9% physiological saline, administered subcutaneously) was administered, and rotational movement was recorded for 30 minutes using an overhead camera starting 3 minutes after injection. Total rotation in both ipsilateral and contralateral directions was quantified, and net rotation was calculated.

[0104] <<Structural Analysis Using Cryo-Electron Microscopy>> GD51, an anti-GluD1 nanobody, and His-tagged GluD1 ATD were mixed with nickel-bonded beads. After washing the beads, the complex of GD51 and His-tagged GluD1 ATD was eluted with imidazole-containing buffer, and the complex was purified by size exclusion chromatography (SEC). After concentrating the purified complex, it was rapidly frozen in liquid ethane, and the complex was placed on a mesh (Au mesh R1.2 / 1.3, M2955A-1, QUANTIFOIL) to prepare a grid for observation using a cryo-electron microscope. The grid was photographed using a cryo-electron microscope, Keios G4 (Thermofisher), and approximately 10,000 images were obtained.

[0105] Regarding the acquired images, Cryosparc software (Structura Bio, Cryosparc ＴＭWe performed analysis using ( ) and created a volume map. First, we corrected and denoised each image. Next, we set the size of the particles to be picked from the images (60-120 Å in this case) and picked up particles of that size. We performed 2D classification (2D classification) on the images of the picked-up particles (millions of particles). Based on the results of the 2D classification, we selected images that captured the characteristics of the molecule whose structure we wanted to see, and based on these, we constructed a rough 3D initial structure from 2D images from different angles. We refined the obtained 3D structure and created a high-resolution volume map. Based on the obtained volume map, the protein structure was modeled using ModelAngelo software (GitHub-3dem / model-angelo: Automatic atomic model building program for cryo-EM maps). The obtained initial modeled structure was refined using coot software (Coot-Cryo-EM-v9.md) and phenix software (Cryo-EM structure solution with phenix) to match the reference volume map.

[0106] <Results> <<Cbln4 is predominantly expressed in the mesolimbic dopamine pathway>> To identify candidate synaptic organizers involved in the formation of the midbrain dopamine circuit, we analyzed a single-cell RNA sequencing atlas of the adult mouse brain. In doing so, we focused on dopamine clusters defined by Dopamine Transporter (DAT, human gene name: Slc6a3) and Tyrosine Hydroxyrase (TH). As a result, among the C1q family organizers, Cbln4 was abundant in DAT-positive clusters and showed minimal expression in adjacent glutamatergic and GABAergic populations, suggesting that it is closely related to the identity of dopaminergic neurons. Next, we investigated the in vivo expression of the Cbln family using HA-tagged knock-in (KI) mice against C1q family molecules.

[0107] In HA-Cbln1 KI mice, HA-Cbln1 labeling was prominent in ventral tegmental area (VTA)-rostral linea nucleus (RLi) neurons, but did not overlap with thoracic neurons (TH). On the other hand, in the lateral substantia nigra (SNL), HA-Cbln1 co-localized with TH-positive neurons (Figure 1A, B). This is consistent with its potential role in SNL-derived projections. In HA-Cbln2 KI mice, HA-Cbln2 immunoreactivity was not detected in the VTA or SNc (Figure 1C, D).

[0108] In contrast, in HA-Cbln4 KI mice, HA-Cbln4 was strongly expressed in TH-positive ventral tegmental area (VTA) neurons (particularly in the interfascicular (IF) and paranucleus nigra (PN) subregions), but its expression was remarkably low in substantia nigra pars compacta (SNc) / substantia nigra pars lateralis (SNL) (Figure 1E, F). These data suggest that Cbln4 is involved in synapse formation, maintenance, and differentiation of the mesolimbic dopamine pathway.

[0109] Consistent with this, pitted staining of HA-Cbln4 was abundantly observed along TH-positive fibers in both the core and shell of the nucleus accumbens (NAc) (Figure 1G, H). This suggests that Cbln4 is located on dopaminergic axons within the primary target region of the VTA.

[0110] Figures 1A-D show the expression and localization of Cbln1 and Cbln2 in the midbrain. Figure 1A shows the staining of HA-Cbln1 (green) and TH (red) in HA-Cbln1 KI mice. Scale bar: 1 mm. Figure 1B shows high-magnification images of the frontal linear nucleus (RLi), substantia nigra compacta (SNc), and lateral substantia nigra (SNL) regions of Figure 1A. Scale bar: 10 μm. Figure 1C shows the staining of HA-Cbln2 (green) and TH (red) in HA-Cbln2 KI mice. Scale bar: 1 mm. Figure 1D shows high-magnification images of the interfascicular fossa (IF), parabrainstem pigment zone (PBP), substantia nigra compacta (SNc), and lateral substantia nigra (SNL) regions of Figure 1C. Scale bar: 10 μm.

