KR20230065860A - Composition for prevention, improvement or treatment of diabetes-mediated Alzheimer's disease comprising a retromer stabilizer - Google Patents

Abstract

The present invention provides a composition for preventing, alleviating or treating diabetes-mediated neurodegenerative diseases including a retromer stabilizer, and use thereof. According to the present invention, it is confirmed that high concentration of glucose causes hyperphosphorylation of amyloid precursor protein (APP) and tau protein through VPS26a reduction-mediated retromer dysfunction, and the hyperphosphorylation can be inhibited when a retromer stabilizer is administered to diabetic model neurons or mice. Therefore, regulation of VPS26a and use of the retromer stabilizer can be applied to a method for preventing, alleviating, or treating diabetes-mediated neurodegenerative diseases or to a field of medicines related to neurodegenerative diseases.

Description

[0001] Composition for prevention, improvement or treatment of diabetes-mediated Alzheimer's disease comprising a retromer stabilizer}

The present invention provides a composition containing a retromer stabilizer for preventing, improving or treating diabetes-mediated neurodegenerative diseases and uses thereof.

Retromers play an important role in neurobiology as protein complexes involved in retrograde transport of cargo from endosomes to the trans-Golgi network or recycling of cargo from endosomes to the cell surface. Impairment of endosomal sorting by retromer dysfunction causes synaptic dysfunction, neuroinflammation, and major pathological features of Alzheimer's disease (AD). The retromeric complex is composed of a cargo recognition core (CRC) composed of a vacuolar protein sorting-associated protein 35 (VPS35)/VPS26/VPS29 trimeric complex and a sorting annexin (SNX). Microarray profiles showed that VPS35 is downregulated in the endocortex of Alzheimer's disease patients. VPS35 is known to directly interact with β-secretase in mouse pyramidal neurons and an early-onset AD-like phenotype can be induced by hemizygous deletion of VPS35 in the Tg2576 mouse model of AD. Downregulation of VPS35 in a mouse model of tauopathy exacerbated tau accumulation and cognitive impairment through modulation of cathepsin D (CTSD) availability. VPS35 is also decreased in the hippocampus of a mouse model of type 2 diabetes. In addition, whole-genome linkage studies identified sortilin-related VPS10 domain-containing receptor 1 (SORCS1), a retromer binding protein, as a key locus involved in the pathogenesis of type 1 and type 2 diabetes. Mice lacking one of the other retromeric binding components, Sorcs1 and the Wiskott-Aldrich syndrome protein and SCAR homologue (Wash), exhibited abnormal glycemic control.

Retromers have also been shown to be regulators in the pathogenesis of diabetes (DM), but no studies have been published that have investigated the role of retromer complexes in DM-mediated AD pathogenesis in APP processing and tau phosphorylation.

KR 10-2010-0105154 (2010-10-27)

As a result of intensive efforts to confirm the effect of high glucose on the retromer complex, the present inventors found that vacuolar protein sorting-related protein 26a (VPS26a) regulates amyloid precursor protein processing (APP processing) and tau phosphorylation, thereby preventing diabetes-mediated neurodegeneration. It was confirmed that the onset of the disease could be suppressed, and the present invention was completed.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing, improving or treating diabetes-mediated neurodegenerative diseases, including a retromer stabilizer.

In addition, another object of the present invention is a method for providing information for diagnosis of diabetes-mediated neurodegenerative diseases using the expression level of VPS26a (Vacuolar protein sorting-associated protein 26a) or a drug for preventing, improving or treating diabetes-mediated neurodegenerative diseases to provide a screening method.

The present invention provides a pharmaceutical composition for preventing, improving or treating diabetes-mediated neurodegenerative diseases, including a retromer stabilizer.

According to a preferred embodiment of the present invention, the retromer is VPS35 (Vacuolar protein sorting ortholog 35), VPS26a (Vacuolar protein sorting-associated protein 26a), VPS26b (Vacuolar protein sorting 26 homolog B), VPS29 (Vacuolar protein sorting 29) , SORCS1 (Sortilin Related VPS10 Domain Containing Receptor 1), SORL1 (Sortilin Related Receptor 1), and WASHC1 (WASH Complex Subunit 1).

According to a preferred embodiment of the present invention, the retromer stabilizer is at least one selected from the group consisting of R33 and R55.

According to a preferred embodiment of the present invention, the diabetes-mediated neurodegenerative disease is at least one selected from the group consisting of diabetes-mediated Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, stroke, and multiple sclerosis.

