WO2015076589A1

WO2015076589A1 - Nerve-cell-regenerating or nerve-cell growth-promoting pharmaceutical composition containing vax protein as active ingredient

Info

Publication number: WO2015076589A1
Application number: PCT/KR2014/011208
Authority: WO
Inventors: 김진우; 김남석
Original assignee: 한국과학기술원
Priority date: 2013-11-20
Filing date: 2014-11-20
Publication date: 2015-05-28

Abstract

The present invention relates to a nerve-cell-regenerating pharmaceutical composition containing Vax protein as an active ingredient, and, more specifically, the Vax protein can advantageously be used as a nerve-cell-regenerating pharmaceutical composition as it has been confirmed that Vax1 is secreted from a ventral hypothalamus (vHT) section isolated from the mouse and that, after the secreted Vax1 has been bound to extracellular sugar groups of heparan sulphate proteoglycans (HSPGs) present in retinal ganglion cell (RGC) axons of a co-cultured retinal section such that the product penetrates as axoplasm (axonplasm), the synthesis of local protein is activated and the growth of retinal ganglion cells (RGCs) is promoted.

Description

【Specification】

[Name of invention]

Pharmaceutical composition for promoting neuronal regeneration or neuronal growth containing ^ Vax protein as an active ingredient _;

ί Technical Field]

The present invention relates to a pharmaceutical composition for promoting neuronal regeneration or neuronal growth containing Vax protein as an active ingredient, and a method for screening candidate substances for promoting neuronal regeneration or neuronal growth using Vax protein.

Background Art

The mammalian binocular visual system is a retinal ganglion cell in the neurons of the dorsal lateral geniculate nucleus (dLGN) of the brain and in the superior colliculus (SC) of the brain. , RGC) axons are formed in topographic synaptic connections. Visual paths including optic disc (0D), optic bottle (optic stalk, OS), optic chiasm (0C), and optic tract (0T) for retinal ganglion cell axons to contact synaptic targets By recognizing the nerve axon growth induction cues expressed in the optic pathway structure, they grow out of the retina and grow in a selective direction (Lemke and Reber, 2005; Petros et al., 2008). Retinal ganglion cell axon induction signals include cell surface ligands such as semaphorin present in the ocular disease and ephrinB2 present in the optic nerve crossing, or netrin -l in the optic nerve disc and There is a secreted factor such as Slitl in the periphery of the optic nerve crossing (Erskine and Herrera, 2007). However, only about 3% of retinal ganglion cell axons derived from the ventral and temporal regions of the mouse retina proceed to the ipsi lateral cerebral hemisphere, and most mouse retinal ganglion cell axons are ventral hypothalamus with optic nerve crossing. hypothalamus) across the midline to the contralateral brain hemisphere.

The ventral hypothalamic cells express molecules that determine the direction of retinal ganglion cell axons in the optic nerve crossing. For example, ventral hypothalamic glial cells express ephrin B2, which binds to EphBl receptors expressed in ventral temporal retinal ganglion cell axons, leading to retraction of axons directed to the ipsilateral visual field. (Nakagawa et al., 2000; Williams et al., 2003). In contrast, vascular endothelial growth factor 164 (VEGF164) and neuronal cell adhesion molecule (Nr CAM) expressed in the ventral hypothalamus are neurophilin UNeurophi Hn 1 and flexin A Plexin Al, respectively. ) To induce retinal ganglion cell axon growth across the ventral hypothalamus liner (Erskine et al., 2011; Kuwaj ima et al., 2012; Williams et al., 2006). Retinal ganglion cell axons must pass through the ventral-lateral diencephalic area where high levels of repulsive induction signals such as Slit and semaphorin are expressed to receive directional induction signals of these molecules in the optic nerve cross. (Erskine and Herrera, 2007). However, the identity of the ventral hepatic region molecules that overcome these regression induction signals and induce retinal ganglion cell axons to grow into the midline is not well known. VaxKventral anterior homeobox 1) includes medial and lateral geniculate eminence (MGE and LGE), abdominal septum, anterior entopeduncular area (AEP), and anterior optic nerve area (pre Dtic area, P0A). And homeodomain transcription factors expressed in various ventral-medial forebrain-derived structures, including ventral hypothalamus and ocular disease (Bertuzzi et al., 1999; Hall one t et al., 1998). Genetic inactivation of Vaxl in humans and rats is characterized by the lack of "coloboma of the eye, as well as multiple central structures of the brain, including anterior commissure, corpus cal losum, and optic nerve crossing. Causes ageesis (Bertuzzi et al., 1999; Hal l one t et al. ᅳ 1999; Slavot inek et al. , 2012). Retinal ganglion cell axons in Vaxl-deficient mice can grow through ocular disease but fail to contact the ventral hypothalamus and eventually do not form optic nerve crossings. Vaxl also plays an important role in retinal ganglion cell axon growth and fasci culat ion but is not expressed in retinal ganglion cells (Bertuzzi et al., 1999). Therefore, the present inventors tried to find a function related to the induction of Vax retinal ganglion cell axon growth and molecules that mediate it. As a result, Vaxl is secreted from ventral hypothalamus (vHT) fragments isolated from mice. The secreted Vaxl binds to the extracellular sugar group of heparan sul fate proteoglycans (HSPGs) present in the axon of retinal ganglion cells (RGC) of cocultured retinal sections. By infiltrating the axonplasm and activating local protein synthesis to promote retinal ganglion cell axon growth, thereby regenerating neurons or regenerating nerve cells, including the retinal ganglion cells. The present invention has been completed by revealing that it can be usefully used as a pharmaceutical composition for promotion.

[Detailed Description of the Invention]

[Technical Challenges]

An object of the present invention is to provide a pharmaceutical composition for promoting neuronal regeneration or neuronal growth containing Vax protein as an active ingredient.

Technical Solution

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for promoting neuronal regeneration or neuronal growth containing the Vax protein as an active ingredient.

In addition, the present invention is a nerve cell regeneration or nerve cell growth containing a vector or a cell containing a polynucleotide encoding Vax protein as an active ingredient Provided is a pharmaceutical composition for promotion.

In addition, the present invention

1) treating the cells obtained from the subject with the test substance and measuring neuronal axon growth;

2) measuring the binding level of Vaxl and hapharan sulfate proteoglycans in the cells of step 1);

3) It provides a method for screening a candidate substance for neuronal regeneration or neuronal growth promotion comprising the step of selecting a test substance to increase the Vaxl and heparan sulfate proteoglycan binding level of step 2) compared to the untreated control.

The present invention also provides a method for regenerating neurons comprising administering a pharmaceutically effective amount of Vax protein to a subject injured in a neuronal cell. In addition, the present invention provides a method for promoting neuronal growth comprising administering a pharmaceutically effective amount of Vax protein to a subject injured in a neuronal cell.

In addition, the present invention provides a method for regenerating neurons comprising administering a vector or cell comprising a polynucleotide encoding a pharmaceutically effective amount of Vax protein to a subject injured in a neuron.

The present invention also provides a method for promoting neuronal growth, comprising administering a vector or cell comprising a polynucleotide encoding a pharmaceutically effective amount of Vax protein to an individual injured with neurons.

The present invention also provides the use of Vax protein for use as a pharmaceutical composition for promoting neuronal regeneration or neuronal growth.

The present invention also provides the use of a vector or cell comprising a polynucleotide encoding a Vax protein for use as a pharmaceutical composition for promoting neuronal regeneration or neuronal growth.

Advantageous Effects The present invention relates to a pharmaceutical composition for nerve cell regeneration containing Vax protein as an active ingredient, Vaxl is secreted from ventral hypothalamus (vHT) fragments isolated from embryonic mice, and the secreted Vaxl is Penetrates into the axonplasm by binding to extracellular sugar groups of heparan sulfate proteoglycans (HSPGs) present in retinal ganglion cells (RGC) axons of cocultured retinal sections. After that, by activating local protein synthesis to promote retinal ganglion cell axon growth, the secreted Vax protein can be usefully used as an active ingredient of a composition for promoting neuronal regeneration or neuronal growth. _.

【Brief Description of Drawings】.

La shows neural progenitor cells (NPCs) of Vaxl normal mice (Vaxl + / +) and Vaxl deficient mice (Vaxl − / −) with embryonic 13.5 (E 13.5) days, post-mitotic neuronal ) Is a diagram showing the expression of Vaxl protein in neurons.

FIG. Lb shows the expression of Vaxl protein in optic chiasm (0C) forming cells of E13.5 Vaxl + / + and Vaxl − / − mice.

Figure 2a is a schematic diagram illustrating a method for measuring the orientation of retinal ganglion cells (RGC) axon (axon).

FIG. 2B shows retinal ganglion cell axons growing in neural retinal (NR) fragments co-cultured with ventral hypothalamic (vHT) isolated from E13.5 Vaxl + / + or Vaxl − / − mice It is also.

FIG. 2C shows the orientation of retinal ganglion cell axon tips toward the ventral hypothalamus in neural retinal sections co-cultured with ventral hypothalamus isolated from E13.5 Vaxl + / + or Vaxl − / − mice :

WT: Vaxl + / +; And

K0: Vaxl-/-.

2D shows the ventral hypothalamus isolated from E13.5 Vaxl + / + or Vaxl − / − mice Diagram showing the direction of the retinal ganglion cell axon shaft to the ventral hypothalamus in cocultured neuroretinal sections:

WT: Vaxl + / +; And

K0: Vaxl-/-.

FIG. 2E is a diagram illustrating defects in the ventral hypothalamus and retinal ganglion cell axons isolated from E13.5 Vaxl + / + or Vaxl − / − mice:

Vegfa: vescular endothelial growth factor, VEGF164;

ISH: In situ hybridization;

NrCAM: neuronal cell adhesion molecule; And

IF: immunof luorescence staining (scale bar: 100 μηι).

3A is a diagram showing retinal ganglion cell axons growing in neural retinal sections co-cultured with Vaxl, transcription inactive Vaxl mutants (Vaxl (R152S)) and C0S7 cells overexpressing Vax2.

3B is a diagram showing the direction of retinal ganglion cell axons directed to C0S7 cells in neural retinal sections co-cultured with Vaxl, transcriptionally inactive Vaxl mutants (Vaxl (R152S)) and Vax2 overexpressed C0S7 cells.

Figure 3c is a diagram showing the detection of Vax protein present in the growth medium of Vaxl, transcription inactive Vaxl mutation (Vaxl (R152S)) and C0S7 cells overexpressed Vax2. '

4A shows the location of Vax protein in C0S7 cells (right) treated with growth medium of HEK293T cells (left) overexpressing Vaxl, GFP-Vaxl and GFP-Vax2:

►: GFP immunostained intact C0S7 cells; And

→ ： GFP immunostained dead cell debris.

4B is a diagram showing Vaxl protein present in the third ventricle of E13 mouse brain injected with growth medium of Myc-Vaxl overexpressed HEK293T cells:

► ： Cells in which Myc-Vaxl exists.

4C shows E14.5 normal (WT) and Vaxl deficient (Vaxl-/-) ventral hypothalamic sections In cell lysates (cel l lysate, CD and growth media (growth media eu GM) for a diagram showing the existence of Vaxl protein. ^"

4D shows the presence of Vaxl protein in cell lysates (CL) of ventral hypothalamic explants, and supernatants (S3) and dead cell debris (P2) of cerebrospinal fluid (cerebrospinal f luid, CSF) in E14.5 mice It is also.

Figure 4e is a retinal ganglion cell ^axon, the, wherein the non-immune (pre-immune) Rabbit (rabbi t) immunoglobulin (rblgG), Rabbit -Vaxl to verify the functionality of the secreted Vaxl Vaxl in C0S7 cells overexpressing in the growth induction Retinal ganglion cell axons that grow toward C0S7 in a retinal segment (NR) co-cultured with C0S7 cells treated with a polyclonal antibody (-Vaxl):

Myc—where the tagged mouse Vaxl is located (scale bar: 500 (left), scale bar: 100 (right).

5A shows pre-immune ^' rabbi t immunoglobulin (rblgG), rabbit anti-Vaxl polyclonal antibody (α-Vaxl) and rabbit anti—Vax2 polyclonal antibody (a-Vax2) Is a diagram showing retinal ganglion cell axons growing toward ventral hypothalamic explants (NR) cocultured with ventral hypothalamic explants (vHT) treated with:

→ ： As the enlarged area _ᅵ Vaxl (green) in the enlarged area appears as yellow colored superimposed two colors when distributed on NF160 (red) labeled retinal ganglion cell axons (scale bar: 500 ΙΜ \)

FIG. 5B shows co-administration with ventral hypothalamic explants (vHT) by treatment of non-immune rabbit immunoglobulin (rblgG), rabbit anti-Vaxl polyclonal antibody (a-Vaxl) and rabbit anti-Vax2 polyclonal antibody (a-Vax2) Directional diagram of retinal ganglion cell axons from the cultured neural retinal segment (NR) to the ventral hypothalamic segment.

FIG. 5C shows co-operation with ventral hypothalamic explants (vHT) treated with non-immune rabbit immunoglobulin (rblgG), rabbit anti-Vaxl polyclonal antibody (a -Vaxl) and rabbit anti-Vax2 polyclonal antibody (a— Vax2) Figure showing the number and thickness of retinal ganglion cell axons from the cultured neural retinal segment (NR) to the ventral hypothalamic segment. FIG. 5D shows a recombinant protein labeled with peptide (100 ng / ml) or His-tagged Vaxl labeled with fluorescein isothiocyanate (FITC) at 6X-His on the retinal fragment. After incubation with (500 ng / ml), this is confirmed:

► ： Enlarged area.

FIG. 5E is a diagram showing immunostaining after incubation with 6X-His peptide (100 ng / ml) or Vaxl-His-FITC protein (500 ng / ml) in retinal sections:

→ Vaxl and His protein positions.

FIG. 5ί is treated with 6X-His peptide (25 ng / ml, white bar) or Vaxl-His protein (100 ng / ml, black bar) in the quadrant of the retinal section, measuring the changed axon length after 24 hours. Is shown:

DN: Dosal Nasal;

DT: Dosal Tempor l;

VN: VentraT Nasal; And

VT: Ventral Temporal.

Figure 5g (A) is a diagram showing the histo-tagged Vax2 (100 ng / ml) added to the retinal fragments, or without the back-side staining cultured for 24 hours, (B) is retinal ganglion cells A graphical representation of measuring axon length:

→ ： Position of Vax2-His (scale bar: 500 (top), scale bar: 100 (bottom).

FIG. 6A shows the growth of retinal ganglion cell axons in the third ventricle (V3) of E13.5 mice injected with non-immune rabbit immunoglobulin (rblgG), rabbit anti-Vaxl polyclonal antibody (α-Vaxl):

A: anterior;

P: posterior;

M: medial;

L: lateral; And

* ： Optic chiasm. FIG. 6B shows retinal ganglion cell axons growing in neural retinal sections treated with Vaxl recombinant protein, or Vaxl recombinant protein and rabbit anti-Vaxl polyclonal antibody (α-Vaxl):

→: As an enlarged site, when Vaxl (green) in the enlarged area is distributed on retinal ganglion cell axons labeled with NF160 (red), the two colors appear yellow with overlap (scale bar: 500 μηύ.