[0111] Figures 1E-1H show the expression and localization of HA-Cbln4 in dopaminergic neurons of the midbrain tegmental area and in the striatal projections of these neurons in HA-Cbln4 KI mice. Figure 1E shows the immunoreactivity of HA-Cbln4 (green) and TH (red) in HA-Cbln4 KI mice. Scale bar: 1 mm. Figure 1F shows high-magnification images of the interfascicular fields (IF) and parabrainstem pigment bands (PBP) of the ventral tegmental area (VTA) and the substantia nigra pars compacta (SNc) portion of Figure 1E. RLi: anterior linear nucleus; SNL: lateral substantia nigra. Scale bar: 10 μm. Figure 1G shows the expression of HA-Cbln4 (green) in the dorsal striatum (caudate nucleus-putamen) and nucleus accumbens (NAc) together with tyrosine hydroxylase (TH; red). The NAc core and shell are shown by dotted lines. Scale bar, 1 mm. Figure 1H shows the expression of HA-Cbln4 in the nucleus accumbens (NAc). HA-Cbln4 (green) colocalizes on TH-positive axons (red) in both the NAc core (left) and NAc shell (right). Scale bar, 5 μm.

[0112] <<Cbln4 regulates the morphology of dopaminergic axons and presynaptic formation in the nucleus accumbens (NAc)>> Cbln proteins are known to act extracellularly and regulate synaptic connections. Therefore, the inventors first investigated the relationship between HA-Cbln4 and presynaptic components in the nucleus accumbens (NAc). The staining signal of HA-Cbln4 was closely adjacent to the staining signal of vesicular monoamine transporter 2 (VMAT2), a vesicular monoamine transporter that takes up dopamine, along TH-positive axons (Figure 2A). On the other hand, the staining signal of HA-Cbln4 showed weak overlap with the staining signals of the vesicular glutamate transporters Vesicular glutamate transporter 2 (VGluT2) and Vesicular glutamate transporter 3 (VGluT3) (Figure 2B, C). Particularly noteworthy was the alignment of the staining signal of HA-Cbln4 with that of Rab3-interacting molecules 1,2 (RIM1 / 2), which are active region proteins essential for dopamine release from dopaminergic nerve axons. This is consistent with Cbln4's involvement in organizing dopamine release sites (Figure 2D).

[0113] Next, we evaluated the morphology of dopaminergic axons in Cbln4 knockout (KO) mice. The density of TH-positive axons in the nucleus accumbens (NAc) was reduced, and the axonal splenium was also reduced and smaller (Figure 2E). Similarly, the expression of presynaptic markers, including VMAT2 and RIM, was also reduced in the nucleus accumbens (NAc) of Cbln4 KO mice (Figure 2F-H). These data suggest that Cbln4 is essential for the normal structure of dopaminergic axons and the organization of the presynaptic region in the nucleus accumbens (NAc).

[0114] Figures 2A-H show that Cbln4 is involved in the morphology of dopaminergic axons and the organization of the presynaptic region in the nucleus accumbens (NAc). Figure 2A shows the distribution of HA-Cbln4 and VMAT2 along TH-positive axons in the NAc. Figure 2B shows the distribution of HA-Cbln4 and VGluT2 along TH-positive axons in the NAc. Figure 2C shows the distribution of HA-Cbln4 and VGluT3 along TH-positive axons in the NAc. Figure 2D shows the distribution of HA-Cbln4 and RIM1 / 2 along TH-positive axons in the NAc. Scale bars in Figures 2A-D: 5 μm. Figure 2E shows stained images of TH-positive axon morphology in wild-type (WT) mice and Cbln4 KO mice. For both WT mice and Cbln4 KO mice, enlarged views of the regions enclosed by A-C in the leftmost figure (left figure) are shown to the right. Scale bars: 10 μm (left figure) and 1 μm (enlarged views A-C). Figure 2F shows the distribution of VMAT2 along TH-positive axons in WT mice and Cbln4 KO mice. Figure 2G shows the distribution of VGluT2 along TH-positive axons in WT mice and Cbln4 KO mice. Figure 2H shows the distribution of VGluT3 along TH-positive axons in WT mice and Cbln4 KO mice. Figure 2I shows the distribution of RIM1 / 2 along TH-positive axons in WT mice and Cbln4 KO mice. Scale bars for Figures 2F-I: 5 μm.

[0115] <<GluD1 functions as a postsynaptic receptor for Cbln4 in dopaminergic synapses>> Cbln proteins can bind to the amino-terminal domain (ATD) of GluD family receptors, and GluD1 is known to be highly expressed in striatal neurons. Therefore, the inventors investigated whether GluD1 functions as a postsynaptic receptor for Cbln4 in the nucleus accumbens (NAc). As a result, the distribution of GluD1 was prominent in both the nucleus accumbens (NAc) and the dorsal striatum (Figure 3A). Furthermore, in high-resolution observation, the staining signal of HA-Cbln4 was frequently arranged in parallel with the staining signal of GluD1 along TH-positive axons in the shell region of the nucleus accumbens (NAc) (Figure 3B). This indicates that Cbln4 and GluD1 are codistributed in dopaminergic contact sites.