The present invention also provides an information providing method for diagnosing diabetes-mediated neurodegenerative diseases, comprising checking the expression level of VPS26a (Vacuolar protein sorting-associated protein 26a) in a sample obtained from the subject.

According to a preferred embodiment of the present invention, the information providing method further includes a step of classifying as a diabetes-mediated neurodegenerative disease if the confirmed expression level of VPS26a is lower than that of a normal sample.

According to a preferred embodiment of the present invention, the method for providing information is to check the levels of amyloid precursor protein (APP) and phosphorylated tau in addition to VPS26a.

According to a preferred embodiment of the present invention, if the levels of APP and phosphorylated tau are higher than normal samples, a step of classifying as a diabetes-mediated neurodegenerative disease is additionally included.

The present invention also includes i) treating a candidate drug to a sample obtained from a patient with diabetes-mediated neurodegenerative disease; and

ii) checking the expression level of VPS26a (Vacuolar protein sorting-associated protein 26a) in the sample treated with the drug;

It provides a drug screening method for the prevention, improvement or treatment of diabetes-mediated neurodegenerative diseases comprising a.

According to a preferred embodiment of the present invention, the screening method includes: iv) classifying as a drug for preventing, improving or treating diabetes-mediated neurodegenerative diseases if the expression level of VPS26a identified is higher than before drug treatment; is to additionally include.

According to a preferred embodiment of the present invention, in step ii), the levels of APP (amyloid precursor protein) and phosphorylated tau are checked together in addition to VPS26a.

According to a preferred embodiment of the present invention, if the level of the APP and phosphorylated tau is reduced than before drug treatment, it can be classified as a drug for preventing, improving or treating diabetes-mediated neurodegenerative diseases.

In the present invention, high concentration glucose induces hyperphosphorylation of amyloid precursor protein (APP) and tau protein through VPS26a reduction-mediated retromer dysfunction, and when a retromer stabilizer is administered to diabetic model neurons or mice, this It was confirmed that suppression was possible. Therefore, the use of VPS26a modulators and retromer stabilizers can be applied to methods for preventing, ameliorating or treating diabetes-mediated neurodegenerative diseases or pharmaceuticals related to neurodegenerative diseases.