Figure 6c is a diagram showing the length of retinal ganglion cell axon over time in the retinal explants treated with Vaxl recombinant protein, or Vaxl recombinant protein and rabbit anti-Vaxl polyclonal antibody (α-Vaxl).

6D is a diagram showing the number, thickness and length of retinal ganglion cell axons in neural retinal sections treated with Vaxl recombinant protein, or Vaxl recombinant protein and rabbit anti-Vaxl polyclonal antibody (a -Vaxl).

7A is a diagram showing the expression of Vaxl mRNA in the retina of Vaxl + / + and VaxllacZ / lacZ E14.5 mice.

7B is a diagram showing expression of Vaxl protein and beta galactosidase (β-galactosidase, β-gal) in the optic pathway structure of Vaxl + / lacZ and VaxllzcZ / lacZ E14.5 mice:

GCL: ganglion cell layer; And

NBL: neuroblast layer.

7C shows in the retina of Vaxl + / + and VaxllacZ / lacZ E14.5 mice

Figure shows the Vaxl protein:

► ： Sites immunostained with NF160 (neurofilament), a retinal ganglion cell axon marker; And

→: Sites immunostained with NF160 and Vaxl.

7D shows Vaxl proteins present in the retina and optic nerve of Vaxl + / + and VaxllacZ / lacZ E18.5 mice:

RGC: retinal ganglion cell;

OS APC: ocular disc astrocyte precursor cell; ► in RGC: Vaxl protein present in intracellular vesicles (endocytic vesicle); And

→ in RGC: Vaxl protein bound to the extracellular surface of the RGC plasma membrane;

► in OS APC: Vaxl protein present in transport vesicles (trafficking vesicle); And

→ in OS APC: Vaxl protein associated with chromatin in the nucleus. 、

7E (A) shows Mui et al using [33P1-CTP-labeled ant i sense Vaxl probes in brain sections prepared from Vaxl + / + (WT) and Vaxl − / − E14.5 mice. , In situ RNA hybridization by the method described in 2005 to visualize the radiation intensity of Va33 mRNA expression of the [33P] -CTP—labeled antisense Vaxl probe. (B) is a diagram showing the brain sections obtained from E14.5 Vaxl + / lacZ and VaxllacZ / lacZ mice and visualized by immunostaining:

HCC ^'' -Hypothalamic cell cord;

ON: Optic nerve; And

0C: Optic chiasm.

8 shows Ptc-gal4> Vaxl-EGFP, Ds-Red Drosophila Pt c-ga 14> Vaxl-EGFP, Ds-Red, Sdc23 Drosophila, Ptc-gal4> Vaxl-EGFP, Ds-Red, Sdc Drosophila, and Ptc- Gal4> Vaxl_EGFP, Ds-Red, Dip This diagram shows the distribution of green fluorescent protein labeled Vaxl protein and red fluorescent protein (dsRed) expressed in the same cells in the imaginal disc.

Figure 9a is a diagram showing the results of the coimmunoprecipitation (Co-i 醒 unoprecipitation) of the binding of Vaxl and Sindecan 2 (Sdc2) in the optic nerve of the post-natal day 0, P0 mice.

9B shows coimmunoprecipitation of the binding of Vaxl, and Vaxl, and Sindecane l (Sdcl) or Sindecane 2 (Sdc2) in Vaxl and HEK293T cells overexpressing GFP-Sdcl or GFP_Sdc2 It is a figure which shows the result of investigation.

Figure 9c is a diagram showing the results of investigating the binding of Vaxl, and Sdcl or Sdc2 in Vax2, and GFP-Sdcl or GFP-Sdc2 overexpressed HEK293T cells by the immunoimmunoprecipitation method.

Figure 9d shows Vaxl, and HEK293T cells overexpressed with GFP-Sdc2-N or GFP-Sdc2-C.

Fig. 3 shows the results of investigating the binding of Vaxl and Sdc2-N or Sdc2-C by co-immunoprecipitation method.

Figure 9e is a diagram showing the results of the coimmunoprecipitation method of the protein binding to Vaxl in the E14.5 mouse optic nerve treated with heparin (heparin).

9F shows heparan sulfate (HS) or CS coated saparose 4B (sepharose).

4B) is a diagram showing the binding of resin and Vaxl protein.

10A shows Vaxl protein, Vaxl protein and jellyfish Kheparinase I), and

A diagram showing retinal ganglion cell axons growing in neural retinal sections treated with Vaxl protein and chondroitinase ABC (ChnaseABC):

→: As an enlarged region, VaxK green in the enlarged region appears as a yellow color with two colors overlapping when distributed on NF160 (red) labeled retinal ganglion cell axons (scale bar: 500 um).

10B shows biotin labeled Vaxl KR-peptide (KR-bio), Vaxl protein and two important sugar binding residues 101 which encode Vaxl amino acid sequences 101-112 having homology with Vaxl protein and 0tx2 sugar binding motif. Replacing lysine and arginine 102 (Lysl01-Argl02 (KR)) with alanine (Ala-Ala (AA))

A diagram showing retinal ganglion cell axons growing in neural retinal sections treated with Vaxl (KR / AA) mutant proteins substituted with AA-bio peptide or Lysl01-Argl02 (KR) with Ala-Ala (AA):

→: As an enlarged area, when VaxK green in the enlarged area is distributed on retinal ganglion cell axons labeled with NF160 (red), the color appears to be yellow with two colors overlapping (scale bar: 500 η).

10c shows Vaxl protein, Vaxl protein and Heparinase I, and Vaxl protein and Growth of retinal ganglion cell axons in neural retinal sections treated with chondroitinase ABC, Vaxl protein and Vaxl KR-peptide (KR-bio), Vaxl protein and AA-bio peptide, and Vaxl (KR / AA) mutant protein Is a diagram showing.

Figure 11a is a diagram showing the results of Western blotting (Vaxl) protein in the growth medium of heparin-treated Vaxl and Vaxl (KR / M) overexpressing HEK293T cells.

FIG. Lib shows the results of investigation of the co-immunoprecipitation method of Vaxl protein binding to Sdc2 in GFP-Sdc2 and Vaxl or Vaxl (KR / AA) overexpressing HEK293T cells.

Figure 11c is a diagram showing the Vaxl protein present in C0S7 cells to which the growth medium of Myc— Vaxl or Myc_Vaxl (KR / AA) overexpressing HEK293T cells was added.

12A shows Ser-Arg amino acid residues 147 tryptophan (Trp) and 148 phenylalanine (Phe) (WR) having homology with important residues of GFP-Sdc2, and the cell-penetrating region of antennapedia, Antp. Fig. 2 shows the results of coimmunoprecipitation of Vadc protein binding to Sdc2 in Myc-Vaxl (WF / SR) mutants substituted with (SR) or HEK293T cells overexpressing Myc-Vaxl.

12B is a diagram showing the results of Western blotting of Vaxl protein present in C0S7 cells added with growth medium of Myc-Vaxl or Myc-Vaxl (WF / SR) overexpressing HEK293T cells.

12C shows retinal ganglion cell axons growing toward C0S7 cells in neural retinal (NR) fragments co-cultured with Myc-Vaxl or Myc-Vaxl (TOVSR) overexpressing C0S7 cells.

12D is a diagram showing the direction of retinal ganglion cell axons directed to C0S7 cells in neural retinal (NR) fragments co-cultured with Myc-Vaxl or Myc-Vaxl (WF / SR) overexpressing C0S7 cells.

Figure 13a is a diagram showing the proteins constituting the Vaxl protein complex in the cytoplasm (cytoplasm) fraction in HEK293T cells overexpressed GST and GST- Vaxl.

Figure 13b is cultured in a medium containing Vaxl or Vaxl (WF / SR) protein The newly synthesized protein in the neural retinal fragment is labeled with green fluorescence.

Figure 13c is a diagram showing the label using green fluorescence of a newly synthesized protein from axons isolated from neural retinal sections cultured in a medium containing Vaxl or Vaxl (WFYSR) protein.

Figure 13d is in a medium containing Vaxl or Vaxl (WF / SR) protein. Figure showing growth of axons isolated from cultured neural retinal sections.

Figure 14a (A) shows the growth of retinal ganglion cell axons in the third ventricle of VaxllacZ / lacZ embryonic mice injected with His-peptide, Vaxl-His, Vaxl (WF / SR) -His, Robol-Fc segment, etc. It is a figure shown by immunostaining, (B) is a graph which shows the fluorescence intensity measured.

Figure 14b (A) is a diagram showing the retinal ganglion cell axons growing in the neural retinal section co-treated with Vaxl and Slit2 by concentration, retinal ganglion grown in the neural retinal section co-regulated by Vaxl and Slit2 by concentration A graph showing the length of cell axons.

14C (A) shows that after incubating the E13.5 mouse neural retinal segment and Vaxl-knockout ventral hypothalamus section together with or without Robo 1-Fc for 24 hours, visualization and immunostaining were performed. (B) is a graph showing the direction coefficient of retinal ganglion cell axon:

+: Forward direction;

0: neutral; And

-: Reverse direction.

FIG. 14D shows immunostaining of retinal ganglion cell axons grown in neural retinal sections co-treated with Vaxl and Slit2 by concentration, and shows the fluorescence intensity thereof graphically (scale bar: 20 μηύ.

FIG. 14E is a diagram showing Vaxl protein infiltrating into retinal ganglion cell axon in Vaxl or neural retinal sections treated with Vaxl and S1 2 proteins. 14F treated with Vaxl, Vaxl (R152S) and Vaxl (WF / SR). Cerebral Figure 1 shows the results of immunofluorescence staining of Vaxl protein infiltrated into axons of cort ical explants.

Figure 14g is a diagram showing a model of retinal ganglion cell axon growth regulation by secreted Vaxl protein. The best form for the implementation of the invention

Hereinafter, the present invention will be described in detail. The present invention provides a Vax ventral anterior homeobox _. It provides a pharmaceutical composition for promoting neuronal regeneration or neuronal growth containing white matter as an active ingredient.

The Vax protein is any one selected from the group consisting of a Vaxl protein having an amino acid sequence as set forth in SEQ ID NO: 1 or a Vax2 protein having an amino acid sequence as set forth in SEQ ID NO: 2, but not limited thereto.

The nerve cells are vaxl-deficient optic (opt ic nerve), cort ical commi ssural nerve, hippocampal commissur l nerve, hypothalamic commissural nerve, trigeminal nerve (tr igeminal nerve) nerve) is preferably any one selected from the group consisting of, but is not limited thereto.

The Vaxl is preferably bound to heparan sul fate proteoglycan (HSPG), but is not limited thereto. In specific embodiments of the present invention, the present inventors have identified VaxllacZ / + and VaxllacZ / lacZ knock-in and Vaxl-/-knockout to confirm the function of Vaxl protein in the growth of retinal ganglion cell axons. -out) mice were prepared and neural ret ina (NR) and ventral hypothalamic (vHT) explants were isolated and cultured in the obtained mice. In addition, Ptc-Gal4> UAS— Vaxl-EGFP, UAS-DsRed overexpressing Vaxl; +,

Pt c-Ga 14> UAS-Vax 1-EGFP, UAS-DsRed; Sdc23,

Ptc-Gal 4> UAS-Vax 1-EGFP, UAS-DsRed; Sdc,

Pt c-Ga 14> UAS- Vax 1-EGFP, UAS-DsRed; D 1 p Drosophila were prepared.

In addition, the present inventors have performed immunostaining (i 薩 unostaining) on the cross-optic cells of Vaxl + / + (T) and Vaxl − /-mice to determine the effect of Vaxl on the opt ic pathway structure. As a result, Vaxl was found in Sox2 (SRY box 2) -positive neural stem cells and nestin (nest in, RC2) -positive glial cells. In addition, it was confirmed that neural stem cells and radioglial cells, which are optic nerve cross-constituting cells, normally develop in Vaxl − / − mice. Therefore, Vaxl is expressed in the optic nerve crossings present in neural stem cells and radioglial cells, and it was confirmed that abnormal development of optic nerve crossings in Vaxl-/-mice was not caused by incorrect formation of ventral hypothalamic cells related to optic nerve cross-sectional formation ( See la and lb).

In addition, we assessed the effect of Vaxl on the development of optic nerve cross-linking in neural retinal explants in (E13.5) Vaxl + / + (WT) and Vaxl-/-(K0) mice. Observation and orientation of retinal ganglion cell axons by co-culture of ventral hypothalamic explants showed that retinal ganglion cell axons from Vaxl + / + (WT) neural retinal sections to Vaxl-/-(K0) ventral hypothalamic explants. By decreasing the number of, it was confirmed that Vaxl derived from the ventral hypothalamic section directly or indirectly induced retinal ganglion cell axon growth in the retina. Thus, it was confirmed that Vaxl regulates retinal ganglion cell axon growth via axon inducing molecules secreted by non-cel autonomous methods (see FIGS. 2A-2E).

In addition, the inventors of the present invention share the same homeomai (homeodomai.n) as Vaxl, transcriptionally inactive Vaxl mutation (Vaxl (R152S)), and Vaxl, to determine whether Vaxl transcriptional activity affects retinal ganglion cell axon growth. Immunostaining, Retinal Ganglion Cells Using C0S7 Cells Overexpressing Vax2 As a result of directional measurement of axon growth and Western blotting of proteins in media, the number of retinal ganglion cell axons growing toward Vaxl and Vaxl (R152S) overexpressed C0S7 cells in control group Compared to the remarkable increase, interestingly, the Vaxl and Vaxl (R152S) proteins were also observed in retinal ganglion cell axons that did not express the protein. In addition, by confirming that both Vaxl and Vaxl (R152S) proteins are found in the cell lysates (cel l lysate, CD and growth media (GM)) of the Vaxl and Vaxl (R152S) overexpressed C0S7 cells, the Vaxl Irrespective of this transcriptional activity, it was secreted out of the cells to induce retinal ganglion cell axon growth (FIGS. 3A to 3C).

In addition, the inventors of the present invention, in order to confirm Vaxl secretion in retinal ganglion cell axon growth induction, C0S7 cells and Vaxl overexpressing HEK293T cell growth medium with Vaxl or Vax2 overexpressing HEK293T cell growth medium injected into E13.5 mouse ventricle Immunostaining and Western blotting using sections (sect ions) confirmed that Vaxl proteins in Vaxl-overexpressing HEK293T cell growth media were found in in vitro C0S7 cells and in ventral diencephal ic (vDC). . In addition, by confirming that Vaxl protein is found in cell lysates and growth media of ventral hypothalamic explants, and cerebrospinal fluid (CSF), the Vaxl protein is a protein secreted from cells in the body as well as in vitro culture. Therefore, it was confirmed that the Vaxl protein is a protein secreted from the ventral hypothalamus regardless of transcriptional activity (see FIGS. 4A to 4E).