[0116] Next, to investigate whether synapse formation in dopaminergic neurons depends on Cbln4, Cbln4-flux mice were injected with AAV-EF1α-Cre-mCherry to conditionally deficiency (cKO) Cbln4 in ventral tegmental area (VTA) neurons. This procedure resulted in a decrease in immunoreactivity to GluD1 in the nucleus accumbens (NAc) (Figure 3C). This suggests that Cbln4 originating from the ventral tegmental area (VTA) contributes to the maintenance of GluD1-related structures at dopaminergic contact sites.

[0117] On the other hand, in GluD1 knockout (GluD1 KO) mice, TH-positive axons and VMAT2 signaling in the nucleus accumbens (NAc) were reduced (Figure 3D). This result supports the idea that GluD1 is required for dopamine innervation and / or presynaptic formation.

[0118] For further verification, synaptic assays were performed (Figure 3E). Specifically, GluD1-ATD, or the Cbln-binding deficient mutant GluD1-ATD ΔＣｂｌｎHEK293 cells expressing GluD1-ATD were co-cultured with primary dopaminergic mice midbrain neurons (Figure 3E). When Cbln4 was added to the culture medium, local accumulation of VMAT2-positive vesicles was induced in DAT-positive axons at the contact site between HEK293 cells expressing wild-type GluD1-ATD and primary dopaminergic neurons. On the other hand, GluD1-ATD, a mutant that cannot bind to Cbln4, ΔＣｂｌｎ Local accumulation of VMAT2-positive vesicles within DAT-positive axons was not induced at the contact sites between HEK293 cells expressing GluD1 and primary cultured dopaminergic neurons (Figure 3E). Similar contact dependence between Cbln4 and GluD1 in synapse formation was reproduced in experiments using human iPS cell-derived primary cultured dopaminergic neurons (Figure 3F). These results support the role of GluD1 as a postsynaptic receptor that enables the formation of characteristic structures of the Cbln4-dependent presynaptic dopaminergic region.

[0119] Figures 3A-3F show that GluD1 functions as a postsynaptic receptor for Cbln4 in dopaminergic synapses. Figures 3A and 3B show the distribution of HA-Cbln4 and GluD1 in the striatum. Figure 3A shows that HA-Cbln4 and GluD1 are distributed together in the dorsal striatum and nucleus accumbens (NAc). Scale bar: 1 mm. Figure 3B shows a high-magnification image showing the colocalization of HA-Cbln4 and GluD1 along TH-positive dopaminergic axons within the NAc shell. Scale bar: 5 μm. Figure 3C shows a reduced distribution of GluD1 in the nucleus accumbens (NAc) of Cbln4 conditional knockout (Cbln4 cKO) mice. AAV-EF1α-mCherry (control) or AAV-EF1α-Cre-mCherry (for GluD1 cKO) was injected into the midbrain tegmentum (VTA) of Cbln4-flox mice. Scale bar: 5 μm. Figure 3D shows the attenuation of dopaminergic neural pathways in GluD1 knockout (GluD1 KO) mice. Immunoreactivity of TH and VMAT2 is reduced in the ventral tegmental area (NAc) of GluD1 KO mice. Scale bar: 5 μm. Figure 3E is a schematic diagram and staining image of an artificial synapse assay demonstrating that Cbln4 can induce dopaminergic synapses from primary cultured dopaminergic mice in a GluD1-dependent manner in vitro. Scale bar: 5 μm. Upon addition of Cbln4, dopaminergic vesicles (VMAT2) accumulated along DAT-positive axons in contact with wild-type GluD1-expressing cells. On the other hand, mutant GluD1 (GluD1-ATD) that could not bind to Cbln4 accumulated. ΔＣｂｌｎ No accumulation of VMAT2 was observed in cells expressing ) . Figure 3F is a schematic diagram and staining image of a synaptic assay showing that Cbln4 can induce dopaminergic synapses from human iPS cell-derived dopaminergic neurons in a GluD1-dependent manner in vitro. Cbln4 accumulated dopamine vesicles (VMAT2) along TH-positive axons in contact with HEK293 cells expressing wild-type GluD1, but mutant GluD1 (GluD1-ATD) that could not bind to Cbln4 was not observed. ΔＣｂｌｎIn cells expressing HEK293, no accumulation of dopamine vesicles (VMAT2) was observed along TH-positive axons in contact with HEK293 cells. Scale bar: 5 μm.

[0120] <<DCC functions as a presynaptic receptor for Cbln4 in dopaminergic synapses>> Most Cbln proteins bridge and form synapses by binding to neurorexin present in the presynaptic terminal and GluD receptors present in the postsynaptic terminal, but Cbln4 is known to have weak binding to neurorexin. On the other hand, Cbln4 was known to interact with DCC, an induction / adhesion receptor present in the presynaptic terminal. Therefore, considering the information that DCC functions in the development of dopaminergic neurons, the inventors investigated whether DCC functions as a presynaptic receptor for Cbln4.