Figure 1 shows a schematic diagram of the mechanism by which the decrease in VPS26a expression in high glucose induces dysregulation in amyloid precursor protein processing and tau hyperphosphorylation.
Figure 2 shows the animal's body weight and blood glucose level and in vivo flow chart. Blood glucose and body weight were measured at 9 and 18 weeks of age. Data are expressed as mean ± SD (two-way ANOVA with Tukey's multiple comparison test).
Figure 3 shows the characterization of iPSC-ND cells. iPSC-ND cells were immunostained with MAP2 and NeuN specific antibodies and counterstained with DAPI (scale bar = 20 μm).
Figure 4a shows that only the expression level of VPS26a among retromeric components decreased in the hippocampus of STZ-induced diabetic mice (n=5).
Figure 4b shows that only VPS26a expression levels among retromeric components decreased in the hippocampus of STZ-induced diabetic mice as a result of immunostaining tissue slides for IHC with VPS35, VPS26a, and VPS29-specific antibodies and counterstaining with DAPI (scale bar=20 μm, n=5).
Figure 4c shows the down-regulation of VPS26a as a result of treating iPSC-NDs with D-glucose (25 mM) for 24 hours (n = 5).
Figure 4d shows that treatment of SH-SY5Y with high glucose (25 mM) in a time-dependent manner and analysis by quantitative real-time PCR showed that mRNA and protein expression levels of VPS26a were reduced after 12 hours (n = 5).
Figure 4e shows that iPSC-NDs were treated with high glucose (25 mM) for 24 hours, immunostained with Rab5 and VPS35-specific antibodies, and counterstained with DAPI, reducing the degree of colocalization between Rab5 and VPS35, early endosome markers. (Scale bar = 20 μm, n = 6). *p < 0.05, **p < 0.01 vs control. Quantitative data are presented as mean ± SD. All blot and immunofluorescence images are representative.
Figure 5a shows that SH-SY5Y was treated with high glucose (25 mM) in a time-dependent manner, and VPS35, VPS26a, VPS26b, VPS29, SORCS1, SORL1, WASHC1, and β-actin were detected by Western blot. decrease in exposed SH-SY5Y (n = 5, one-way ANOVA using Dunnett's multiple comparison test).
Figure 5b shows the results of treatment with D-glucose (25 mM) or L-glucose (25 mM) in SH-SY5Y for 24 hours, and detection of VPS35, VPS26a, VPS29 and β-actin by Western blot (n = 5, One-way ANOVA with Dunnett's multiple comparison test).
Figure 5c shows the results of SH-SY5Y treated with high glucose (25 mM) for 24 hours immunostained with Rab5 and VPS35 specific antibodies and counterstained with DAPI (scale bar = 3 μm, n = 6). *p < 0.05, **p < 0.01 vs control. Quantitative data are presented as mean ± SD. All blot and immunofluorescence images are representative.
6A shows that STZ-induced diabetic mice have higher hippocampal DNMT1 levels compared to vehicle-treated mice (n = 5).
Figure 6b shows the results of immunostaining with an NF-κ-specific antibody and counterstaining with DAPI after cells were incubated with NAC (4 mM) for 30 minutes before high glucose treatment (25 mM) for 12 hours (n = 6, Scale bar = 3 μm).
Figure 6c shows the results of DNMT1 mRNA and protein expression levels analyzed by quantitative PCR and Western blot, respectively, after cells were treated with Bay 11-7082 (10 μM) for 30 minutes before high glucose treatment (25 mM) for 24 hours ( n = 5).
Figure 6d shows SH-SY5Y was transfected with NT siRNA or DNMT1 siRNA for 12 hours before high glucose treatment (25 mM) for 24 hours, and then the methylation status of -118 and -346 CpG regions of the VPS26A gene of gDNA was confirmed by MSP analysis indicates Relative methylation levels of VPS26A were plotted relative to unmethylated forms (n = 5).
Figure 6e shows the results of SH-SY5Y transfected with NT siRNA or DNMT1 siRNA for 12 hours before high glucose treatment (25 mM) for 24 hours, and the mRNA and protein expression levels of VPS26a were analyzed by quantitative PCR and Western blot, respectively. (n = 5). *p < 0.05, **p < 0.01 vs control, #p < 0.05, ##p < 0.01 vs high glucose. Quantitative data are presented as mean ± SD. All blot and immunofluorescence images are representative.
Figure 7a is a result of SH-SY5Y treated with high glucose (25 mM) in a time-dependent manner and then analyzed by quantitative PCR and Western blot. As a result, only the mRNA expression of DNMT1 was increased by high glucose, and the protein expression level of DNMT1 was also high. Shows a time-dependent increase upon glucose treatment (n = 5, one-way ANOVA using Dunnett's multiple comparison test).
Figure 7b shows that the ROS/NF-κ-mediated DNMT1 pathway induces DNA hypermethylation in high glucose. After cells were incubated with NAC (4 mM) for 30 minutes before high glucose treatment (25 mM) for 12 hours, NF-κα-tubulin and Lamin A/C were detected by Western blot in cytoplasmic and nuclear fraction samples (n = 5 ). *p < 0.05, **p < 0.01 vs control, #p < 0.05, ##p < 0.01 vs hyperglycemia. Quantitative data are presented as mean ± SD. All blot and immunofluorescence images are representative (two-way ANOVA with Tukey's multiple comparison test).
Figure 7c shows that cells were treated with Bay 11-7082 (10 μM) for 30 minutes before high glucose treatment (25 mM) for 24 hours, then immunostained with a DNMT1-specific antibody and counterstained with DAPI. As a result, increased by high glucose It shows that the protein level of DNMT1 reduced by Bay 11-7082 (Scale bar = 3 μm, n = 6).
Figure 7d shows SH-SY5Y was transfected with NT siRNA or DNMT1 siRNA for 12 hours before high glucose treatment (25 mM) for 24 hours, then immunostained with a 5mC-specific antibody and counterstained with DAPI, resulting in 5-mC staining A high glucose-mediated increase in intensity was reduced by DNMT1 silencing (scale bar = 5 μm, n = 6, two-way ANOVA with Tukey's multiple comparison test).