In addition, to determine whether the secreted Vaxl protein is a secreted protein that induces retinal ganglion cell axon growth towards the ventral hypothalamic explants, the rabbit anti-Vaxl polyclonal antibody and anti Vax2 polyclonal Retinal ganglion cell axon growth direction and length measurements of co-cultivated neuroretinal and ventral hypothalamic fragments with extracellular Vax protein were isolated. Anti-Vaxl antibody-treated neural retinal fragments Decrease in the number and thickness of retinal ganglion cell axons growing toward the ventral hypothalamic section in By confirming, the extracellular Vaxl plays an important role in the axon's fasciculat ion. As a result of using Vaxl-His-FITC protein, it shows a strong stimulatory effect on the growth of retinal ganglion cell axons. In addition, it was confirmed that a strong stimulatory effect on the growth of retinal ganglion cell axons, and in the case of Vax2 was confirmed to induce the growth of retinal ganglion cell axons almost the same as Vaxl (see FIGS.

In addition, the present inventors have used E13.5 mouse brain slices in which anti-Vaxl polyclonal antibody was implanted into the third ventricle to confirm the role of extracellular Vaxl in retinal ganglion cell axon growth in vivo. Di axon chromosome detection and immunostaining resulted in a decrease in retinal ganglion cell axons directed to the optic nerve crossover in mice implanted with anti-Vaxl polyclonal antibodies, axons stopped at the outer wall of the ventral hepatic brain, and retinal ganglion cell axons. It was confirmed that the distribution of intra Vaxl protein is reduced.

In addition, the present inventors treated the Vaxl recombinant protein or the anti-Vaxl polyclonal antibody to the neural retinal explant culture, and then immunostained, retinal ganglion cell axon growth, axon thickness measurement, the nerve treated with Vaxl recombinant protein The number, thickness, and length of retinal ganglion cell axons in retinal sections increased, whereas the number, thickness, and length of retinal ganglion cell axons in neural retinal sections treated with retinal Vaxl recombinant protein and anti-Vaxl polyclonal antibody. By confirming the decrease, it was confirmed that the recombinant Vaxl protein directly showed the retinal ganglion cell axon growth stimulating effect (see FIGS. 6A to 6D).

In addition, the present inventors have performed RNA in situ using the retina and brain sections of Vaxl + / +, VaxllacZ / lacZ and Vaxl + / lacZ mice to confirm that Vaxl mRNA and protein are expressed in the retina. As a result, Vaxl + / + mice were found to have Vaxl mRNA in the optic disc (0D), eye disease (OS), and optic nerve cross-section (P0A) except for the retina. In addition, Vaxl + acZ mouse eye fragments expressing Vaxl and beta-galactosidase (lacZ) in each homologous chromosome were treated with anti-Vaxl multiple antibodies. As a result of immunostaining using beta-galactosidase antibody, Vaxl + acZ mice with co-expressing beta-galactosidase (OS) astrocytoblasts (astrocyte precursor cel l, APC) was confirmed. Separately, Vaxl protein was also detected in retinal ganglion cells that do not express beta-galactosidase. Investigation of intracellular distribution of Vaxl protein using immunoelectron microscopic analysis revealed that Vaxl protein binds to the extracellular surface of the retinal ganglion cell plasma membrane in Vaxl + / + mice and endocyt ic vesicels Vaxl was expressed in and secreted from ophthalmic glial cells other than the retina, and the secreted Vaxl protein migrated into retinal ganglion cells (see FIGS. 7A to 7E).

In addition, the inventors of the present invention are syndecan (Sdc), a heparan sul fate proteoglycan (HSPG), which regulates the intercellular migration of Vaxl and regulates the intercellular migration of Vaxl. Ptc-Gal4> UAS-Vaxl-EGFP, UAS-DsRed, in which Vaxl was overexpressed to determine if involved; +

Pt c-Ga 14> UAS-Vaxl-EGFP, UAS-DsRed; Sdc23

Pt c-Ga 14> UAS-Vaxl-EGFP, UAS-DsRed; Sdc,

Immunostaining using Pt c-Ga 14> UAS-Vaxl-EGFP, UAS-DsRed; D 1 p Drosophila showed that red fluorescent protein coexpressed with Vaxl-EGFP protein While the number of Vaxl-expressing cells increases in front of the missing imaginal disc, co-expressing Dip, a Drosophila homologous protein of syndecane and HSPG glypi can (Glp), with Vaxl-EGFP red fluorescent protein By confirming that the number of cells expressing only Vaxl in front of the fly lamellar plate of the Drosophila was reduced, it prevented the synthecan and Dip from binding to the Vaxl protein and dispersed into neighboring cells. Thus, the intracellular migration of Vaxl was hepatic. It was confirmed that lan sulfate was mediated by proteoglycans (see FIG. 8). In addition, the inventors of the present invention, in order to determine the role of play ¾ in the movement of Vaxl, post-natal day 0 P0 of the optic nerve and Vaxl, syndecan KSdcl), Cincan 2 (Sdc2), Cincan 2 (Sdc2-C) lacking the N-terminal extracellular domain, Cincan 2 (Sdc2-N) lacking the C-terminal cytoplasmic domain, and HEK293T cells overexpressed As a result of immunoprecipitation and Western blotting, it was confirmed that Vaxl was precipitated by Sdc2 in the retinal optic nerve and HEK293T cells, and that Vaxl was precipitated by Sdc2-N. It was confirmed that it interacts with the extracellular domain of decane 2 (see FIGS. 9A-9D).

In addition, the present inventors performed immunoprecipitation using the optic nerve of E14.5 mice to which high concentrations of heparin were added in order to confirm that Vaxl binds to the heparan sulfate (HS) side chain of syndecane 2. Heparin-treated optic nerves did not precipitate Sdc2, Sdc3, Glpl except Pax2 by anti-Vaxl antibodies, and HS-safarose 4B resin using heparin-coated sapharose 4B resin. By confirming that the Vaxl protein was precipitated, the Vaxl competed with heparin and confirmed binding to the heparan sulfate side chain of the heparan sulfate proteoglycan protein (see FIGS. 9E and 9F).

In addition, the present inventors have found that Vaxl stimulates the retinal ganglion cell axon growth

To determine whether Vaxl is mediated by binding to heparin oligosaccharides, Vaxl was treated in the presence of jellyfishase Khepar inase I) or chondroitinase ABCCchondro inase ABC, ChnaseABC). It was confirmed that Vaxl accumulation in axons was inhibited by jellyinase I treatment (see FIG. 10A).

In addition, the present inventors have carried out a biotin-labeled Vaxl encoding this site to determine whether the binding to the heparan sulfate proteoglycan of Vaxl is mediated by Vaxl amino acid sequences 101-112 having homology with the 0tx2 sugar binding motif. KR-peptide (KR-bio) or M-bio peptide, or Lysl01, substituted with alanine (Ala-Ala (AA)) for the two and important sugar binding residues 101 lysine and 102 arginine (Lysl01-Argl02 (KR)) Immunostaining and retinal ganglion cell axon growth were measured using neural retinal fragments treated with Vaxl (KR / AA) -His mutant protein substituted with -Agl02 (KR) with Ala-Ala (AA). KR-bio Peptides Treated Together Vaxl is a sugar-binding motif by reducing the growth of retinal ganglion cell axons in neural retinal sections and reducing the growth of retinal ganglion cell axons in neural retinal sections treated with Vaxl (KR / AA) mutant proteins. It was confirmed that the stimulation of the growth of retinal ganglion cell axons in combination with haeparan sulfate using (see FIG. 10C). .

In addition, the inventors of the present invention, VaxKKR / AA) mutant protein secretion and the binding force between the syndecan 2, Vaxl or Vaxl (KR / AA) Vaxl extracted in the growth medium after addition of heparin to the growth medium of overexpressing HEK293T cells And the amount of Vaxl (KR / AA) protein was compared with heparin-free growth medium, and immunoprecipitation and Western blotting were performed using Sdc2 and Vaxl or Vaxl (KR / M) overexpressing HEK293T cells. KR / M) protein and Sdc2 interaction (see Figure lib). In addition, immunostaining was performed using C0S7 cells containing 293T cell growth medium overexpressing Vaxl or Vaxl (KR / AA). Vaxl (KR / AA) was observed in C0S7 cells containing VaxKKR / AA) overexpressing HEK293T cell growth medium. ), The sugar binding motif of Vaxl binds to heparan sulfate of heparan sulfate proteoglycan, moves into retinal ganglion cell axon and induces axon growth (FIG. 11c).

In addition, to determine whether Vaxl stimulates retinal ganglion cell axon growth by regulating cytoplasmic reaction after intracellular infiltration, or activates a subsignal of heparan sulfate proteoglycan, Sdc2, and Immunoprecipitation and Western blotting were performed using HEK293T cells overexpressing Vaxl (WF / SR) mutations that could bind to Vaxl or cell surface heparan sulfate proteoglycans but could not penetrate target cells. WF / SR) Immunostaining using C0S7 cells with HEK293T overgrowth growth medium, and immunostaining and retinal retinal tissue using co-cultured retinal explants of Vaxl or VaxKWF / SR) overexpressing C0S7 cells Orientation measurements of ganglion cell axons showed that Vaxl (WF / SR) binds to synthecan 2 but not in C0S7 cells. Vaxl (WF / SR) through the undiscovered The mutations were able to bind heparan sulfate proteoglycans but were unable to penetrate into the cells. In addition, it was confirmed that the number of retinal ganglion cell axons that grow toward the Vaxl (WF / SR) and expressing C0S7 cells in the neural retinal fragments decreased, thereby confirming that Vaxl penetrated into the retinal ganglion cells to induce axon growth. 12A-12 (see 1).

In addition, the present inventors purified the protein complex interacting with the GST-Vaxl protein overexpressed in 293T cells in order to confirm the function of Vaxl in the cytoplasm, and confirmed the protein by MALDI-T0F after staining (si lver staining) Xl Ribosomal proteins (RPs) Lll, L23A, 126, S14 and S16, Vaxl and ribosomal ^`` r ibosomal component ^'' , elF (eukaryot ic translat ion init iat ion factor, translating ion regulator) By identifying 3B and 3C, and HSPA1A (chaperon heat shock 70-KD protein 1A), Vaxl was found to be involved in local protein synthesis, a mechanism similar to cytoplasmic En2 that regulates retinal ganglion cell axon growth ( 13a).

In addition, the present inventors have used immunostaining and retinal ganglion cells using neural retinal fragments of E13.5 mice cultured in a medium containing Vaxl or Vaxl (VSR) mutant protein to determine whether Vaxl is involved in local protein synthesis. As a result of measurement of axon growth, the protein synthesis rate and growth of retinal ganglion cell axons in Vaxl overexpressed neural retinal sections increased, whereas the proteins of retinal ganglion cell axons of Vaxl (WF / SR) overexpressed neural retinal sections were increased. By confirming the decrease in synthesis rate and growth, it was confirmed that Vaxl migrated into cells to promote local protein synthesis and stimulate the growth of retinal ganglion cell axons (see FIGS. 13A and 13D).

In addition, the inventors of the present invention, in order to determine whether the retinal ganglion cell axons damaged in the Vaxl-/-mice are recovered by extracellular Vaxl protein, VaxllacZ / lacZ collagen gel containing the recombinant protein, Vaxl WF / SR) After transplantation into the third brain ventricle of the mouse and immunostaining, the retinal ganglion cells of the mouse transplanted with VaxKWF / SR) protein were different from those of the Vaxl protein. We found that the axon's hypothalamus approached and grew poorly, and Robo

The growth of retinal ganglion cell axons in the ventral hypothalamus showed a stronger effect when transplanted with a collagen gel containing Vaxl-His and Robo 1-Fc. The Vaxl protein was confirmed to infiltrate the axoplasm and stimulate the growth of retinal ganglion cell axons independent of the expression of axon inducing molecules (see FIG. 14A). In addition, the present inventors performed immunostaining and retinal ganglion cell axon length measurement using neural retinal sections treated with Vaxl and Sl it2 proteins by concentration, in order to confirm the relationship between Vaxl and Sl it2, Vaxl alone. Retinal ganglion cell axon growth was inhibited, axon length was decreased, and Vaxl confirmed that infiltration into retinal ganglion cell axons was not significantly affected by Sl i t2 in the group treated with Sl i t2. When the culture was treated with Vaxl and Robo 1-Fc together, the growth of axons of retinal ganglion cells was remarkably increased, and once again, the group treated with Vaxl and Sl i t2 competed with each other. ， The Vaxl and Sl i t2 confirms that reciprocal antagonism with Sl i t2 without competitive binding to heparan sulfate proteoglycans Was (see Fig. 14b through 14e). In addition, Vaxl, Vaxl (R152S) and Vaxl (WF / SR) were treated on the cerebral explants and immunostained. As a result, Vaxl protein was confirmed to induce the growth of cerebral nerve axons as well as retinal ganglion cell axons. The transmembrane midline passage of various neuronal axons in gene deficient mice and humans was also confirmed to be regulated by Vaxl protein similarly to retinal ganglion cell axons (see FIG. 14F).

In addition, the present inventors are expressed in the radioglia and neural stem cells of the ventral hypothalamus of the mammalian brain through the above results and secreted out of the cells, and the secreted Vaxl binds to the blue sea sulfate sulfate proteoglycan including syndecan and retinal ganglion cells. After infiltrating into the axon, it was confirmed that the retinal ganglion cell axon growth was stimulated by promoting local protein synthesis in the axon morphology. Reference).

Thus, Vaxl secreted from the ventral hypothalamus and cerebral septum of the present invention binds to the extracellular sugar group of heparan sulfate proteoglycan present in retinal ganglion cells and cerebral cross nerve axons and infiltrate into the axonplasm. After that, by activating local protein synthesis to promote retinal ganglion cell axon growth, it can be usefully used as an active ingredient of a pharmaceutical composition for promoting neuronal regeneration or neuronal growth. The present invention also provides a pharmaceutical composition for promoting neuronal regeneration or neuronal growth containing a vector or cell comprising a polynucleotide encoding Vax protein as an active ingredient.

The Vax protein is any one selected from the group consisting of a Vaxl protein having an amino acid sequence as set out in SEQ ID NO: 1 or a Vax2 protein having an amino acid sequence as set out in SEQ ID NO: 2, but is not limited thereto.

The vector is preferably, but not limited to, linear DNA, fulllasmid DNA or recombinant viral vector.

The recombinant virus is from the group consisting of retrovirus, adenovirus, adeno-associated virus (AVA), herpes simplex virus, lentivirus. It is preferably any one selected, but is not limited thereto.

Preferably, the cell is any one selected from the group consisting of hematopoietic stem cells, dendritic cells, autologous tumor cells, and established tumor cells. It is not limited to this.

Vax protein secreted from the ventral hypothalamus and cerebral septum of the present invention is heparan sulfate present in retinal ganglion cells and cerebral cross-neuronal axon After binding to the extracellular sugar group of the proteoglycan and penetrating into the axon morphology, it activates local protein synthesis to promote retinal ganglion cell axon growth.

Vectors or cells comprising polynucleotides encoding Vax proteins may be usefully used as active ingredients of pharmaceutical compositions for promoting neuronal regeneration or neuronal growth. The composition of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. ^'

The composition of the present invention can be administered parenterally, when parenteral administration, external skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method to select a solid body transplant It is preferable, but it is not limited to this.