[0121] In the ventral tegmental area (VTA), both DCC and HA-Cbln4 expression were observed in TH-positive neurons in HA-Cbln4 knock-in (HA-Cbln4 KI) mice (Figure 4A). In the nucleus accumbens (NAc), punctate staining of HA-Cbln4 and DCC was located near the GluD1 signaling pathway (Figure 4B). Conditional knockout of Cbln4 in the ventral tegmental area (VTA) (Cbln4 cKO) reduced the immunoreactivity of DCC and GluD1 in the NAc (Figure 4C). Furthermore, conditional knockout of DCC in ventral tegmental area (VTA) neurons (DCC cKO) reduced the density of TH-positive axons and the number of splenic areas in the nucleus accumbens (NAc) (Figure 4D). The results in these DCC cKO mice were very similar to the major structural defects observed in Cbln4 cKO mice.

[0122] Furthermore, in primary cultured dopaminergic cells, beads bound to Cbln4 induced local clustering of endogenous DCCs along DAT-positive axons, while control beads without Cbln4 binding did not (Figure 4E, F). These findings strongly suggest that DCCs are presynaptic receptors involved in Cbln4-dependent organization of dopaminergic axons and presynaptic structure.

[0123] Figures 4A-4F illustrate that DCC functions as a presynaptic receptor for Cbln4 in dopaminergic synapses. Figure 4A shows DCC expression in the ventral tegmental area (VTA) near the striatum. HA-Cbln4 and DCC are codistributed in TH-positive neurons. Scale bar: 10 μm. Figure 4B shows DCC expression in the nucleus accumbens (NAc). HA-Cbln4 signaling and DCC-positive signaling are adjacent to GluD1 signaling. Scale bar: 5 μm. Figure 4C shows reduced DCC signaling in the nucleus accumbens (NAc) of Cbln4 conditionally knockout (Cbln4 cKO) mice. DCC, GluD1, and TH signaling are reduced in Cbln4 cKO mice. Scale bar: 5 μm. Figure 4D shows a reduction in dopaminergic axons in the nucleus accumbens (NAc) of DCC conditionally knockout (DCC cKO) mice. TH-positive axons and the ampulla are reduced in DCC cKO mice. Scale bar: 5 μm. Figure 4E shows a schematic diagram of a clustering assay using Cbln4-binding beads. Figure 4F shows that Cbln4 recruits endogenous DCC and VMAT2 in vitro in dopaminergic neurons. Scale bar: 5 μm.

[0124] <<Cbln4 is essential for reward-related and social behavior in the ventral tegmental area (VTA)>> The ventral tegmental area (VTA)-nucleus accumbens (NAc) pathway is involved in reward evaluation, motivation, and social behavior. Therefore, to investigate the effect of Cbln4 on behavior in ventral tegmental area (VTA) neurons, AAV-EF1α-Cre-mCherry was injected into the ventral tegmental area (VTA) of Cbln4-flux mice (control: AAV-EF1α-mCherry) to conditionally eliminate Cbln4, and the mice were evaluated for social and reward-related tasks after 30 days (Figure 5A). Male Cbln4 conditionally knockout (Cbln4 cKO) mice showed reduced interaction time in contact tests with female mice (Figure 5B). In the social cognition test, male Cbln4 cKO mice did not show preference for new same-sex individuals (female mice were normal) (Figure 4C). In the sucrose preference test, sucrose preference was reduced in both sexes (Figure 5D). These data indicate that Cbln4 is necessary for normal reward processing and various aspects of social behavior in ventral tegmental area (VTA) neurons.

[0125] Figures 5A-D show that mice in which Cbln4 was specifically knocked out in the ventral tegmental area (VTA) exhibited impairments in reward-related and social behaviors. Figure 5A shows the experimental design to investigate the involvement of Cbln4 in social interaction, social cognition, and reward sensitivity. AAV-EF1α-mCherry (for the control group) or AAV-EF1α-Cré-mCherry (for Cbln4 cKO mice) were injected into the ventral tegmental area (VTA) of Cbln4-flux mice. Figure 5B shows the results of a contact test (social approach) between male Cbln4 cKO mice and wild-type female mice. Interaction time with the novel female mice was quantified (mean ± standard error; P < 0.05 vs. control group). Figure 5C shows the results of a social cognition test. Control mice increased their interaction with new same-sex individuals, but male Cbln4 cKO mice did not show a social preference for new same-sex individuals. On the other hand, female Cbln4 cKO mice showed no difference between known and new same-sex individuals (mean ± standard error; P < 0.05 vs. control group). Figure 5D shows the results of the sucrose preference (reward sensitivity) test. After acclimatization, the mice were given 1% sucrose water and water alternately in a bottle for 2 hours each day for 3 days. Both male and female Cbln4 cKO mice showed decreased sucrose preference compared to the control group (mean ± standard error; P < 0.05 vs. control group).