Figure 7e shows the result of detecting the expression level of 5-mC in cell gDNA by dot blot analysis after SH-SY5Y was transfected with NT siRNA or DNMT1 siRNA for 12 hours before high glucose treatment (25 mM) for 24 hours, The high glucose-mediated increase in 5-mC staining intensity was reduced by DNMT1 silencing (n = 5, two-way ANOVA with Tukey's multiple comparisons test).
Figure 7f shows SH-SY5Y was transfected with NT siRNA or DNMT1 siRNA for 12 hours before high glucose treatment (25 mM) for 24 hours, and then cells were immunostained with a VPS26a-specific antibody and counterstained with DAPI. Downregulated mRNA and protein expression levels of VPS26a in glucose are shown to be reversed by DNMT1 silencing (scale bar=3 μm, n=5, two-way ANOVA using Tukey's multiple comparisons test). *p < 0.05, **p < 0.01 vs control, #p < 0.05, ##p < 0.01 vs hyperglycemia. Quantitative data are presented as mean ± SD. All blot and immunofluorescence images are representative.
Figure 8a shows the results of Western blot analysis after cells were treated with R33 (5 μM) for 30 minutes before high glucose treatment (25 mM) for 24 hours (n = 5).
Figure 8b shows the results of immunostaining of iPSC-ND and SH-SY5Y with EEA1-specific antibody and counterstaining with DAPI after cells were treated with R33 (5 μM) for 30 minutes before high glucose treatment (25 mM) for 24 hours. (Scale bar = 20 μm). Average EEA1-puncta diameters were measured in independent experiments (n = 7).
Figure 8c shows the results of iPSC-ND and SH-SY5Y immunostained with an EEA1-specific antibody and counterstained with DAPI after cells were treated with R33 (5 μM) for 30 minutes before high glucose treatment (25 mM) for 24 hours. (Scale bar = 3 μm). Mean EEA1-puncta diameters were measured in independent experiments (n = 6).
Figure 8d shows the results of detecting the protein expression levels of APP, C99 and β-Actin in iPSC-ND by Western blot after cells were treated with R33 (5 μM) for 30 minutes before high glucose treatment (25 mM) for 24 hours and iPSC Aβ1-42 from the cell culture medium of -ND was measured by human Aβ1-42 specific ELISA assay (n = 5).
Figure 8e shows the protein expression levels of p-Tau Ser396, p-Tau Thr181, t-Tau and β in iPSC-ND after cells were treated with R33 (5 μM) for 30 minutes before high glucose treatment (25 mM) for 48 hours. The results of detection by Western blot are shown (n = 5). *p < 0.05, **p < 0.01 vs control, #p < 0.05, ##p < 0.01 vs hyperglycemia. Quantitative data are presented as mean ± SD. All blot and immunofluorescence images are representative.
9A shows the protein expression levels of APP, C99, and β in SH-SY5Ys by western blot (n = 5), and B shows that Aβ1-42 from the cell culture medium of SH-SY5Y is human Aβ1- 42 shows the result measured by specific ELISA assay (n = 5), and C shows the result of detecting the protein expression levels of p-Tau Ser396, p-Tau Thr181, t-Tau and β in SH-SY5Y by Western blot represents (n = 5). (Two-way ANOVA with Tukey's multiple comparison test) **p < 0.01 vs control, #p < 0.05, ##p < 0.01 vs high glucose. Quantitative data are presented as mean ± SD. All blot and immunofluorescence images are representative.
Figure 10a shows that when iPSC-NDs were treated with R33 (5 μM) for 30 min before high glucose treatment (25 mM) for 24 h, high glucose increased the colocalization of APP and the early endosome marker EEA1, which was related to R33. (Scale bar = 20 μm, n = 6).
Figure 10b shows that when iPSC-NDs were treated with R33 (5 μM) for 30 min before high glucose treatment (25 mM) for 24 h, the colocalization of APP and trans-Golgi marker TGN38 was reduced at high glucose, and R33 treatment Recovered (scale bar = 20 μm, n = 6).
Figure 10c shows that SH-SY5Y was transfected with pcDNA3.1-eGFP or pcDNA3.1-VPS26A-eGFP plasmid for 24 hours prior to high glucose treatment (25 mM) for 24 hours, and then cells were immunostained with EEA1 and APP specific antibodies. and counterstaining with DAPI showed that the colocalization of APP and EEA1 was reduced by VPS26a overexpression (scale bar = 5 μm, n = 5).
Figure 10d shows that SH-SY5Y was transfected with pcDNA3.1-eGFP or pcDNA3.1-VPS26A-eGFP plasmid for 24 hours before high glucose treatment (25 mM) for 24 hours, and then cells were visualized with TGN38 and APP specific antibodies Counterstaining with DAPI shows that the colocalization of APP and TGN38 is higher than that of high glucose treated cells with control pcDNA3.1-eGFP transfection (scale bar = 5 μm, n = 5).
10E shows that VPS26a upregulation reversed retention of APP to early endosomes by high glucose reducing Aβ. APP, C99 and β actin were detected by Western blot (n = 5). SH-SY5Y was transfected with pcDNA3.1-eGFP or pcDNA3.1-VPS26A-eGFP plasmid for 24 hours before high glucose treatment (25 mM) for 48 hours. Aβ1-42 from the cell culture medium was then measured by a human Aβ1-42 specific ELISA assay (n = 5). *p < 0.05, **p < 0.01 vs control, #p < 0.05, ##p < 0.01 vs hyperglycemia. Quantitative data are presented as mean ± SD. All blot and immunofluorescence images are representative.
11 shows that high glucose did not significantly change the expression level of proteins related to tau phosphorylation. SH-SY5Y was analyzed by Western blot after treatment with high glucose (25 mM) in a time-dependent manner (n = 5, one-way ANOVA using Dunnett's multiple comparison test). Quantitative data are presented as mean ± SD.