The composition of the present invention may be used in the form of external preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. Carriers, excipients, and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbbi, manny, xili, erysri, malty, starch, acacia rubber, alginate, gelatin, calcium phosphate. Calcium silicate, salose, methyl cellulose, microcrystalline cellulose, polyvinyl pyridone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using a diluent or excipient such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants which are commonly used. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories. As the non-aqueous solvent and the suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as evolved oil, injectable esters such as ethyl oleate, and the like can be used. As a base of suppositories, wi tepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the compositions of the present invention are determined by the condition and weight of the patient, Depending on the degree, drug form, route of administration, and duration, it may be appropriately selected by those skilled in the art. However, for the desired effect, the composition is preferably administered at 0.0001 to 1 g / kg, preferably 0.001 to 200 mg / kg per day, but is not limited thereto. The administration may be administered once a day, or may be divided several times. The dosage does not limit the scope of the invention in any aspect. In addition, the present invention

3) Vaxl and heparan sulphate proteoglycan binding level of the step 2) non-treated control. Provides a method for screening a candidate substance for nerve cell regeneration or neuronal growth promotion comprising the step of selecting a test substance to increase compared to the group. do.

The binding level is preferably any one selected from the group consisting of immunofluorescence, mass spectrometry, protein chips, western blotting and ELISA, but is not limited thereto.

Vaxl secreted from the ventral hypothalamus ^' and cerebral enlargement of the present invention binds to the extracellular sugar group of the heparan sulfate proteoglycan present in retinal ganglion cells and cerebral cross nerve axons, infiltrate into the axon morphology, and activate local protein synthesis. Since it promotes the growth of retinal ganglion cell axons, it can be usefully used as a screening candidate for neuronal regeneration or neuronal growth promotion. The present invention also provides a method for regenerating neurons comprising administering a pharmaceutically effective amount of Vax protein to a subject injured in a neuronal cell. In addition, the present invention provides a method for promoting neuronal growth comprising administering a pharmaceutically effective amount of Vax proteinol neurons to a damaged individual.

In addition, the present invention provides a method for regenerating neurons comprising administering a vector or cell comprising a polynucleotide encoding a pharmaceutically effective amount of Vax protein to a subject injured.

Vax protein secreted from the ventral hypothalamus and cerebral septum of the present invention binds to the extracellular sugar group of heparan sulfate proteoglycan present in retinal ganglion cells and cerebral cross nerve axons, infiltrate into the axon morphology, and then activate local protein synthesis. By promoting the growth of retinal ganglion cell axons, a vector or cell containing the Vax protein, or a polynucleotide encoding the Vax protein, is administered to an individual injured with nerve cells, which is useful as a method for promoting nerve cell regeneration or nerve cell growth. Can be used. The present invention also provides the use of Vax protein for use as a pharmaceutical composition for promoting neuronal regeneration or neuronal growth.

Vax protein secreted from the ventral hypothalamus and cerebral septum of the present invention binds to the extracellular sugar group of heparan sulfate proteoglycan present in retinal ganglion cells and cerebral cross nerve axons, infiltrate into the axon morphology, and activate local protein synthesis. By promoting the growth of retinal ganglion cell axons, the vector or cell comprising the Vax protein, or polynucleotide encoding the Vax protein is a pharmaceutical composition for promoting neuronal regeneration or neuronal growth. It can be usefully used for the purpose. Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are only to illustrate the present invention,

It is not limited by the embodiment.

Example 1 Fabrication of Vaxl Over Express Ion, Knock-out, and Knock-in Animal Models

<1-1> Isolation and culture of neural retina (NR) and ventral hypothalamic (νΗΓ) fragments from Vaxl knock-out mice

VaxllacZ / + and Vaxl-/-knock-out and VaxllacZ / lacZ knock-in mice are described in Bertuzzi et al. , 1999; Hal l one t et al. , According to the method described in 1999. Neural ret ina (NR) and ventral hypothalamic (vHT) explants of the obtained mice were obtained from Sato et al. , Obtained by the method described in 1994. The obtained neural retinal or ventral hypothalamic fragment was added to a collagen (col lagen) mixture, and the collagen mixture containing the neural retinal or ventral hypothalamic fragment was added to the poly-L-lysine at 10 / g / m. (poly-L-lysine) and laminin of 10 «g / ηι were added to the plate, and then gelled by incubating for 1 hour at 37 ° C. It was then incubated in Neurobasal medium containing B27 supplement (Invi trogen).

<1-2> Vaxl Overexpression Drosophila Production

Ptc-Gal4, UAS-DsRed, UAS-Sdc, UAS-Dlp, and sdc23 Drosophila were obtained from the Bloomington stock center. In addition, the mouse Vaxl (SEQ ID NO: 1) was cloned into a pUAS-EGFP vector to obtain a pUAS- Vaxl-EGFP vector, and the vector was injected into a fruit fly to obtain a UAS-Vaxl-EGFP Drosophila. Ptc-Gal4> UAS-Vaxl-EGFP, UAS-DsRed; Third instar larvae of + were obtained by cross-crossing Pt c-Ga 14>UAS-DsRed; TM6B Drosophila and UAS-Vaxl-EGFP Drosophila. Tertiary larvae of Ptc-Gal4> UAS-Vaxl-EGFP, UAS-DsRed; Sdc23 were obtained by cross-crossing Pt c-Ga 1>UAS-DsRed; Sdc23 Drosophila and UAS-Vaxl-EGFP Drosophila. Ptc-Gal4> UAS-Vaxl-EGFP, UAS-DsRed; Tertiary larvae of Sdc include Ptc-Gal4>UAS-DsRed; Sdc Drosophila and UAS-Vaxl-EGFP Drosophila were obtained by cross crossing. Ptc-Gal4> UAS-Vaxl-EGFP, UAS-DsRed; Tertiary larvae of Dlp include Pt c-Ga 14>UAS-DsRed; Dip Drosophila and UAS-Vaxl-EGFP Drosophila were obtained by cross crossing.

Example 2 Non-cel l-automonous Retinal Ganglion Cell (RGC) Axon Growth Regulation of Vaxl

Development of normal optic nerve crossing (0C) forming cells in <2-l> Vaxl-deficient mice (Vaxl-/-)

Vaxl is expressed in cells located in opt ic pathway structures, such as opt ic stalk (OS) and optic nerve cross (opt ic chiasm, 0C), and fasciculat ion of retinal ganglion cell axons. And an important role in the formation of optic nerve crossing (Bertuzzi et al., 1999; Hal l one t et al., 1999). Therefore, in order to confirm the effect of Vaxl on the visual pathway structure, immunostaining was performed using optic cross-crossing cells of Vaxl deficient mice (Vaxl − / −).

Specifically, brain sections extracted from Vaxl + / + and Vaxl − / − embryonic 14.5 (E14.5) mice obtained by the method described in Example <1-1> were cut into brain sections (vertical ion) (vertical). (coronal): 12). The brain sections were washed with 1 XPBS and fixed with 4¾ (v / v) paraformaldehyde (PFA) / PBS for 2-16 hours at 4 ^° C. and 20¾ (w / v for 16 hours at 4 ^° C.). After incubation with sucrose / PBS solution, the cells were infiltrated with OCT medium and frozen. The frozen brain section was 0.2% Tr itonX-100, 5% normal for 1 hour. Rabbit anti-Vaxl antibodies for 16 hours at 4 ^° C after incubation with blocking solution containing normal donkey serum and 2% bovine serum albumen (BSA) (Mui et al., 2005) (Abstract), a goat anti-Sox2 antibody (Santa Cruz Biotechnology) (red) to visualize Sox2, a neural progenitor cell (NPC) marker, and the developing post-divisional nerve After reaction with mouse anti-Tujl Antibody (Covance) (blue) to visualize the post-mitotic neuronal marker, tubulin-beta I II (tubul in-betal 11), wash three times with PBS. It was. Next, the phases were reacted with Alexa488, Cy3 or Cy5 'conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Glove, PA, USA) diluted 1: 1000 for 2 hours at silver, washed with PBS and Zeiss LSM710 confocal microscope. Visualization using (Fig. La). In addition, the brain sessions were fixed, infiltrated and blocked as described above, and immunostained using anti-Vaxl antibody (green), anti-estin (nest in, RC2) antibody (red) (Millipore) Visualized (Figure lb).

As a result, as shown in FIGS. La and lb, Nestin-positive glia cells in which Vaxl provided Sox2 (SRY box 2) -positive neurons i and RC2, which provided retinal ganglion cell axon guidance signals. ), But not in Tujl-positive neurons (Fig. La and lb, row 1), but by confirming that optic nerve cross-forming cells develop normally in Vaxl-/-mice (Fig. La and lb, row 2). Vaxl is expressed in optic nerve crossings present in Sox2-positive neural stem cells and Nestin-positive radioglial cells, and abnormal development of optic nerve crossing in Vax 1 deficient mice is caused by incorrect formation of optic nerve cross-forming ventral hypothalamic cells. It was confirmed that it is not (Fig. La and lb).

<2-2> Confirmation of Axon Growth Regulation of Non-ceU-autonomous Retinal Ganglion Cells by Vaxl

In order to confirm the effect of Vaxl on the development of cross-optic nerve, directional observation and measurement of retinal ganglion cell axon growth was performed by co-cultured ventral hypothalamus and neural retinal fragments isolated from Vaxl-/-mice. Specifically, the ventral hypothalamus isolated from E13.5 mice of Vaxl normal mice (Vax + / +, WT) and Vaxl deficient mice (Vax-/-, K0) obtained by the method described in Example <1-1>. (vHT) and neural retinal (NR) fragments were co-administered for 48 hours with a combination of Vaxl + / + vHT / Vaxl + / + NR, Vaxl + / + vHT / Vaxl-/-NR and Vaxl-/-vHT / Vaxl + / + NR After incubation, mouse anti-NF160 antibody (DSHB) for fixation with 4% PFA / PBS at 4 ^° C. for 2 to 16 hours and for visualizing NF160, a retinal ganglion cell axon marker, as described in Example <2-1>. (Red) and immunostained and visualized with DAP1 (4 ′, 6-diamidino-2-phenyl indole) (blue) ol to visualize nuclei of sections (FIG. 2B), followed by retinal ganglion cell axon growth. Was measured. As shown in the schematic diagram of FIG. 2A, a region between 20 mm and 40 m of the outer surface of the neural retinal explant, which is a significant retinal axon growth site, is indicated by a yellow ring, and a line parallel to the end of the retinal axon / bundle and nerve Measure the clockwise angle formed by the line connecting the centers of the retinal segment (NR) and the ventral hypothalamic segment (vHT), positive if the angle value is between 0 ^° and 60 ^{° and} between 301 ° and 360 ^° . Red) direction, defined as neutral (0; green) direction between 61 ^° and 120 ° and between 241 ^° and 300 ^° , negative (ᅳ; blue) direction between 121 ° and 240 ^° . t ip) (FIG. 2C) and the orientation of the retinal ganglion cell axon shaft (FIG. 2D) at the yellow ring site (FIG. 2C and FIG. 2D).

As a result, as shown in FIGS. 2B-2D, the number of retinal ganglion cell axons from the retinas of Vaxl + / + and Vaxl − / − mice toward the ventral hypothalamic explants of Vax + / + mice increases, while Vaxl− It was confirmed that the number of retinal ganglion cell axons directed to the dorsal hypothalamic explants decreased, indicating that Vaxl derived from the ventral hypothalamic explants induced retinal ganglion cell axon growth in the retina (FIGS. 2B-2D). .

<2-3> Confirmation by In situ Hybridizat Ion (ISH) and Immunof Luorescence Staining (IF) by Vaxl Vascular endothelial growth factor, a retinal ganglion cell axon growth factor, was used to identify the effect of Vaxl on the development of optic nerve cross-linking. vescular endothelial growth factor (VEGF164) and neuronal adhesion molecule (NrCAM) were examined.

Specifically, embryonic heads isolated from E14.5 mice of Vaxl normal mice (Vax + / +, WT) and Vaxl deficient mice (Vax-/-, K0) obtained by the method described in Example <1 ᅳ 1>. After fixation with 4% PFA / PBS at 4 ° C. for 2-16 hours and prepared again as frozen sections, the expression of Vegfa mRNA present in these sections was confirmed by in situ hybridization. Confirmed by fluorescence staining (scale bar: 100 μ ^).

As a result, as shown in FIG. 2E, NrCAM and Vegfa, which are signals of optic nerve crossing cells and optic nerve crossing parts, were expressed in Vaxl-/-mice, and the expression sites thereof were somewhat widened (FIG. 2E).

Thus, the results of Example 2 confirmed that Vaxl regulates retinal ganglion cell axon growth by a non-cel i autonomous method. Example 3 Confirmation of Extracellular Vaxl Secretion Independently of Transcription Activity

<3-1> Vaxl Transcriptional Activity Independent Retinal Ganglion Cell Axon Growth Stimulation Confirmation Vaxl (R152S) mutations that are transcriptionally inactivated are associated with ptosis, coloboma. It has been reported in patients with cleft palate and ages of corpus cal losum (ACC), a phenotype similar to mutations in Vaxl-deficient mice (Slavot inek et al., 2012). Thus, to determine whether Vaxl transcriptional activity affects retinal ganglion cell axon growth, C0S7 overexpressing Vaxl, a transcriptionally inactive Vaxl mutation (Vaxl (R152S)), and Vax2, which shares the same homeodomain as Vaxl The cells were subjected to immunostaining, directional measurement of retinal ganglion cell axon growth, and western blotting.

Specifically, Myc-tagged mouse Vaxl, Myc-tagged mouse transcriptional inactive Vaxl mutant (Vaxl (R152S) and Myc-tagged mouse Vax2 encoded by replacing serine (S) with 152 arginine (R) After constructing the construct, the construct was transformed into C0S7 cells (ATCC) by the calcium phosphate method, and the neural retina obtained by the method described in Example <1-1>. After incubation for 48 hours with the fragment, immunostaining using anti-Myc antibody (Santa Cruz Biontechnology) (green), anti-NF160 antibody (red) and DAPU blue, as in Example <2-1>. And visualized. The centers of the two sections were connected by a dotted red line (scale bar: 500 1 (1 row), scale bar: 100 J I (2 rows and 3 rows) (FIG. 3A).

In addition, the direction of the retinal ganglion cell axon was graphed by measuring the angle based on the red dotted line connecting the center of the neural retinal fragment and the C0S7 cells as in Example <2-2>. Values in the graph were averaged and error bars were obtained as SD (control (n = 21), Vaxl (n = 17), Vaxl (R152S) (n = 8), Vax2 (n = 7)). ρ-value was calculated by AN0VA (0.01 <ρ <0 · 005) (FIG. 3B).

In addition, Western blotting was performed to confirm the presence or absence of Vax protein in the growth medium of Vaxl, Vaxl (R152S) or Vax2 overexpressed C0S7 cells. The growth medium and cells of the prepared Myc-Vaxl, Myc-Vaxl (R152S) or Myc-Vax2 transformed C0S7 cells were obtained. The obtained cells were lysed with lysis buffer containing 10 mM Tris-HCA (pH 7.4), 200 nM NaCl and 1¾ NP-40. In addition, the obtained growth medium was centrifuged twice at 500 × g for 10 minutes, followed by further centrifugation at 2,000 × g for 15 minutes to obtain a supernatant (S3) fraction. The same amount of 3 M trichloroacetic acid (TCA) solution was mixed with the obtained supernatant to precipitate macromolecules, and the TCA precipitate was washed twice with 100% acetone (acetone) and dried to pellet. (pellet) was obtained. The obtained cell lysate and pellets The reaction was terminated by adding 2 XSDS sample buffer, separated using SDS-PAGE gel, and transferred to a PVDF membrane (Polyvinyl idene fluoride membrane) (Mi 1 ipore, USA). After reacting with anti-Myc antibody with the primary antibody, HRP-conjugated secondary antibody was attached to the primary antibody attached to the membrane, and this was confirmed by using ECL (Pierce chemical co, USA) (FIG. 3C).