[0126] <<Introduction of Cbln4 into the striatum enhances substantia nigra-striatal dopamine function and reduces post-6-OHDA injury damage>> Cbln4 expression is low in SNc neurons (Figure 1E, F), but DCC is present in SNc, and GluD1 is expressed throughout the dorsal striatum (Figure 3A). This suggests that exogenous Cbln4 may activate this receptor pair in the adult substantia nigra-striatal circuit. Therefore, to verify this possibility, Cbln4 was injected into one dorsal striatum of adult mice, and saline was injected into the opposite dorsal striatum. After one day, Cbln4 increased TH-positive axons containing VMAT2-positive vesicles. On the other hand, Cbln4 binding-deficient mutants to GluD1 or DCC (Cbln4) ΔＧｌｕＤ１ and Cbln4 ΔＤＣＣ) did not increase TH-positive axons containing VMAT2-positive vesicles (Figure 6A). Two days after protein injection, apomorphine administration selectively induced significant rotational movement in mice injected with Cbln4. This suggests that Cbln4 injection enhances dopaminergic output (Figure 6B). On the other hand, Cbln4 binding-deficient mutants (Cbln4 ΔＧｌｕＤ１ and Cbln4 ΔＤＣＣ In mice injected with ), no induction of rotational movement by apomorphine administration was observed (Figure 6B).

[0127] To be sure, the binding activity of Cbln4 binding-deficient mutants to GluD1 and DCC was confirmed in vitro. Recombinant HA-tagged Cbln1 (control), Cbln4, Cbln4 ΔＧｌｕＤ１ (GluD1 binding deficiency), or Cbln4 ΔＤＣＣ (DCC binding deficiency) was added to HEK293 cells co-expressing the GluD1 amino-terminal domain (ATD) and GFP, and incubated. Then, the HA immunoreactivity on the GFP-positive (GluD1) region was quantified. As a result, Cbln4 ΔＧｌｕＤ１ It was confirmed that it does not indeed bind to the ATD of GluD1 (Figure 6C).

[0128] HA-tagged Cbln1 (control), Cbln4, Cbln4 ΔＧｌｕＤ１ (GluD1 binding deficiency), or Cbln4 ΔＤＣＣ (DCC-binding deficiency) cells were incubated with HEK293 cells expressing DCC fibronectin type III domains 4-6 (FN4-6) and GFP. HA immunoreactivity on the GFP-positive (DCC) region was then quantified. The result showed that Cbln4 ΔＤＣＣ It was confirmed that it does not indeed couple to DCC's FN4-6 (Figure 6D).

[0129] Next, we investigated whether Cbln4 delivery improved motor impairment in a hemi(unilateral)PD model induced by unilateral 6-OHDA injury. After selecting mice with comparable baseline apomorphine-induced rotation (day 2), Cbln4 or saline (control) was injected into the injured striatum (day 3). On day 6, Cbln4 reduced apomorphine-induced rotation (Figure 6E, F) and increased the survival rate of TH-positive neurons in the substantia nigra on day 7 (Figure 6G). These results suggest that activation of the GluD1-Cbln4-DCC module in the adult striatum may enhance dopaminergic terminal organization and restore dopaminergic neuronal function after substantia nigra-striatal tract injury.

[0130] Figures 6A-D show that Cbln4, in vivo, works in coordination with DCC and GluD1 to induce functional dopaminergic synapses in the striatum. Figure 6A shows that injection of Cbln4 into the putamen can induce dopaminergic synapses in a GluD1 and DCC-dependent manner. One day after injection of Cbln4 protein, wild-type Cbln4 increased TH (red) positive axons containing VMAT2 (gray) positive vesicles. ΔＤＣＣ and Cbln4Δ ＧｌｕＤ１ It was not possible to increase TH (red) positive axons containing VMAT2 (gray) positive vesicles. Scale bar: 10 μm. Figure 6B shows that Cbln4 enhances apomorphine-induced rotational motility. Cbln4, Cbln4 ΔＤＣＣ , or Cbln4 ΔＧｌｕＤ１ Two days after unilateral administration, apomorphine (0.5 mg / kg, subcutaneous administration) was administered, and rotational movement over 3 minutes was quantified. Cbln4 ΔＤＣＣ ya Cbln4 ΔＧｌｕＤ１ Rotational movement was not induced in the mutant Cbln4, but it was induced in the wild-type Cbln4, showing an increase in dopaminergic output. Figure 6C shows the binding activity of wild-type and mutant Cbln4 proteins to GluD1 ATD. Recombinant HA-tagged Cbln1 (control), Cbln4, Cbln4 ΔＧｌｕＤ１ (GluD1 binding deficiency), or Cbln4 ΔＤＣＣThe (DCC-binding-deficient) protein was incubated with HEK293 cells co-expressing the GluD1 amino-terminal domain (ATD) and GFP, and the HA immunoreactivity on the GFP-positive (GluD1) region was quantified. Figure 6D shows the binding activity of wild-type and mutant Cbln4 proteins to DCC FN4-6. Four HA-tagged proteins, the same as those in Figure 6C, were incubated with HEK293 cells expressing DCC fibronectin type III domains 4-6 (FN4-6) and GFP, and the HA immunoreactivity on the GFP-positive (DCC) region was quantified.