Figure 12a shows iPSC-ND treated with R33 (5 μM) for 30 minutes before high glucose treatment (25 mM) for 24 hours, and then cells were visualized with EEA1 and CI-MPR specific antibodies and counterstained with DAPI. High glucose in -ND increased the colocalization of CI-MPR and EEA1, which was reversed by R33 pretreatment (scale bar = 20 μm, n = 6).
Figure 12b shows the results of iPSC-ND treated with R33 (5 μM) for 30 minutes before high glucose treatment (25 mM) for 24 hours, and then immunostained with TGN38 and CI-MPR specific antibodies and counterstained with DAPI, Colocalization of CI-MPR and TGN38 was reduced in cells treated with high glucose and recovered in cells pretreated with R33 (scale bar = 20 μm, n = 6).
Figure 12c shows iPSC-ND treated with R33 (5 μM) for 30 minutes before high glucose treatment (25 mM) for 24 hours, and then the cells were visualized with a CTSD-specific antibody and stained with Lysotracker red and DAPI. Cells in a show lower colocalization of lysosomes and CTSD than cells in normal glucose conditions recovered by R33 pretreatment (Scale bar = 20 μm, n = 6).
Figure 12d shows that SH-SY5Y was transfected with pcDNA3.1-eGFP or pcDNA3.1-1 VPS26A-eGFP plasmid for 24 hours prior to high glucose treatment (25 mM) for 24 hours, and then cells were incubated with EEA1 and CI-MPR specific antibodies. As a result of visualization and counterstaining with DAPI, in SH-SY5Y transfected with pcDNA3.1-eGFP or pcDNA3.1-VPS26a-eGFP, the colocalization of CI-MPR and EEA1 increased by high glucose was reduced by VPS26a overexpression. decrease (scale bar = 5 μm, n = 7).
Figure 12e shows that SH-SY5Y was transfected with pcDNA3.1-eGFP or pcDNA3.1-1 VPS26A-eGFP plasmid for 24 hours prior to high glucose treatment (25 mM) for 24 hours, then cells were incubated with TGN38 and CI-MPR specific antibodies. As a result of immunostaining and counterstaining with DAPI, cells transfected with pcDNA3.1-VPS26a-eGFP at high glucose show higher colocalization of CI-MPR and TGN38 than cells transfected with pcDNA3.1-eGFP at high glucose. (Scale bar = 5 μm, n = 7).
Figure 12f shows that SH-SY5Y was transfected with pcDNA3.1-eGFP or pcDNA3.1-1 VPS26A-eGFP plasmid for 24 hours before high glucose treatment (25 mM) for 24 hours, then cells were visualized with CTSD-specific antibody and Lysotracker As a result of staining with red and DAPI, the colocalization of CTSD with lysosomes was reduced by high glucose using pcDNA3.1-eGFP and restored by pcDNA3.1-VPS26a-eGFP transfection (Scale bar = 5 μm, n = 5).
Figure 12g shows that SH-SY5Y was transfected with pcDNA3.1-eGFP or pcDNA3.1-1 VPS26A-eGFP plasmid for 24 hours before high glucose treatment (25 mM) for 24 hours, Pro-CTSD, Mat-CTSD, CI- MPR and β are Western blots, and CTSD activity of cell lysates is a CTSD activity fluorescence measurement assay kit (n = 5).
Figure 12h shows that SH-SY5Ys was transfected with pcDNA3.1-eGFP or pcDNA3.1-VPS26A-eGFP plasmid for 24 hours and then treated with high glucose (25 mM) for 24 hours to p-Tau Ser396, p-Tau Thr181, t- Tau and β represent the results detected by Western blot (n = 5). *p < 0.05, **p < 0.01 vs control, #p < 0.05, ##p < 0.01 vs hyperglycemia, $p < 0.05 vs pcDNA3.1-VPS26a-eGFP + hyperglycemia. Quantitative data are presented as mean ± SD. All blot and immunofluorescence images are representative.
Figure 13a shows that the level of VPS26a was reduced and restored in the hippocampus of STZ-diabetic mice by R33 treatment (n = 5).
Figure 13b shows hippocampal slides for IHC immunostained with Rab5-, Synaptophysin-, PSD95- and GFAP-specific antibodies, respectively, and counterstained with DAPI. higher (scale bar = 20 μm, n = 5).
13c shows the results of detecting hippocampal APP, C99, p-Tau Ser396, p-Tau Thr181, t-Tau, Pro-CTSD, Mat-CTSD, CI-MPR, and β-actin by Western blot, and hippocampal Aβ1-42 As a result measured by rat and mouse Aβ1-42 specific ELISA assay, R33 treatment shows upregulated levels of APP, C99, Aβ phosphorylated tau and pro-CTSD and downregulated levels of mature-CTSD ( n = 5).
Figure 13d shows the results of hippocampal synaptophysin, PSD95, GFAP, and β-actin detected by Western blot. In STZ mice, the level of PSD95 decreased, but the level of hippocampal synaptophysin did not decrease, and the level of GFAP increased, all of which were R33 treatment reversed (n = 5).
13e shows that hippocampal slides for IHC were immunostained with Rab5-, Synaptophysin-, PSD95- and GFAP-specific antibodies, respectively, and counterstained with DAPI. As a result, the level of PSD95 was decreased in STZ mice, but the level of synaptophysin in the hippocampus was not decreased. and GFAP levels increased, all of which were reversed by R33 treatment (scale bar = 20 μm, n = 5).
Figure 13f shows the results of immunostaining of hippocampal slides for IHC with Rab5-, Synaptophysin-, PSD95- and GFAP-specific antibodies, respectively, and counterstaining with DAPI. GFAP levels increased, all of which were reversed by R33 treatment (scale bar = 20 μm, n = 5).
Figure 13g shows that hippocampal slides for IHC were immunostained with Rab5-, Synaptophysin-, PSD95- and GFAP-specific antibodies, respectively, and counterstained with DAPI. As a result, the level of PSD95 decreased in STZ mice, but the level of hippocampal synaptophysin did not decrease. GFAP levels increased, all of which were reversed by R33 treatment (scale bar = 20 μm, n = 5).
13H shows the results of the Y-maze test and the NOR test to evaluate the spatial and object working memory functions of mice, respectively (n = 6). *p < 0.05, **p < 0.01 vs control, #p < 0.05, ##p < 0.01 vs STZ treated mice. Quantitative data are presented as mean ± SD. All blot and immunofluorescence images are representative.