As a result, as shown in Figs. 3a and 3b, the number of neurofibrillary fibers 160 kDaCneurof i lament 160 kDa, NF 160) co-cultured in the neuronal retinal explants co-cultured with Vaxl overexpressing C0S7 cells control In contrast, the number of retinal ganglion cell axons growing toward co-cultured wild type C0S7 cells and Vax2 overexpressing C0S7 cells was the same. On the other hand, when the C0S7 cells expressing the transcriptionally inactivated Vaxl (R152S) mutant were co-cultured, the number of growing retinal ganglion cell axons increased, similar to the co-culture of wild-type (WT) Vaxl overexpressing C0S7 cells, In addition, by confirming that both Vaxl and Vaxl (R152S) proteins are also found in NF160-positive retinal ganglion cell axons from cocultured retinal fragments, retinal ganglion cell axon growth stimulation activation is specific for Vaxl and the Vaxl is a transcriptional non- It was confirmed to induce retinal ganglion cell axon growth dependently (FIGS. 3A and 3B).

In addition, as shown in Figure 3c, Vaxl and Vaxl (R152S) proteins are found in the growth medium (GM) of transformed C0S7 cells, while the Vax2 protein was confirmed by confirming that the Vaxl transcription Irrespective of activity, it was secreted out of cells to induce retinal ganglion cell axon growth (FIG. 3C).

<3-2> Confirmation of Vaxl Secretion in In Vitro and In Vitro Cells _r

In order to confirm Vaxl secretion in retinal ganglion cell axon growth induction, immunostaining and western blotting were performed using in vitro and in vivo cells. Specifically, after constructing a constructor encoding EGFP, EGFP tagged -Vaxl, EGFP tagged -Vax2, transformed into HEK293T cells (ATCC) as in Example <3-1>, and after 48 hours Growth medium was obtained, centrifuged and cells removed to obtain supernatant (S3) containing secreted protein and extracellular membrane vesicles. After the obtained supernatant was added to non-transformed C0S7 cells and incubated for 12 hours, HEK293T cells and C0S7 cells were mouse anti-GFP antibody (Santa Cruz Biotechnology) (Abstract) as in Example <2-1>. And DAPK blue) and immunostained and visualized (scale bar: 20 Hz) (FIG. 4A). In addition, MyC or Myc-Vaxl construct was transformed into HEK293T cells as in Example <3-1>, and the supernatant (S3) of the growth medium was obtained after 48 hours. The supernatant (S3) of the obtained growth medium was concentrated using a Centricon filters (cut-off limit 20 kDa; MiUipore) according to the manufacturer and the procedure, and the concentration including 50 // g protein. One supernatant 2 ^ was injected into the lateral ventricle of the in utero E13.5 mouse brain. After 12 hours, the brain was removed and brain sections (horizontal: 20 mi) were obtained as in Example <2-1>, and then immunostained and visualized using mouse anti-Myc antibody and DAP1ol (Fig. 4b).

In addition, after incubating for 24 hours the ventral hypothalamic fragments isolated from E13.5 mice obtained by the method described in Example <1-1>, the growth medium (GM) Pellets and cell lysates were obtained, and the obtained cell lysates and pellets were subjected to western blotting using anti-Vaxl antibodies and anti-tubulin-beta III antibodies (FIG. 4C). In addition, a cerebrospinal fluid (cerebrospinal fluid, CSF) is obtained from the lateral ventricle prepared from E14.5 mice, and the supernatant solution is obtained in the same manner as the method of obtaining a supernatant from the growth medium of Example <3-1>. S3) was isolated from dead cell debris (P2). The obtained supernatant (S3), dead cell debris (P2), and ventral hypothalamic explant cell lysate (CL) were subjected to anti-Vaxl and anti-leucine-beta III antibodies as in Example <3-1>. Western blotting was performed using (FIG. 4D). As a result, as shown in Figures 4a and 4b, the viability of EGFP, EGFP-Vaxl, EGFP-Vax2 transformed HEK293T cells are similar to each other, Vaxl protein in the growth medium of Vaxl-transformed HEK293T cells is cultured in vitro ( in vi tro) The Vaxl protein was transformed by confirming that it was found not only in C0S7 cells (FIG. 4A, line 2) but also in in vivo ventral diencephal ic (vDC) (FIG. 4B, line 2). It was confirmed that they migrate to secreted and cocultured retinal ganglion cell axons from HEK293T cells (FIGS. 4A and 4B).

In addition, as shown in FIGS. 4C and 4D, the expression level of intracellular Vaxl expressed in the ventral hypothalamic fragment was similar to the expression level of Vaxl (FIG. 3C) overexpressed in C0S7 cells, and the growth medium of the ventral hypothalamic fragment ( 4C, GM) and cerebrospinal fluid (CSF) were found (Fig. 3D, CSF; S3), it was confirmed that the Vaxl is a protein secreted from cultured cells as well as in vitro culture.

<3-3> Confirmation of Vaxl Secretion in In Vitro Cells

In order to confirm Vaxl secretion in retinal ganglion cell axon growth induction, immunostaining was performed using in vitro cells.

Myc-tagged mouse Vaxl was transformed into C0S7 cells by calcium phosphate method and co-cultured with neural retinal sections obtained by the method described in Example <1-1> for 24 hours. . Then, after co-culture for another 24 hours in a medium to which rb-IgG (l / g / m «) or anti -Vaxl antibody (-Vaxl, 1 / / m« added), the above <Example 2> Immunostained and visualized using anti-Myc antibody (green), anti-NF160 antibody (red) and DAP1 (blue) as shown in the box on the left is shown on the right and the arrow is labeled Myc- on the retinal ganglion cell axon. Indicates where tagged mouse Vaxl is present (scale bar: 500 (left), scale bar: 100 (right)).

As a result, as shown in FIG. 4E, the axon Vaxl immunostaining signal was significantly reduced in the presence of anti-Vaxl antibody (α-Vaxl) (FIG. 4E).

Therefore, through the results of the above <Example 3> Vaxl regardless of the transcriptional activity The protein was secreted from the ventral hypothalamus.

Example 4 Confirmation of Retinal Ganglion Cell Axon Growth Stimulation by Secreted Vaxl Protein

<4-1> Retinal ganglion cell axon growth induction induced by Vaxl secreted from ventral hypothalamic section

To determine whether the secreted Vaxl protein can induce retinal ganglion cell axon growth towards the ventral hypothalamic fragment, extracellular Vax protein was isolated using a rabbit anti-Vaxl polyclonal antibody and an anti-Vax2 polyclonal antibody. Immunostaining, directionality of RGC axon growth and RGC axon length measurements were performed.

Specifically, preimmune rabbit IgG (rblgG) l obtained in the ventral hypothalamic explants and neural retinal explants obtained by the method described in Example <1-1>.

After treatment with anti-Vaxl polyclonal antibody (a -Vaxl) 1 g / m or anti -Vax2 polyclonal antibody (a -Vax2) and incubated for 48 hours, the rabbit anti-Valk as in Example <2-2> Immunostained and visualized using -Vaxl antibody (green) and anti-NF160 antibody (red). The centers of the two sections were connected by red dotted lines and the arrows were enlarged (scale bar: 500) (FIG. 5A). ^"Further, the direction of the retinal ganglion cell axons by measuring the angle with respect to the red dotted line as shown in Example <2-2>, were graphed. The values in the graph were averaged and the error bars were obtained as SD (rbIgG (n = 17), a -Vaxl (n = 19), a -Vax2 (n = ll)). The p-value was determined by AN0VA (0.001 <p <0.005) (FIG. 5B).

In addition, pixels (pixe l) of the NF160 immunofluorescence image were calculated to graph the relative number and thickness of axons / bundles emerging from the retina. After converting the NF160 immunofluorescent image to a binary image, the number of pixels was measured using the histogram function of Image-J software. The relative number of axons / bundles is shown by subtracting the number of pixels inside the neural retinal segment from the total number of segments including the axons / bundles. The thickness is represented by measuring the number of pixels between 3 and 40 rn in one axon / bundle each. Graph The values were averaged, the rblgG treatment group was set to 100%, and the error bars were obtained with SD (FIG. 5C).

In addition, after culturing the retinal fragments obtained by the method described in Example <1-1> for 24 hours, peptide (peptide) labeled with fluorescein isothiocyanate (FITC) on 6X-His. ) LOO ng / ml) or His-tagged Vaxl was incubated for 24 hours with the addition of FITC-labeled recombinant protein (500 ng / ml). During each of these culture periods, neuronal axons growing in culture retinal sections and FITC fluorescence signals present in these neuronal axons were detected and visualized. Red arrows show magnified images (FIG. 5 (1). After washing the retinal sections with PBS, the rabbit a-Vaxl (green) and anti-His antibodies (by the method described in Example <2-1> above) Immunostained in red), the right box shows the enlarged image portion on the left, and the arrow shows the location of Vaxl and His proteins in retinal ganglion cell axons (FIG. 5E) (scale bar: 100 μηύ. In the quadrant of the retinal fragment obtained by the method described in Example <1-1>, 6X-His peptide (25 ng / ml, white bar) or Vaxl-His protein (100 ng / ml, black bar) was used. Axons lengths changed 24 hours after treatment were plotted and graphed, the values in the graph averaged and the error bars obtained as SD, the number of bars used is the number of axons analyzed and the number of analyte fragments. Is shown at the top P-value was obtained by t-test (** ， θ.001). The results obtained in two independent experiments (Fig. 5f).

In addition, His-tagged Vax2 (100 ng / ml) was added to the retinal fragments obtained by the method described in Example <1-1>, or after culturing for 24 hours without the above Example < Visualization was performed by immunostaining with rabbit anti-Vax2 antibody (green), mouse anti-NF160 antibody (red) and DAPI (blue) by the method described in 2-1>. The upper box represents the lower magnified image portion, and the arrow indicates the location of His-tagged Vax2 on the retinal ganglion cell axon (scale bar: 500 μπι (top), scale bar:: LOO ni (bottom) (Fig. 5g-A), and its axon length was measured and plotted, the values of the graphs being averaged and the error bars calculated as SD. The number of rods is the number of axons analyzed, and the number of fragments analyzed is 6X-His (n = 5) and Vax2 (n = 4). The p-value was obtained by t-test (**, pO.001) (FIG. 5G -B).

As a result, as shown in FIGS. 5A and 5B, unlike the treatment with the rabbit IgG and the anti-Vax2 polyclonal antibody, when treated with the anti-Vaxl polyclonal antibody, Vaxl was not found in the retinal ganglion cell axon ( Figure 5a), confirming the reduction of retinal ganglion cell axons directed to the ventral hypothalamic explants (Figure 5b), inhibiting the migration of Vaxl to retinal ganglion cell axons and growth of axons under the influence of anti-Vaxl polyclonal antibodies. Confirmed.

In addition, as shown in Figure 5c, when the rabbit IgG and anti-Vax2 polyclonal antibody treatment, the number and thickness of retinal ganglion cell axons / bundles are similar, whereas when treated with anti-Vaxl polyclonal antibody axons / bundle By confirming the decrease in the number and thickness of the cells, it was confirmed that extracellular Vaxl plays an important role in the axon fibrillation (Fig. 5c).

In addition, as shown in Figures 5d to 5f, when the FITC-labeled recombinant protein was added to Hi x tagged Vaxl, it was confirmed that exhibits a strong stimulating effect on retinal ganglion cell axon and growth (Fig. 5d to Fig. 5). 5e), it was confirmed that the same appears in the quadrant of the retinal section (Fig. 5ί).

In addition, as shown in Figure 5g, in the case of Vax2 was confirmed to induce the growth of retinal ganglion cell axons almost the same as Vaxl (Fig. 5g).

<4-2> Retinal Ganglion Cell Axon Growth Stimulation by Extracellular Vaxl in Vitro Culture

To determine the role of extracellular Vaxl in retinal ganglion cell axon growth in in vivo, immunostaining, thickness and length measurement of retinal ganglion cell axons in mice implanted with collagen gel lagen gel Was performed.

Specifically, in order to label retinal ganglion cell axons as shown in FIG. 6A (above), a lipophilic fluorescent dye (l ipophi lic f luorescent) was applied to the left eye of an E13.5 mouse. dye) After injecting Dil (25 μM), the dorsal half of the brain and the lower part of the mouth are cut and transferred to the culture slide cambers with the dorsal side down. And mouse brain slabs including midbrain structures. Next, a collagen solution containing non-immune rabbit IgG (rblgG) 1 jg / n or rabbit anti-Vaxl polyclonal antibody (a -Vaxl) 1 / «/ ι was implanted into the third ventricle of the slab and filled with culture medium. Incubated for 12 hours at 37 ^° C, 7% C02 incubator with humidity. Thereafter, brain slabs were cut as in Example <2-1> to obtain brain sections (horizontal cut: 12; I), fixed with 4% PFA / PBS, and frozen in OCT medium. The frozen brain sections were visualized by Olympus FV1000 confocal microscopy of the surface fluorescence (epifluorescence) of the DU to confirm Dil-labeled axon growth in the ventral hypothalamus (Figure 1, row 1). In addition, the brain sections containing the frozen optic nerve cross were immunostained and visualized using anti-Vaxl antibody (green) and anti-NF160 antibody (red) as in Example <2-1> (scale bar: 200 m) (2 rows below FIG. 6). In addition, after incubating the retinal sections obtained by the method described in Example <1-1> for 24 hours, Vaxl- was treated with or without the rabbit anti-Vaxl polyclonal antibody of 1 / g / ni. His recombinant protein 0.1 «/ was treated and further incubated for 24 hours. Then, after 24 hours, immunostaining and visualization using anti—Vaxl antibody (green), anti-NF160 antibody (red), and DAPI (blue) as in Example <2-1>, and magnified the arrow area. (Scale bar: 500 / πι) (FIG. 6B). In addition, the change in the length of retinal ganglion cell axons in real time, 24 hours before immunostaining was video taken and graphed (Fig. 6c). In addition, the number, thickness, and length change of retinal ganglion cell axons / bundles for 24 hours after immunostaining were graphed as in Example <4-1> (Fig. 6 (1). Error bars were obtained in SD (no treatment (n = ll), Vaxl (n = 10), Vaxl + a -Vaxl (n = 12)).

As a result, as shown in Fig. 6A, compared with the case of non-immune IgG treatment, mice transplanted with anti-Vaxl polyclonal antibody reduced retinal ganglion cell axons directed to the optic nerve crossing (Figs. 6A, 1, 1). DU-labeled in dark areas ^"Retinal ganglion cell axons), wherein eu Vaxl implant times in his mouse, to show a large number of retinal ganglion cells Vaxl immune banung (immunoreact ivi ty) the axonal reduction similar to that found in Vaxl mice stop in the outer wall of the ventral diencephalon It was confirmed that there is (Fig. 6a, line 2).