[0131] Figures 6E-6G show that Cbln4 suppresses rotational movement and dopaminergic neuron loss in a unilateral Parkinson's disease model. Figure 6E is a schematic diagram showing the method for creating a unilateral Parkinson's disease model. 6-OHDA (2 μg) was injected into the lateral dorsal striatum to induce a slowly progressive unilateral lesion. After selecting mice with comparable apomorphine-induced rotation (day 2), Cbln4 (5 μg) or saline was injected into the lesioned lateral striatum (day 3). Rotational movement was evaluated on day 6. On day 7, surviving dopaminergic neurons were analyzed immunohistochemically. Figure 6F shows the effect of Cbln4 protein administration on a unilateral Parkinson's disease model. Figure 6G shows the effect of Cbln4 administration on preventing the loss of dopaminergic neurons (TH-positive neurons) in the substantia nigra pars compacta (SNc) treated with 6-OHDA. Scale bar: 100 μm.

[0132] <<Designs of Two Types of Dopaminergic (DA) Synapse Connectors>> Figure 7 is a schematic diagram showing the designs of two types of DA artificial synapse connectors. In the antibody-based format, nanobodies (VHHs) specific to the extracellular domains of DCC and GluD1, which are expressed on the surface of the presynaptic (dopaminergic axon) and postsynaptic (striatal) parts of the DA synapse, are created, and by crosslinking the two VHHs, a bispecific VHH for DCC and GluD1 is created. This approach provides a DA synapse connector that is more active than wild-type Cbln4 and can be delivered to the brain, due to the high epitope specificity of VHHs and their relatively small molecular weight that allows them to cross the blood-brain barrier.

[0133] In a natural (protein-based) format, we develop DA synaptic connectors with superior activity compared to wild-type Cbln4 by manipulating Cbln4, an endogenous DCC / GluD1 binding protein, to improve its binding affinity to DCC and / or GluD, as well as its pharmacokinetics. This approach offers potential advantages such as safety, as the DA synaptic connector is derived from the endogenous protein Cbln4.

[0134] <<Implementation Example of Antibody-Based DA Synapse Connector>> Figure 8 is a schematic diagram showing an implementation example of an antibody-based DA synapse connector (antibody-type synapse connector). It was fabricated by conjugating isolated anti-DCC nanobodies (e.g., DA1 (SEQ ID NO: 3)) and anti-GluD1 nanobodies (e.g., GD51 (SEQ ID NO: 4)) using a linker of appropriate length. As a prototype, DA1-GD51-Fc (SEQ ID NO: 5) was fabricated by fusing a human IgG Fc domain to enhance binding affinity through the avidity effect and extend the serum half-life. Figure 9 shows the schematic structure and amino acid sequence of the bispecific antibody DA1-GD51-Fc.

[0135] <<Analysis of the binding epitopes of GD51 and GluD1>> Using a cryo-electron microscope (KriosG4, Thermofisher), we analyzed the structure of the complex formed by the binding of GD51, an anti-GluD1 nanobody, to the amino-terminal domain (ATD) of mouse GluD1. As a result, the epitopes of each molecule involved in the binding of GD51 and GluD1 ATD were revealed (Figure 10). Although the data is not shown, the epitope on GluD1 ATD to which GD1, another anti-GluD1 nanobody different from GD51, binds was completely different from that of GD51.

[0136] Figure 10 shows the three-dimensional structure of the anti-GluD1 nanobody, GD51, and the GluD1 ATD complex, as well as their respective binding epitopes.

[0137] <<Antibody-type synaptic connector, DA1-GD51-Fc, induces DA synapse formation in vitro>> HEK293 cells expressing GluD1 and primary cultured mouse dopaminergic neurons were co-cultured in DIV12 in the presence of 100 nM Cbln4 or DA1-GD51-Fc. Cbln4 is a positive control. Immunocytochemical staining revealed VMAT2 accumulation in DAT-positive axons at the interface with GluD1-expressing HEK293 cells in the presence of DA1-GD51-Fc, similar to the presence of Cbln4 (Figure 11).

[0138] Figure 11 shows that the antibody-type synaptic connector, DA1-GD51-Fc, induces DA synapse formation in vitro. Scale bar: 50 μm.

[0139] When using DA1-GD1-Fc, an antibody-type synaptic connector in which GD51 is replaced with GD1, DA synapse formation was induced in vitro, similar to DA1-GD51-Fc.

[0140] <<DA1-GD51-Fc induces DA synapse formation in vivo>> 5 μg of DA1-GD51-Fc was injected into one side of the striatum of 6-week-old male wild-type mice. As a control, physiological saline was injected into the opposite side of the striatum (Figure 12, upper left). Immunohistochemical staining of striatal sections performed one day after injection (Figure 12, upper right) showed an increase in TH-positive DA axons containing VMAT2-positive vesicles on the side injected with DA1-GD51-Fc compared to the side injected with physiological saline. In a similar experiment, two days after injecting DA1-GD51-Fc into the right side of the striatum, apomorphine (0.5 mg / kg, s.c.) was administered to mice to evaluate the enhancement of DA synapse function. As a result, mice administered DA1-GD51-Fc to the right striatum exhibited clockwise rotational behavior (Figure 12, bottom). Similarly, rotational behavior was observed in mice treated with DA1-GD1-Fc during apomorphine administration (data not shown).