To identify the basic mechanism for the relationship between hyperglycemia-induced retromer dysfunction impairing intracellular transport and Alzheimer's disease, we studied human induced pluripotent stem cell-derived neural differentiated cells exposed to high glucose and SH- SY5Y cells were used. To elucidate whether retromers contribute to Alzheimer's disease-like pathology, we used streptozotocin-induced diabetic mice. As a result, it was confirmed that vacuolar protein sorting-associated protein 26a (VPS26a) was decreased in the hippocampus of diabetic mice and human neurons treated with high glucose. High glucose downregulated VPS26a through ROS/NF-κmethyltransferase 1-mediated promoter hypermethylation. VPS26a withdrawal blocked the retention of APP and cation-independent mannose-6-phosphate receptors in endosomes and promoted their transport to the trans-Golgi, reducing Aβ levels and improving cathepsin D activity, thereby reducing p-tau levels, respectively . Retromer enhancement improved synaptic deficits, astrocyte hyperactivation and cognitive impairment in diabetic mice.

In conclusion, VPS26a of the present invention could suppress DM-related Alzheimer's disease by regulating APP processing and tau phosphorylation.

Hereinafter, the present invention will be described in more detail.

The term 'prevention' in the present invention refers to all activities that suppress or delay the onset of diabetes-mediated neurodegenerative diseases or related diseases due to the retromer stabilizer or the candidate drug screened according to the present invention.

The term 'improvement' or 'treatment' of the present invention means that parameters related to diabetes-mediated neurodegenerative diseases or related diseases, for example, the severity of symptoms, are improved or It means any action that is beneficial.

'Diagnosis' of the present invention may mean to judge the actual condition of a patient's disease in all aspects in a broad sense. The content of judgment is the name of the disease, etiology, type of disease, severity, detailed condition of the disease, and presence or absence of complications. Diagnosis in the present invention is preferably to determine diabetes-mediated neurodegenerative diseases or related diseases and the risk of developing them.