In addition, as shown in Figure 6b to 6d, the addition of recombinant Vaxl protein to the growth medium increases the number, thickness and length of retinal ganglion cell axons, while adding the recombinant Vaxl protein and anti-Vaxl polyclonal antibody together In this case, anti-Vaxl antibodies co-cultured with recombinant Vaxl protein antagonized each other in axon position and axon growth. It was confirmed that the recombinant Vaxl protein exhibits a strong retinal ganglion cell axon growth stimulation effect (Figs. 6B to 6D). Therefore, the results of Example 4 confirmed that extracellular Vaxl secreted out of the cells directly stimulate the growth of retinal ganglion cell axons.

Example 5 Confirmation of Intercellular Transfer of Vaxl Protein In Vivo

Vaxl mRNA was detected in optic nerve discs (0D), eye disease (OS), and E14.5 mice. It has been reported to be expressed in retinal ganglion cell axon-related structures, including the preoptic area (POA) and the ventral hypothalamus (vHT) (Bertuzzi et al., 1999; Hal lonet et al., 1999). Therefore, in order to confirm that Vaxl mRNA and protein are also expressed in the retina, in situ RNA hybridization, immunostaining and qRT-PCR were performed.

Specifically, retinal [33P] -CTP-labeled antisense Vaxl probes prepared from Vaxl + / + (WT) and VaxllacZ / lacZ E14.5 mice obtained by the method described in Example <1-1>. (probe) using Mui et al. In vitro RNA localization was performed by the method described in 2005, to visualize Vaxl mRNA expression (scale bar: 200 / m) (FIG. 7A).

Further, E14.5 VaxllacZ / + obtained by the method described in Example <1-1> and Brain sections were obtained from VaxllacZ / lacZ mice as in Example <2-1>, rabbit anti-Vaxl antibodies (green), mouse anti-beta-galactosidase antibody (DSHB) (red) ) And DAPI (blue) and immunostained and visualized. i and iii visualized neural retina (NR), ii and iv visualized eye disease (OS) (scale bar: 200 1 (columns 1 and 2)), scale bar: 20 1 (columns 3 and 4) ) (FIG. 7B).

Furthermore, brain sections were obtained from Vaxl + / + and VaxllacZ / lacZ E14.5 mice obtained by the method described in Example <1-1> as in Example <2-1>, and the rabbit anti-Vaxl antibody ( Abstract), immunostained and visualized using mouse anti-NF160 antibody (red) and DAPK blue). i and iii visualized neural retina (NR), ii and iv visualized eye disease (OS) (scalebar: 200 1 (column 1), scalebar: 20 (columns 2 and 3)) (FIG. 7C) ).

Further, brain sections were obtained from Vaxl + / + (WT) or VaxllacZ / lacZ E18.5 mice obtained by the method described in Example <1-1>, as in Example <2-1>, and the retina (stomach) ) And optic nerve (0N) (bottom) were immunostained with rabbit anti-Vaxl antibody and gold (250 nm) -labeled anti rabbit IgG and visualized by electron microscopy (EM). (Scale bar: 0.5 (column 1), scale bar: 0.2 1 (columns 2 and 3)) (FIG. 7D).

[33P] -CTP-labeled antisense Vaxl probes in brain sections prepared from Vaxl + / + (WT) and Vaxl − / − E14.5 mice obtained by the method described in Example <1-1> above. (probe) using Mui et al. , RNA in situ was performed by the method described in 2005 to visualize Vaxl mR A expression, and immunostained with anti-Vaxl antibody to visualize it (scale bar: 50 Hz) (FIG. 7E-A). A brain section was obtained from E14.5 Vaxl + / lacZ and VaxllacZ / lacZ mice obtained by the method described in <1-1> as in Example <2x1>, and the rabbit anti-Vaxl antibody (green) _' and the mouse were obtained. Immunostained and visualized using anti-beta-galactosidase antibody (DSHB) (red). The left box shows the magnified image position on the right (scale bar: 50; wm) (FIG. 7E-B). As a result, as shown in FIG. 7A, Vaxl mRNA was expressed in the optic nerve disc (0D), the eye disease (OS), the optic cross section (P0A), and the ventral hypothalamus, but not in the retina. By confirming that the Vaxl was expressed in the retinal ganglion cell axon-related structure including the optic disc disc and the optic nerve cross-section except the retina (FIG. 7A).

In addition, as shown in FIG. 7B, Vaxl protein is expressed in retinal cells of VaxllacZ / + mice, and Vaxl and beta-galactosidase are simultaneously expressed in ophthalmic (OS) astrocyte precursor cel ls (APCs). However, Vaxl protein is mainly present in the nuclei (Fig. 7b, line 1), and Vaxl is perfect in retinal ganglion cells and ocular astrocytoblasts (OS APCs) of VaxllacZ / lacZ heterozygous knock-in mice. By confirming that it is not expressed (Fig. 7b, the bottom row), it was confirmed that the Vaxl is specific for retinal ganglion cells of VaxllacZ / + mice (Fig. 7b).

. In addition, as shown in FIG. 7C, Vaxl and NF160 proteins were simultaneously expressed in the nucleus of Vaxl + / + mice with astrocytosis i (FIG. 7C, line 1), whereas NF160 was present in the ocular and neural retinas of VaxllacZ / lacZ mice. By confirming that the protein is expressed but not the Vaxl protein (FIG. 7C, line 2), the Vaxl is present in the retinal ganglion cell axon of Vax + / + mouse optic nerve, but in defibrated VaxllacZ / lacZ mouse retinal ganglion cell axon. It was confirmed that it did not appear (FIG. 7C).

In addition, as shown in FIG. 7D, the Vaxl protein binds to the extracellular surface of the retinal ganglion cell plasma membrane and is present in endocytic vesicles (FIG. 7D, Line 1), eye blast glial progenitor cells (OP APC) were found to exist with transport vesicles and chromatin in the nucleus (Fig. 7D, line 2) (Fig. 7D). In addition, as shown in FIG. 7E, mRNA of Vaxl is expressed in the hypothalamus cell code, optic nerve, and optic nerve cross-section, and VaxllacZ / lacZ _. In mice, it was confirmed that it is not expressed in the brain (FIG. 7E). Therefore, the results confirmed that Vaxl is expressed in the retinal ganglion cell axon-related structures other than the retina, and Vaxl protein secreted from the ophthalmic or ventral hypothalamus seats on the retinal ganglion cell axon and moves into retinal ganglion cells. Example 6 Confirmation of Vaxl Intercellular Migration Control by Heparan Sulfate Proteoglycan (HSPG)

Intercellular migration has been reported in homeodomain transcr ipt ion factors such as En2 (engrai Ied-2) and 0tx2 (orthodent icle homeodomain 2) (Brunet et al., 2007; Brunei et al., 2007). 2005; Sugiyama et al., 2008), the regulatory mechanisms for the transport of homeodomain transcription factors are not well known. We also found syndecan (Sdc), a transmembrane heparan sulfate proteoglycan (HSPG) protein, as a gene encoding protein capable of modifying intracellular transport of Vaxl in Drosophila (Spr ing et al., 1994). . Thus, the arc flies that the movement control of the inter-cell regulatory mechanisms and Vaxl of moving between cells of the Vaxl Meo domain transcription factor in order to ensure that the new decane ^'involved, expressing Ptc-induced Gal4- Vaxl-EGFP and Ds-Red ， Drosophila expressing Ptc-Gal4-induced Vaxl-EGFP, Ds-Red and Sdc or Sdc mutants (sdc23), and Ptc-Gal4-induced Vaxl-EGFP, Ds-Red and heparan sulfate proteoglycan glypican (glypican, Glp Immunostaining was performed using a Drosophila expressing Dlp (dal ly l ike protein), a Drosophila homology protein.

Specifically, Pt c-ga 14> Vaxl-EGFP, Ds-Red Drosophila, Ptc-gal4> Vaxl-EGFP, Ds-Red, Sdc23 Drosophila, Pt c- obtained by the method described in Example <1-2> ga 14> Va-EGFP, Ds-Red, Sdc Drosophila, and Ptc-gal4> Vaxl-EGFP, Ds-Red, Dip Drosophila wing imaginal discs with 4% PFA / PBS In order to visualize extracellular Vaxl, the reaction was reacted with anti-Vaxl antibody diluted 1: 10 at 4 ^° C for 10 minutes and washed three times with PBS. The phases were then reacted with Cy5-conjugated secondary antibodies diluted 1: 1000 for 1 hour at, followed by Ptc—Gal4-induced green fluorescence signal and Ds-Red protein of extracellular Vaxl, EGFP and Vaxl-EGFP proteins. Red Fluorescence signals were visualized by confocal microscopy (Olympus FV1000). As a result, as shown in Fig. 8, the number of cells expressing only Vaxl-EGFP was increased in front of the wing plate of the Sdc mutant-expressing Drosophila, while sdc (Figs. 8 and 3) and Dip (Figs. 8 and 4) Expression of Drosophila in front of the wings of the Drosophila is confirmed that the number of Vaxl-EGFP-expressed cells decreases, so that the overexpressed syndecan (Sdc) in the superpara wing membrane membrane is co-expressed with the Vaxl protein, which is dispersed in neighboring cells. It was confirmed to interfere. Thus, these results confirm that the Vaxl protein migrates to neighboring cells in the Drosophila wing lamellar plate, similar to the mammalian system, and the intracellular migration of Vaxl is mediated by heparan sulfate proteoglycans (HSPGs) (FIG. 8). .

Example 7 Confirmation of Vaxl and Hafaran Sulfate Dependent Hafaran Sulfate Proteoglycans

<7-1> Confirmation of Vaxl and Haparan Sulfate Proteoglycans

In order to confirm the role of syndecane in the movement of Vaxl, immunoprecipi tat ion and Western blot ¾ were performed.

Specifically, optic nerves prepared from post-natal day 0, P0 mice were treated with 10 mM Tr i s-HCl (pH 7.4), 200 mM NaCl, 1% Tr i ton X-100, and 1% NP— Dissolve with lysis buffer containing 40. Cell lysates were centrifuged at 12,000 × g for 10 minutes at 4 ° C. to obtain supernatant. The obtained supernatant was treated with anti-Vaxl and anti-Sdc2 antibodies (Santa Cruz Biotechnology) and reacted at 4 ° C. for 16 hours, followed by addition of Protein A-agarose bead (4 ° C.). The reaction was further precipitated for 16 hours at. After washing 5 times with lysis buffer and adding 2 X SDS sample buffer, the reaction was terminated, followed by Western antibody using anti-Sdc2 and anti-Vaxl antibodies as the primary antibody as in Example <3-1>. Blotting was performed (FIG. 9A). The negative control group was precipitated with rabbit IgG (rblgG) and Western blot was performed as above.

Also, constructs encrypting Vaxl, Vax2, GFP-tagged Sdcl and Sdc2 After construction, Vaxl, and GFP—Sdcl or GFP-Sdc2 were transformed with HEK293T cells as shown in Example <3-1> (FIG. 9B), and Vax2, and GFP-Sdcl or GFP-Sdc2 were transformed. (FIG. 9C). After 2 days, the cells were lysed as described above to obtain supernatant, immunoprecipitated with anti-GFP antibody, and then the anti-Vaxl antibody (Fig. 9b) or anti Western blotting was performed with -Vax2 antibody (FIG. 9C) (FIGS. 9B and 9C).

As a result, as shown in Figures 9a to 9c, Sdc2 is precipitated by the Vaxl antibody in the E14.5 mouse optic nerve, Vaxl is precipitated by the Sdc2 antibody (Fig. 9a), Vaxl by GFP antibody in HE 293T cells While sedimenting (FIG. 9B), Vax2 did not settle (FIG. 9C), the Vaxl was found in the E14.5 mouse optic nerve as well as in the synthecan l (Sdcl) and synthecan 2 (Sdc2) overexpressed in HEK293T cells. It was confirmed that it interacts with syndecane 2 (FIGS. 9A-9C).

<7-2> Synthecan 2 binding site of Vaxl

Immunoprecipitation and Western methods using Sdc2-C lacking the N-terminal extracellular domain and Sdc2-N lacking the C-terminal cytoplasmic domain to identify sites of syndecan 2 (Sdc2) that interact with Vaxl Blotting was performed.

Specifically, after constructing a construct encoding GFP-tagged Sdc2-N or Sdc2-C, Vaxl, and GFP-Sdc2-N or Sdc2 were used as HEK293T cells as in Example <3-1>. -C constructs were transformed. Then the above embodiment

After lysing the cells to obtain a supernatant of the cell lysate as shown in <7-1>, immunoprecipitating with an anti-GFP antibody was performed, and then the anti-Vaxl antibody was used as the primary antibody as in Example <3-1>. Western blotting was performed using FIG. 9D.

As a result, as shown in FIG. 9D, when GFP-Sdc2-N was overexpressed, Vaxl was precipitated by the GFP antibody, but when GFP-Sdc2-C was overexpressed, Vaxl was not precipitated by the GFP antibody. By doing so, it was confirmed that Vaxl binds to the extracellular domain of syndecane (FIG. 9D). <7-3> Confirmation of Vaxl binding to the side of blue sulfate

The extracellular domain of syndecane is primarily modified by heparan sulfate sugars (Bishop et al., 2007), and thus Vaxl can interact with sugar groups as well as the protein backbone of syndecane. Therefore, immunoprecipitation was performed to confirm that Vaxl binds to the heparan sulfate (HS) side chain of syndecane 2. Specifically, 1 mg of hepar in (Acros Organics) on the E14.5 mouse optic nerve. / me was treated or untreated, and precipitated with anti-Vaxl antibody as in Example <7-1>, and Western clear was performed using each antibody. The negative control group was precipitated with rabbit IgG (rblgG) and Western blot was performed as above. As the primary antibodies, anti-sdc2, anti-Sdc3 (santa cruz Biotechnology), anti-GlpKSanta Cruz Biotechnology, anti-3 2 (111 ^ 1 "1 ^ 0 ^), and anti-Vaxl antibodies were used (FIG. 9E). In addition, 1 g of Vaxl-Hi s protein was added at 4 ^° C with sapharose 4B resin coated with HS or Chondroi t in sul fate at a final concentration of 0.2 mg / m £. for a time and incubated Vaxl protein bound to the resin SDS sample ^• wherein in after processing the buffered solution example after <3 eu 1> electric by 10% SDS-PAGE as shown in the gel, primary antibody - used Vaxl antibody Western blotting was performed The relative intensities of the Vaxl bands were analyzed using image-J software (FIG. 9F).

As a result, as shown in Figure 9e, when heparin is not added, Sdc2, Sdc3 and Glpl is precipitated by the Vaxl antibody, whereas when heparin is added, the Sdc2, Sdc3, Glpl does not precipitate Thus, free heparin and Vaxl compete with heparan sulphate of proteoglancan, and heparan sulphate expressed in retinal ganglion cell axons, except for Pax2, where Vaxl complexes with Vaxl in ocular astrocytoblasts. It was confirmed that it interacts with proteoglycans (FIG. 9E).