[0141] Figure 12 shows that the antibody-type synaptic connector, DA1-GD51-Fc, induces DA synapse formation in vivo. Scale bar: 5 μm.

[0142] When using DA1-GD1-Fc, an antibody-type synaptic connector in which GD51 is replaced with GD1, DA synapse formation was induced in vivo, similar to DA1-GD51-Fc.

[0143] In other words, when using GD51 and GD1, which recognize completely different epitopes on GluD ATD, antibody-type synaptic connectors with DA synapse formation activity were fabricated both in vitro and in vivo. This suggests that any nanobody that binds to GluD ATD is sufficient as an anti-GluD nanobody to be used in the fabrication of antibody-type synaptic connectors.

[0144] <<High affinity synaptic connector, Cbln4 ｈｉｇｈ >> Figure 13 shows a Cbln4 mutant with high affinity for GluD1 (Cbln4 ｈｉｇｈ This figure shows the amino acid sequence (SEQ ID NO: 2) of Cbln4. Based on the cocrystal structure of Cbln1 and GluD2, which are C1q family molecules, the inventors have identified Cbln4 ｈｉｇｈ We designed the following. Specifically, it was known that Cbln1 has a higher affinity for GluD2 than Cbln4. Therefore, by substituting the YQSQT motif of the CD loop of Cbln4, which is involved in binding to the GluD molecule, with the Cbln1 type sequence YNRQT, we designed a high-affinity mutant, Cbln4. ｈｉｇｈ This mutation was created (Figure 13). This mutation was predicted to increase the binding affinity of Cbln4 to GluD1 by approximately 10 times.

[0145] <<Cbln4 ｈｉｇｈ HEK293 cells expressing GluD1 are mixed with 100 nM Cbln4 or a high-affinity Cbln4 mutant (Cbln4) for GluD1 in vitro. ｈｉｇｈ ) in the presence of, and Cbln4 is also Cbln4 ｈｉｇｈDIV12 was co-cultured in a state where neither Cbln4 nor Cbln4 existed (mock). Cbln4 was used as a positive control. Immunocytochemical staining showed that Cbln4 was present in a state where Cbln4 was not present. ｈｉｇｈ In the presence of GluD1, greater accumulation of VMAT2 was observed in DAT-positive axons at the interface with HEK293 cells expressing GluD1 (Figure 14).

[0146] Figure 14 shows the difference between Cbln4 and wild-type Cbln4. ｈｉｇｈ This staining image shows that it more induces dopamine synapse formation in vitro. Scale bar: 50 μm.

[0147] <<Cbln4 ｈｉｇｈ This induces DA synapse formation in vivo >> Next, 5 μg of Cbln4 ｈｉｇｈ The substance was injected into the right striatum of 6-week-old male wild-type mice. Physiological saline (saline) was used as a mock and injected into the opposite striatum (Figure 15, upper left). Immunohistochemical staining of striatal sections performed one day after injection (Figure 15, upper right) showed that Cbln4 was more abundant on the side where physiological saline was injected compared to the side where it was injected. ｈｉｇｈ An increase in tyrosine hydroxylase (TH)-positive DA axons containing VMAT2-positive vesicles was observed on the side where the drug was injected. The scale bar represents 5 μm. As shown in Figure 15 below, two days after administration, apomorphine (0.5 mg / kg, s.c.) was administered to evaluate the enhancement of DA synaptic function. The mice were Cbln4 ｈｉｇｈ The administration showed an increase in clockwise rotational behavior. This result was obtained with Cbln4 ｈｉｇｈ This was consistent with the upregulation of functional DA synapses via [the specified mechanism].

[0148] Figure 15 shows Cbln4 ｈｉｇｈ This figure shows the in vivo effect of DA synapse formation induction.

[0149] Although embodiments of the present invention have been described in detail above, the present invention is not limited to the specific embodiments described above, and various modifications and changes are possible within the scope of the gist of the present invention as described in the claims.

[0150] This international application claims priority under U.S. Patent Application No. 63 / 747,592, filed on 21 January 2025, which is incorporated herein by reference to the entire contents of U.S. Patent Application No. 63 / 747,592.