The 'retromer stabilizer' of the present invention may refer to an agent capable of promoting or restoring an intended function by correcting a misfolded retromer complex to stabilize the three-dimensional structure of the retromer complex. there is.

The 'diabetes-mediated neurodegenerative disease' of the present invention may refer to general neurodegenerative diseases caused by or related to diabetes. Preferably, the diabetes-mediated neurodegenerative disease of the present invention may mean diabetes-mediated Alzheimer's disease.

The present invention demonstrated that retromer dysfunction is a potential mechanism of diabetes (DM)-mediated Alzheimer's disease (AD) pathogenesis. Indeed, in vitro data showed that high glucose-mediated downregulation of VPS26a induced aberrant APP processing and tau phosphorylation in neurons. In vivo data showed that Aβ upregulation, tau phosphorylation, synaptic deficits, astrocyte hyperactivation and cognitive impairment were restored by retromer enhancement in diabetic mice. No studies have investigated changes in the expression levels of numerous retromeric components in diabetes. Interestingly, in the present invention, it was confirmed that only the protein level of VPS26a, not VPS35, SORCS1 or WASHC1, among the retromeric components, was downregulated in the hippocampus of STZ-induced diabetic mice and human neurons exposed to high glucose. VPS35 deficiency is known to reduce the levels of both VPS26 and VPS29, but we showed that high glucose-mediated VPS26a downregulation was not accompanied by decreases in VPS35 and VPS29, which may be due to differences in molecular stability. Moreover, we confirmed that the recruitment of VPS35 to early endosomes is reduced under high glucose conditions. These results indicate that VPS26a is a factor inducing high glucose-specific retromer dysfunction. Genome-wide DNA methylation analysis studies have shown that the VPS26A promoter is hypermethylated in both type 1 and type 2 diabetes, but the mechanisms of hypermethylation and cell type-specific hypermethylation are unknown. In diabetic mice, DNMT1 induces hypermethylation of wound healing-related genes and downregulates the expression of these proteins. Destabilization of TET2 by diabetes-suppressive AMP-activated kinase does not regulate DNA methylation, resulting in loss of TET2's tumor suppressor function. However, the present invention showed that only DNMT1 is increased in diabetic conditions among DNA methylation-related genes. High glucose stimulation Sp1/NF-κp65 complex binds to the promoter region of DNMT1 and upregulates its expression, leading to hypermethylation of podocyte slit diaphragm genes. In addition, we confirmed that high glucose upregulated DNMT1 expression through ROS-mediated NF-κp65 stimulation. In the present invention, we confirmed that DNMT1 induces hypermethylation of the VPS26A promoter containing two CpG sites (-118 and -346), indicating that DNMT1-mediated epigenetic regulation reduced VPS26a expression in neurons under diabetic conditions. suggests

In addition, we confirmed that R33 treatment restored VPS26a levels reduced by high glucose as retromer stabilization. In addition, high glucose-mediated early endosome hypertrophy and increases in APP, Aβ, and tau phosphorylation were downregulated by R33 treatment. These results suggested that retromers are potential regulators of both APP processing and alteration of tau phosphorylation in neurons exposed to high glucose. In the present invention, it was shown that high glucose increases the transport of APP to early endosomes and reduces the transport to the trans Golgi recovered by R33 treatment. Given that early endosomal abnormalities are associated with C99 accumulation, these results suggest that high glucose-mediated retromer dysfunction induces pathogenic APP regulation in early endosomes. Previous studies have investigated the molecular mechanisms of retromer-mediated APP processing. VPS35 directly binds APP in COS-7 cells, and its deficiency increases APP in early endosomes of mouse hippocampal neurons. Engineering the YAQM motif in the SORCS1 cytoplasmic tail increases APP retention in early endosomes and reduces APP transport to the trans Golgi in H4 glioma cells. Overexpression of SORL1, which interacts with APP, induced APP redistribution to the trans-Golgi in human neuroblastoma cells. SNX27 directly binds to the cytoplasmic tail of SORL1 and transports APP to the cell surface of mouse primary neurons. However, gain-of-function experiments show that overexpression of VPS26a promotes APP transport from early endosomes to the trans Golgi and reverses the high glucose-mediated increase in APP, C99 and Aβ. VPS26a is the C-terminal region of SNX3, and a possible mechanism of the beneficial effects of VPS26a overexpression is through functional enhancement of CRC.