In addition, as shown in Figure 9f, by confirming that the recombinant Vaxl protein binds to the blue sulfate-coated resin, the Vaxl is in the retinal ganglion cells It was confirmed that the heparan sulfate side chains of the expressed heparan sulfate proteoglycan protein (FIG. 9F). Therefore, the results of <Example 7> confirmed that Vaxl specifically binds to the extracellular domain of heparan sulfate proteoglycans, in particular, the heparan sulfate side chain of heparan sulfate proteoglycan.

Example 8 Confirmation of Neural Retinal Cell Axon Growth Stimulation by Interaction of Vaxl and Heparan Sulfate Proteoglycans

<8-1> Confirmation of retinal ganglion cell axon growth stimulation by Vaxl bound to heparan sulfate proteoglycan

The effect of Vaxl bound to heparan sulfate proteoglycan on the retinal ganglion cell axon growth stimulation activity, and the effect of Vaxl on the retinal ganglion cell axon growth stimulation was homologous to the motif in 0tx2 as shown in Table 1 below. To determine if it is mediated by sugar binding mot if, .Vaxl and jellyparnase I (chonparit inase ABC, ChnaseABC), biotin in labeled Immunostaining was performed using neural retinal sections treated with Vaxl KR-peptide or M-Mo peptide, and Vaxl (KR / AA) His mutant protein.

Specifically, heparinase Khepar inase D (Sigma) 2.5 U / m £ or chondroitin (not shown) that cuts the heparin and heparan sulfate sugar chains into the neural retinal fragments obtained by the method described in Example <1-1>. chondroi t inase ABC (Chondroi t inase ABC, ChnaseABC) (Sigma) 2.5 U / iM treated for 3 hours to remove sugar chains, and then added Vaxl-His recombinant protein 0.1 / g / me Reaction for 24 hours. Then, to confirm the presence of Vaxl in retinal ganglion cell axons, immunostaining and visualization using anti-Vaxl antibody (green) and anti-NF160 antibody (red) as in Example <2-1>. Arrows are enlarged areas (scale bar: 500) (FIG. 10A).

In addition, precultured for 24 hours obtained by the method described in Example <1-1> After measuring the axon length of the neural retinal fragment as in Example <4 ᅳ 1>, homologous to the binding motif of 0x2 sugar recombinant 0x2 and recombinant Vaxl-His 0.1 g / n in the neural retinal fragment as shown in Table 1 below. Biotin labeled Vaxl KR-peptides (KR-bio; 100 ng / nd) encoding Vaxlamino acids SEQ ID NOs: 101 to 112, Vaxl-His (0.1 μg / mSί) and two important sugar binding residues 101 M-bio peptide (100 ng / l) substituted with alanine (Ala-Ala (AA)) for lysine and 102-arginine (Lysl01-Argl02 (KR)), or Ala-Ala (AA) for Lysl01-Argl02 (KR) Vaxl (KR / AA) -His mutant protein ((hi / i KAnyGen) substituted with) was reacted for 24 hours. The length of the axon was then re-measured as in Example <4-1>. After fixing with 4% PFA / PBS, immunostained and visualized with anti-Vaxl antibody (green) and anti-NF160 antibody (red) as shown in Example <2-1> (Fig. 10B). part The expressed (Scale bar: 500 ηύ.

Table 1

In addition, the retinal ganglion cell axon length during the 24-hour incubation of the heparinase I, chondroitinase ABC, KR-bio peptide, M-bio peptide and Vaxl (KR / M) mutant protein. The change of was measured and graphed as in Example <4-1>. Values were averaged and error bars were analyzed by SD (Vaxl (n = 7), Vaxl + Hepar inasel (n = 6), Vaxl + ChnaseABC (n = 6), Vaxl + RK-bio (n = 6). ), Vaxl + AA-bio (n = 6), Vaxl (KR / AA) (n = 5)). P-values were obtained by student t-test (*, ρ <0.0ΐ; **, ρ <0.001) (FIG. 10C).

As a result, as shown in FIGS. 10A and 10C, the neuroretinal explants treated with chondroitinase ABC were significantly reduced. Heparan sulphate proteoglycan by Heparinase I was confirmed by inhibiting retinal ganglion cell axon growth in heparinase I-treated neural retinal fragments and decreasing axon immunostaining signals. Vascular sugar chains were cleaved to prevent Vaxl from binding to the sulphated sulfate, thus inhibiting growth stimulation of retinal ganglion cell axons (FIGS. 10A and 10C).

In addition, as shown in Figure 10b and 10c, when treated with R-bio peptide or Vaxl (KR / AA) mutant protein, growth of retinal ganglion cell axons is inhibited, while AA-bio peptide treatment, Vaxl only It was confirmed that axons grow similarly to the case of treatment (FIGS. 10B and 10C).

<8-2> Confirmation of retinal ganglion cell axon growth stimulation by binding of Vaxl sugar binding motif and heparan sulfate proteoglycan

To determine the effect of Vaxl (KR / AA) mutant protein on retinal ganglion cell axon growth, immunoprecipitation, Western blotting and immunostaining were performed.

Specifically, after constructing a construct encoding V5-tagged Vaxl or Vaxl (KR / AA), transformed into HEK293T cells as in Example <3-1>, heparin 10 mg for 3 hours After culturing / m treated, cells were obtained and lysed as in Example <3-1>, and the growth medium was centrifuged to obtain a supernatant (S3). The obtained supernatant was mixed with the same amount of 3 M trichloroacetic acid (TCA; 20% final concentration) solution to precipitate macromolecules, washed three times with cold acetone, and dried to obtain pellets. The obtained cell lysate and pellets were subjected to Western blotting using anti—V5 antibody (GenWay Biotech) as the primary antibody as in Example <2-1> (FIG. Lla, above), and relative to the Vaxl band. Intensities were graphed using Image-J software (FIG. Lla, bottom).

In addition, GFP-Sdc2, and Myc-Vaxl or Myc-Vaxl (KR / AA) constructs were transformed into HEK293T cells as in Example <3-1>. Then, the cells were lysed as in Example <7-1> to obtain a supernatant of the cell lysate, followed by immunoprecipitation with an anti-Vaxl antibody, followed by the primary antibody as in Example <3-1>. Anti-GFP antibodies Western blotting was performed using (FIG. Lib).

In addition, the Myc-Vaxl or Myc-Vaxl (KR / AA) construct was transformed into HEK293T cells as in Example <3-1>, and the supernatant (S3) of the cell growth medium was added to C0S7 cells. Incubated for 3 hours. The C0S7 cells were then _{immunostained} and visualized using anti-Myc antibody (green, cell surface), anti-Vaxl antibody (red) and DAPI (blue) (scale bar: 20) (FIG. 11C).

As a result, as shown in Figs. 11a to 11c, the amount of Vaxl and Vaxl (KR / M) precipitated in heparin-treated HEK293T cells is similar, but Vaxl (KR / AA) precipitated in heparin-untreated HE 293T cells. ) Is greater than Vaxl (FIG. 11A). In addition, Vaxl (KR / AA) and Sdc2 did not bind (FIG. Lib), Vaxl (KR / AA) did not move into cells (FIG. Lie), and the number of retinal ganglion cell axons growing in neural retinal sections was small. By confirming (10c), it was confirmed that the Vaxl (KR / AA) mutant protein binds less effectively to heparan sulfate proteoglycans than Vaxl and inhibits retinal ganglion cell axon growth induction (FIGS. 11A-11C and 10C). Therefore, the results of <Example 8> confirmed that the binding of Vaxl to the heparan sulfate proteoglycan is essential for inducing retinal ganglion cell axon growth.

Example 9 Growth Confirmation of Retinal Ganglion Cell Axons by Intracellular Vaxl Infiltration and Local Protein Synthesis Induction

<9-1> Confirmation of Retinal Ganglion Cell Axon Growth by Intracellular Vaxl Infiltration

Example 7 and Example 8 confirmed that extracellular Vaxl not only binds to heparan sulfate proteoglycans but also migrates into the cytoplasm of retinal ganglion cell axons. Thus, in order to determine whether Vaxl stimulates retinal ganglion cell axon growth by regulating cytoplasmic reaction after intracellular penetration or by activating a subsignal of heparan sulfate proteoglycan, it stimulates cell surface haemoran sulfate proteoglycan. Vaxl (WF / SR) mutations that can bind to but cannot penetrate target cells And immunoprecipitation and immunostaining were performed using the same.

Specifically, the amino acid residues of tryptophan (Trp) and 148 phenylalanine (PheXWR) amino acids having homology with the important residues of the GFP-Sdc2 and the cell-penetrating region of the antennapedia (antp) were identified as Ser-Arg (SR). Vaxl (WF / SR) mutant or Vaxl was substituted with HEK293T cells as in Example <3-1>, and then cell lysate was obtained. After immunoprecipitation of the supernatant of the cell lysate with anti-GFP as in Example <7-1>, Western blotting was performed using the anti-Vaxl antibody as the primary antibody as in Example <3-1>. Was performed (FIG. 12A).

In addition, the supernatant (S3) ol of HEK293T cell growth medium overexpressing Vaxl and vaxl (WFVSR) was added to C0S7 cells as in Example <3-1> and incubated for 3 hours. The C0S7 cells were then immunostained and visualized using anti-Myc antibody (green), anti-Vaxl antibody (red) and DAPI (blue) as in Example <3-1> (scale bar: 20 (FIG. 12B).

In addition, Myc-Vaxl (WT) or Vaxl (WF / SR) mutations were transformed into C0S7 cells as in Example <3-1>. Next, after co-culturing the neural retinal fragment obtained by the method described in Example <1-1> and the transformed C0S7 cells for 48 hours, the anti-Vaxl antibody as in Example <2-1>. (Abstract) and visualized by immunostaining with anti-NF160 antibody (red) (scale bar: 500 above), 200 (bottom)) (12c), the orientation was measured (FIG. 12D).

As a result, as shown in FIGS. 12A and 12B, both Vaxl and Vaxl (WF / SR) were precipitated by anti-GFP antibody (FIG. 12A), but not found in cells in the case of Vaxl (WF / SR). By confirming (FIG. 12B), the VaxKWF / SR) mutant was able to bind heparan sulfate proteoglycans but not penetrate into target cells (FIGS. 12A and 12B).

In addition, as shown in Fig. 12c and 12d, by confirming that the growth induction of retinal ganglion cell axons directed to C0S7 cells in the retinal explants co-cultured with Vaxl (WR / SR) overexpressing C0S7 cells is inhibited, the Vaxl Intracellular Infiltration of Vaxl for Intraretinal Ganglion Cell Axon Growth Confirmed required.

<9-2> Confirmation of Vaxl Protein Complex in Cytoplasm

In order to confirm the function of Vaxl in the cytoplasm, the GST-Vaxl protein complex was purified and silver stained, and the protein was identified by MALDI-TOF.

Specifically, after constructing a construct encoding GST and GST-Vaxl was transformed into HEK293T cells as in Example <3-1>. Then, the cell lysate was obtained by lysing the GST and GST-Vaxl overexpressed HEK293T cells. The obtained lysate was centrifuged at 12,000 × g at 4 ^° C. for 10 minutes to obtain cytoplasmic supernatant (S3), followed by glutathione Sepharose 4B resin at 4 ^° C. for 1 hour. Incubated together. It was then washed 5 times with lysis buffer and eluted with 2XSDS sample buffer. The eluted protein was electrophoresed and silver. Staining was performed using the staining kit (Thermo) according to the manufacturer's procedure. In addition, the cells were lysed with PBS solution containing 0.1% SDS and 1% Triton X-100, and a supernatant was obtained from the lysed cells as above and incubated with the resin. Then, washed twice with high salt buffer (10 mM Tris-HCl (H 7.4), 1 M NaCl, 1% Triton X-100 and 1% NP-40) and PBS, and 10 mM glutathione GST-conjugated protein was eluted with the included PBS solution. The samples were subjected to electrophoresis on 10% SDS-PAGE and stained with the Pierce Silver Stain Kit for Mass Spectrometry® (Pierce) following the manufacturer's procedure. The dyed bands were analyzed by MALDI-TOF mass spectrometry by the Korea Basic Science Institute.

As a result, as shown in Figure 13a, the main protein isolated by Vaxl affinity purification is ribosomal proteins (RPs) Lll, L23A, L26, S14 and S16, the translational regulator is a ribosomal component eukaryotic translation initiation factor (elF) 3B and 3C, which are translat ion regulators, and chaperon heat shock 70-KD protein 1A, HSPA1A. By confirming, Vaxl confirmed that cytoplasmic En2 is involved in local protein synthesis, a mechanism similar to the regulation of retinal ganglion cell axon growth (Brunet et al., 2005; Υοο ^η et al., 2012) (FIG. 13A . <9-3> Confirmation of Promoting Local Protein Synthesis by Vaxl Infiltrated by Retinal Ganglion Cell Axons

To determine whether Vaxl is involved in local protein synthesis, immunostaining was performed using Vaxl (WF / SR) mutations.

Specifically, the E13.5 mouse neural retinal sections obtained by the method described in Example <1-1> were cultured for 24 hours, and then transferred to a medium containing Vaxl or Vaxl (WF / SR) for further culture for 24 hours. It was. Then, 30 minutes after replacing the explant culture medium with methionine free medium, bioorthogonal noncanonical amino acid

50 μM of AHA (L-az i dohomoa 1 an i ne, Invi trogen) was added. After 6 hours, the neural retinal sections were washed twice with D-PBS containing \% FBS and darkened by the addition of 30 μΜ DIBO-Al exa Fluor (Invi trogen) in D-PBS containing 1% FBS. Incubated at room temperature for 1 hour. Then, washed 4 times with D-PBS containing FBS, incubated for 15 minutes at 4¾ PFA / D-PBS in silver, and then visualized the fluorescence of the AHA-Alexa488-containing ganglion cell axon (Fig. 13b, Line 2) and the protein synthesis in the cell body (Fig. 13b, line 3) were confirmed (scale bar: 500 m (line 1), scale bar: 100 // m (line 3)) ( 13b).

In addition, to confirm the effect on the nucleus in Vaxl-induced retinal ganglion cell axon growth, retinal nerve fragments were placed on a microscope, and the axons were cut around the cell body using an anatomical knife. After removing only the cell bodies, the axons were separated from the cell bodies, and 50 μM of AHA was added as above, followed by 6 hours of incubation, and the fluorescence of the proteins including AHA-Alexa488 was visualized to confirm the protein synthesis rate (scale bar: 500 (Fig. 13c), the length of the retinal ganglion cell axon grown for 6 hours as in Example <4-1> 0 *, pO.001) (FIG. 13D).

As a result, as shown in FIG. 13B, when WT recombinant Vaxl was expressed, the fluorescence density of Alexa Fluor 488′-labeled protein was remarkably increased in retinal ganglion cell axons (FIG. 13B, column 2). (WF / SR) mutations confirm that inhibition of densely synthesized protein density increases in retinal ganglion cell axons (FIG. 13B, column 3), which exogenous Vaxl penetrates axons and stimulates protein synthesis. It was confirmed (Fig. 13B).