Claims

A synaptic connector that promotes the formation of dopamine synapses, wherein the synaptic connector is a molecule that has the ability to connect the DCC (Deleted in Colorectal Cancer) protein present on the surface of the dopamine presynaptic site with the GluD (Delta-type Glutamate receptor) protein present on the surface of the dopamine postsynaptic site. 　 The synaptic connector according to claim 1, wherein the GluD is GluD1. 　 The synaptic connector according to claim 1, wherein the synaptic connector is a protein, nucleic acid, or small molecule compound. 　 The synaptic connector according to claim 1, wherein the synaptic connector is a protein. 　 The synaptic connector according to claim 1, wherein the synaptic connector is celevelin 4 (Cbln4) or a functional variant thereof. 　 The synaptic connector according to claim 5, wherein the Cbln4 or its functional variant is human Cbln4 or its functional variant. 　 The synaptic connector according to claim 6, wherein Cbln4 or a functional variant thereof is a protein comprising the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO:

2. The synaptic connector according to claim 1, wherein the synaptic connector is a complex of a binding domain that binds to DCC and a binding domain that binds to GluD. 　 The synaptic connector according to claim 8, wherein the synaptic connector is a composite of a nanobody that binds to DCC containing the amino acid sequence represented by SEQ ID NO: 3 and a nanobody that binds to GluD containing the amino acid sequence represented by SEQ ID NO:

4. The synaptic connector according to claim 9, wherein the synaptic connector is a polypeptide comprising the amino acid sequence represented by Sequence ID No.

5. A nucleic acid having a base sequence encoding a synaptic connector according to any one of claims 4 to 10. 　 A dopamine synapse formation promoter comprising the synaptic connector according to any one of claims 1 to 10. 　 The dopamine synapse formation promoter according to claim 12, wherein the synaptic connector is Cbln4 or a functional variant thereof. 　 The dopamine synapse formation promoter according to claim 13, wherein the Cbln4 or a functional variant thereof is translated from a polynucleotide containing the encoded base sequence. 　 The dopamine synapse formation promoter according to claim 14, wherein the polynucleotide is mounted on an expression vector. 　 The dopamine synapse formation promoter according to claim 15, wherein the expression vector is a plasmid vector or a viral vector. 　 The dopamine synapse formation promoter according to claim 16, wherein the viral vector is one of an adeno-associated virus (AAV) vector, an adenovirus vector, and a lentiviral vector. 　 The dopamine synapse formation promoter according to claim 12, wherein the synaptic connector is a complex of a binding domain that binds to DCC and a binding domain that binds to GluD. 　 The dopamine synapse formation promoter according to claim 18, wherein the complex is translated from a polynucleotide comprising a base sequence encoding a binding domain that binds to DCC and a binding domain that binds to GluD. 　 The dopamine synapse formation promoter according to claim 19, wherein the polynucleotide is mounted on an expression vector. 　 The dopamine synapse formation promoter according to claim 20, wherein the expression vector is a plasmid vector or a viral vector. 　 The dopamine synapse formation promoter according to claim 21, wherein the viral vector is one of an adeno-associated virus (AAV) vector, an adenovirus vector, and a lentiviral vector. 　 A pharmaceutical composition comprising a dopamine synapse formation promoter according to any one of claims 12 to 22. 　 A pharmaceutical composition according to claim 23 for the treatment of Parkinson's disease. 　 A method for promoting the formation of dopamine synapses, comprising the step of administering to a synaptic connector according to any one of claims 1 to 10. 　 A method for treating Parkinson's disease, comprising the step of administering a therapeutically effective amount of the synaptic connector described in any one of claims 1 to 10. 　 A synaptic connector according to any one of claims 1 to 10, for use in promoting the formation of dopamine synapses. 　 A synaptic connector according to any one of claims 1 to 10, for use in the treatment of Parkinson's disease. 　 Use of a synaptic connector according to any one of claims 1 to 10 in the manufacture of a dopamine synapse formation promoter. 　 Use of the synaptic connector according to any one of claims 1 to 10 in the manufacture of a pharmaceutical composition for the treatment of Parkinson's disease. 　 A functional variant of CBLN4 containing the amino acid sequence represented by SEQ ID NO:

2. A nucleic acid encoding a functional variant of CBLN4 according to claim 31. 　 A complex comprising a binding domain that binds to DCC containing the amino acid sequence of SEQ ID NO: 3, and a GluD binding domain that binds to the ATD region of GluD1 as shown in SEQ ID NO:

6. The complex according to claim 33, comprising a binding domain that binds to DCC containing the amino acid sequence represented by SEQ ID NO: 3, and a binding domain that binds to GluD containing the amino acid sequence represented by SEQ ID NO:

4. A complex according to claim 33 or 34, comprising a binding domain that binds to DCC, a binding domain that binds to GluD, an immunoglobulin Fc, and a linker sequence. 　 The complex according to claim 35, comprising the amino acid sequence represented by Sequence ID No.

5. A nucleic acid encoding the complex according to any one of claims 33 to 36. A method for producing an artificial synaptic connector that promotes the formation of dopamine synapses, characterized by binding a molecule that binds to DCC protein with a molecule that binds to GluD protein. 　 A method for producing an artificial synaptic connector according to claim 38, wherein the molecule that binds to the DCC protein and the molecule that binds to the GluD protein each have a binding domain that binds to the DCC protein and a binding domain that binds to the GluD protein.

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