In addition, as shown in FIGS. 13C and 13D, retinal axons isolated from cell bodies also increase the density of the synthesized protein when recombinant Vaxl is expressed (13c, 2nd row), and when the VaxKWF / SR) mutation is expressed. The density of the protein was confirmed to be similar to the control (Fig. 13C, column 3). In addition, the retinal axons isolated from the cell body increased axon growth by stimulation of Vaxl, but it was confirmed that VaxKWF / SR) mutant-expressed retinal: axons inhibited growth (FIG. 13D). Therefore, the results of Example 9 confirmed that Vaxl migrates into cells, promotes local protein synthesis, and stimulates retinal ganglion cell axon growth.

<Example 10> Confirmation of retinal ganglion cell axon regulation directed to midl ine by exogenous Vaxl protein

<10-1> Confirmation of growth recovery of damaged retinal ganglion cell axons by extracellular Vaxl protein

In order to confirm that retinal ganglion cell axons damaged in Vaxl − / − mice were recovered by extracellular Vaxl protein, immunostaining was performed using Vaxl recombinant protein and Vaxl (WF / SR) recombinant protein.

Specifically, 0.1 fg 6 x Hi s peptide, Vaxl ᅳ His or Vaxl (WF / SR) -Hi.s protein is mixed in the third ventricle of VaxllacZ / lacZ embryonic mouse brain slab as in Example <4-2>. After transplanting the collagen gel for 24 hours, the surface fluorescence of Di l was visualized and immunostained and visualized using anti-Vaxl antibody (green) and anti-NF160 antibody (red) (FIG. 14A_A). As in <4-2> 1 I Robo 1-Fc segment (12.35 pmol, R & D Systems) and 4.78 I 6X-Hi s peptide (5.69 nmol) or 200 I Vaxl-His (5.69 nmol) protein in the third ventricle of VaxllacZ / lacZ embryonic mouse brain sulfab After transplanting the mixed collagen gel for 12 hours, the upper fluorescence image was visualized with a photodetector, and the lower image was immunostained using anti-Vaxl antibody (green) and anti-NF160 antibody (red). Visualization and fluorescence intensity of immunostained images were also measured and graphed using Image-J software. The values indicate the expression intensity of the samples treated with rb-IgG, the number on the graph is the number of brain slabs analyzed, the error bar was obtained by SD, and the P-value was obtained by AN0VA. <0.001) (FIGS. 14A-B).

As a result, as shown in FIG. 14A, when the Vaxl deficient mouse embryo and the Vaxl (WF / SR) mutant protein were transplanted, the retinal ganglion cell axon approached the ventral hypothalamus and axon growth was poor. A large number of retinal ganglion cell axons were found in the ventral hypothalamus of the Vaxl deficient Mau ^ embryo implanted with collagen gel containing Vaxl protein (+ Vaxl-Hi s). Meanwhile, by confirming that the transplantation of Vaxl and VaxKWF / SR) proteins did not affect the expression of Sl itl in the ventral latral diencephalon and the lack of EphB3 in the medial diencephalon, the Vaxl protein was axon Penetrating into axoplasm was confirmed to stimulate retinal ganglion cell axon growth independent of expression of axon inducing molecules. In addition, the growth of retinal ganglion cell axons in the ventral hypothalamus showed a more enhanced effect when transplanted with collagen gel containing Vaxl-His and Robo 1-Fc (FIG. 14A). <10-2> Confirmation of growth regulation of retinal ganglion cell axon by antagonism between extracellular Vaxl protein and Sl it2

In optic nerve crossing, retinal ganglion cell axon projections are often associated with spinal cord axons directed to the f loor plate (FP). Compared to the projection. Slitl, which is expressed in the medial spinal cord, prevents immature entry into the midline, grows in the ventral direction, and in the same way, the Slitl and ventral-lateral hepatic brain in the anterooptic zone (P0A) Prevent retinal ganglion cell axons from entering the brain before reaching the optic nerve crossing. Spinal cord axons, on the other hand, sense induced signals such as netrin and Shh secreted from the lamination. It has been reported that netrin and Shh attractive signals can compete with the repulsive signal of SHt2, which can be locally accumulated by heparan sulfate proteoglycans (Matsumoto et al., 2007; Wright et al. al., 2012). However, neither netrine nor Shh is required to induce retinal ganglion cell axons into the ventral hypothalamus (Deiner and Sretavan, 1999; Sanchez-Camacho and Bovolenta, 2008; data not shown). Therefore, the present inventors performed immunostaining and retinal ganglion cell axon length measurement by treating Vaxl and slit2 proteins to confirm the relationship between Vaxl and Slit2.

Specifically, after incubating the E13.5 mouse neural retinal sections obtained by the method described in Example <1-1> for 24 hours, Vaxl 0 ng / (left), 10 ng / ra £ (center) and Slit2-His (R & D Systems) 10 ng / ^ was treated at 100 ng / (right) concentration for 24 hours (FIG. 14B-A, top row). In addition, Vaxl-His 10 ng / m £ was treated for 24 hours under the concentrations of Slit2-His 0 ng / m £ (left), 10 ng / i (center) and 100 ng / (right) in contrast to the above (FIG. 14B_A , Bottom line). Then, it was visualized (FIG. 14B-A) and the length of retinal ganglion cell axon changed for 24 hours was graphed as in Example <4-1>. Error bars were obtained with SD, and the numbers on the graph indicate the number of axons analyzed (untreated group (n = 22), VaxKlOng / ml) (n = 10), Vaxl (100 ng / ml) (n = 9), Slit2 (10 ng / ml) (n = 5), Slit2 (100 ng / ml) (n = 5), VaxKlO ng / ml) + Sl it2 (10 ng / ml) (n = 7), VaxKlO ng / ml) + Sl it2 (100 ng / ml) (n = 6), VaxKlO ng / ml) + Sl it2 (10 ng / ml) (n = 13), and Vaxl (100 ng / ml) + SHt2 (10 ng / ml) (n = 7)) (FIG. 14B-B).

In addition, an E13.5 mouse obtained by the method described in Example <1-1>. 100 ng / ml Robo with Neural Retinal Explants and Vaxl-knockout Ventral Hypothalamic Explants

Incubate for 24 hours with or without 1-Fc (FIG. 14C-A) or without (FIG. 14C-B). Then it was visualized (left) or immunostained for visualization (right) (scale bar: 500) (FIG. 14C-A). In addition, the direction of retinal ganglion cell axons was counted and graphed using NF160 immunostaining image pixel, an axon marker, where + is forward, 0 was positive,-was reverse, and error bars were SD. The number on the axis indicates the number of intercepts analyzed.

P-value was calculated as AN0VA (ρθ.01) (FIG. 14C-B).

In addition, the ^'Example <1-1> to an E13.5 mouse neural retina fragment body obtained by the method described in for 24 hours after the culture, Vaxl 0 ng / m £ (left), 10 ng / in.e ( Middle) and 10 ng / of SI it2-His (R & D Systems) for 24 hours under 100 ng / m £ (right) concentration (FIG. 14D, bottom row). Also, contrary to the above

Slit2-His under concentrations of 0 ng / (left), 10 ng / ni £ (center) and 100 ng / m ^ right)

Vaxl-His 10 ng / m £ was treated for 24 hours (FIG. 14D, bottom row). This was then visualized (FIG. 14B-A) and fluorescence intensities were graphed using Image-J software. Error bars were obtained in SD, and the numbers on the graph represent the number of analyzed regions (scale bar: 20) (FIG. 14D). ^"

In addition, to the neural retinal fragment obtained by the method described in Example <1-1>.

Vaxl-HisdOng / n ^), and Vaxl-His (10 ng / ni £) and Slit2-His (10 ng / mO were treated and visualized as above, anti-Vaxl antibody (green) and anti-His antibody ( Red) to visualize by immunostaining as in Example <2-1> (scale bar: 500 ^ m (dark field), scale bar: 10 μπι (dashed box)) (FIG. Me).

In addition, after obtaining the cortical explants (cortical explants) as in Example <1-1>, after processing the Vaxl, Vaxl (R152S) and Vaxl (WF / SR) to the obtained cerebral explants, the implementation As shown in <3-1>, immunostaining and visualization were performed using anti-Vaxl antibody (green), anti-NF160 antibody (red) and DAPK blue) (FIG. 14F).

As a result, as shown in FIG. 14B, the growth of retinal ganglion cell axons was inhibited in the group treated with Slit2 rather than the group treated with Vaxl alone (VaxKlOng / π) and Comparison of Slit2 untreated images with VaxKlO ng / i) and Slit2 (10 ng / i) images, Sl2 2 treated with Vaxl rather than Sl 2 treated group to promote retinal ganglion cell axon growth (Slit2 (10 ng) / mi) and Vaxl untreated images were confirmed to be Slit2 (10 ng / ml) and Vaxl (10 ng / m image comparison) (Fig. 14b-A) Also, compared with the group treated with Vaxl alone Slit2 together By confirming that the length of the axons is generally reduced in the treated group (FIG. 14B-B), Vaxl and Slit2 compete with each other, and Vaxl antagonizes S1 2-induced retinal ganglion cell axon retraction response It was confirmed that (Fig. 14b).

In addition, as shown in Fig. 14c to 14d, when treated with a culture of Vaxl and Robo 1-Fc, it was confirmed that the growth of axon growth of retinal ganglion cells significantly (Fig. 14c), together with Vaxl and Slit2 Treated: The group was once again confirmed to compete with each other (FIG. 14D).

In addition, as shown in FIG. 14E, Vaxl penetrates into the retinal ganglion cell axon, but by confirming that slit2 is caught on the axon surface, the reciprocal antagonism of Vaxl and Slit2 is mediated by heparan sulfate proteoglycans. Not sure (FIG. 14E).

In addition, as shown in Figure 14f, when treated with Vaxl and Vaxl (R152S), by confirming that axon growth is induced in cerebral sections, Vaxl protein induces not only retinal ganglion cell axon but also cerebral nerve axon growth. Thus, it was confirmed that the midline passage of various nerve axons is also regulated by Vaxl protein similarly to retinal ganglion cell axons (FIG. 140.

<10-3> Retinal ganglion cell axon growth control model by secreted Vaxl protein The function of Vaxl as the axon growth factor secreted in the ventral hypothalamus of the mammalian brain through the results of <Example 2> to <Example 9> It was confirmed.

As shown in FIG. 14F, Vaxl is expressed in the ventral hypothalamus, radioglia and neural stem cells (NPCs), secreted out of the cells, forms a concentration gradient, and is present at high concentrations in the midline. Spread to the side. Vaxl binding to heparan sulphate proteoglycans (HSPGs), including the syndecan of retinal ganglion cell axons approaching the ventral hypothalamus, results in a locally increased concentration of Vaxl at the retinal ganglion cell growth site. Vaxl then penetrates into retinal ganglion cell axons to stimulate protein synthesis and thus; Promotes axon growth towards the midline. In addition, retinal ganglion cell axon-induced signals enriched in the ventral hypothalamus, such as induced VEGF164 ᅳ neuphyllin and inhibitory ephrinB2-EphBl, are retinal ganglion in the midline for optic nerve crossing formation. Determine the aroma of the cell axon. To Vaxl protein through the above. Retinal ganglion cell axon growth regulation model was completed.

Claims

【Scope of Claim】

【Claim 1】

A pharmaceutical composition for promoting nerve cell regeneration or nerve cell growth containing VaxCventral anter ior homeobox) protein as an active ingredient.

【Claim 2】

The nerve cell of claim 1, wherein the Vax protein is any one selected from the group consisting of a Vaxl protein having an amino acid sequence shown in SEQ ID NO: 1 or a Vax2 protein having an amino acid sequence shown in SEQ ID NO: 2. Pharmaceutical composition for promoting regeneration or nerve cell growth.

[Claim 3]

The method of claim 1, wherein the nerve cells are an optic nerve, cort ical commissural nerve, hippocampal commissural nerve, or pituitary chiasm that lack Vaxl protein. A pharmaceutical composition for promoting nerve cell regeneration or nerve cell growth, characterized in that it is selected from the group consisting of hypothalamic commissural nerve and trigeminal nerve.

【Claim ⁴ 】. _.

The pharmaceutical composition for promoting nerve cell regeneration or nerve cell growth according to claim 2, wherein the Vaxl protein binds to heparan sulfate proteoglycan (HSPG).

【Claim 5】

The method of claim 2, wherein the Vaxl protein binds to the extracellular domain of Syndecan-2 (Sdc2). Pharmaceutical composition for growth promotion.

【Claim 6】

A pharmaceutical composition for promoting nerve cell regeneration or nerve cell growth containing as an active ingredient a vector or cell containing a polynucleotide encoding the Vax protein.

【Claim 7】

The nerve of claim 6, wherein the Vax protein is any one selected from the group consisting of Vaxl protein having the amino acid sequence shown in SEQ ID NO: 1 or Vax2 protein having the amino acid sequence shown in SEQ ID NO: 2. Pharmaceutical composition for promoting cell regeneration or nerve cell growth.

【Claim 8】

The pharmaceutical composition for promoting nerve cell regeneration or nerve cell growth according to claim 6, wherein the vector is linear DNA, plasmid DNA, or a recombinant viral vector.

[Claim 9]

The method of claim 6, wherein the recombinant virus is any one selected from the group consisting of retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, and lentivirus. Pharmaceutical composition.

【Claim 10】

The pharmaceutical composition for promoting nerve cell regeneration or nerve cell growth according to claim 6, wherein the cells are any one selected from the group consisting of hematopoietic stem cells, dendritic cells, autologous tumor cells, and established tumor cells. .

【Claim 11】

1) treating cells obtained from a subject with a test substance and measuring nerve axon growth;

2) measuring the binding level of Vaxl and heparan sulfate proteoglycan in the cells of step 1);

3) A method for screening candidate substances for promoting nerve cell regeneration or nerve cell growth, comprising the step of selecting a test substance that increases the binding level of Vaxl' and heparan sulcitate proteoglycan in step 2) compared to the untreated control group.

【Claim 12】.

The method of claim 10, wherein the binding level is any one selected from the group consisting of immunofluorescence, mass spectrometry, protein chip, Western blotting, dot-blotting, and ELISA. Method for screening candidate substances for promoting regeneration or neuronal growth.

【Claim 13】

A method of nerve cell regeneration comprising administering a pharmaceutically effective amount of Vax protein to an individual with damaged nerve cells.

[Claim 14]

A method for promoting nerve cell growth comprising the step of administering a pharmaceutically effective amount of Vax protein to an individual with damaged nerve cells.

【Claim 15】

A method of nerve cell regeneration comprising the step of administering a pharmaceutically effective amount of a vector or cell containing a polynucleotide encoding the Vax protein to an individual with damaged nerve cells.

【Claim 16】

A method for promoting nerve cell growth comprising the step of administering a pharmaceutically effective amount of a vector or cell containing a polynucleotide encoding the Vax protein to an individual with damaged nerve cells. ：

【Claim 17】

Vax protein for use as a pharmaceutical composition for promoting nerve cell regeneration or nerve cell growth _. Purpose of query.

【Claim 18】 _.

Use of a vector or cell containing a polynucleotide encoding Vax protein for use as a pharmaceutical composition for promoting new cell regeneration or nerve cell growth.