Since phosphorylated tau is degraded by mature CTSD in lysosomes, lack of CTSD proteolytic capacity leads to abnormal accumulation of phosphorylated tau. Previous studies have shown that CI-MPR directly binds to CTSD and contributes to maturation of CTSD through retromer-mediated transport in HEK293 cells. We confirmed that retromer enrichment by R33 treatment in iPSC-NDs reversed the high glucose-mediated increase in CI-MPR retention in early endosomes via transport to the trans-Golgi. In addition, R33 increased CTSD and lysosomal colocalization, which was downregulated by high glucose conditions. VPS35 knockout induced inappropriate processing of CTSD and reduced its activity through impaired CI-MPR transport from the endosome to the trans Golgi in HeLa cells. Although VPS35 regulates tau phosphorylation through regulation of active CTSD availability in neurons, no study has investigated the effect of VPS26a on CI-MPR trafficking and related CTSD maturation. Our VPS26a overexpression results showed that high glucose-mediated inappropriate CI-MPR and CTSD trafficking and reduced CTSD activity depended on VPS26a. In addition, it was confirmed that the effect of VPS26a on the degradation of phosphorylated tau using Pepstatin A was mediated by the proteolytic ability of active CTSD. Meanwhile, Cdk5, p25, GSK3, and PP2A, which have the potential to regulate tau phosphorylation, are all activated in pancreatic β cells exposed to high glucose. However, the present invention showed that high glucose did not cause significant changes in Cdk5, p25, GSK3 or PP2A levels in neurons. Differences in mechanism of action may be cell type specific and primarily related to regulation. Thus, we propose that VPS26a-mediated retromer dysfunction promotes pathogenic APP processing and tau phosphorylation through inappropriate trafficking under high glucose conditions.

Gain-of-function experiments showed that VPS35 gene transfer in the central nervous system of transgenic Alzheimer's disease mice reduced the Alzheimer's disease phenotype. R33 treatment for 9 months in AD mice restored CRC expression, reduced Aβ and tau phosphorylation levels, and improved learning, memory, and synaptic integrity. It was also shown that R33 increased VPS26a and decreased Aβ and tau phosphorylation levels in diabetic mice. The present inventors confirmed that the STZ-mediated decrease in PSD95, increase in GFAP, spatial and object working memory and cognitive impairment were all restored by R33 treatment. This suggests that pharmacological restoration of retromeric function ameliorates the diabetes-mediated AD-like phenotype. Given that the Vps29 mutation leads to mislocalization of the retromer to the endosome of the soma, which inhibits synaptic transmission, and that genetic deficiency in SORCS2 impairs N-methyl-D-aspartate receptor 2A (NR2A) subunit regulation. When synaptic plasticity trafficking, other retromeric components may contribute to diabetes-mediated AD-like phenotype.

Accordingly, the present invention can provide a pharmaceutical composition for preventing, improving or treating diabetes-mediated neurodegenerative diseases, including a retromer stabilizer.

According to a preferred embodiment of the present invention, the retromer is VPS35 (Vacuolar protein sorting ortholog 35), VPS26a (Vacuolar protein sorting-associated protein 26a), VPS26b (Vacuolar protein sorting 26 homolog B), VPS29 (Vacuolar protein sorting 29) , SORCS1 (Sortilin Related VPS10 Domain Containing Receptor 1), SORL1 (Sortilin Related Receptor 1), and WASHC1 (WASH Complex Subunit 1). Preferably, the retromer may be VPS26a (Vacuolar protein sorting-associated protein 26a).

According to a preferred embodiment of the present invention, the retromer stabilizer may be at least one selected from the group consisting of R33 and R55. Preferably, the retromer stabilizer may be R33.

According to a preferred embodiment of the present invention, the diabetes-mediated neurodegenerative disease may be one or more selected from the group consisting of diabetes-mediated Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, stroke, and multiple sclerosis. . In addition, the diabetes-mediated neurodegenerative disease may be an early diabetes-mediated neurodegenerative disease.

The pharmaceutical composition of the present invention may be in various oral or parenteral dosage forms. When formulating the composition, one or more buffers (eg, saline or PBS), antioxidants, bacteriostats, chelating agents (eg, EDTA or glutathione), fillers, bulking agents, binders, adjuvants (eg, aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrating agents or surfactants, diluents or excipients.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient in one or more compounds, such as starch (corn starch, wheat starch, rice starch, potato) including starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol, cellulose, methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl -It is prepared by mixing cellulose or gelatin. Tablets or dragees may be obtained, for example, by combining the active ingredient with a solid excipient which is then milled and processed into a mixture of granules after adding suitable auxiliaries.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, or syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, or preservatives may be included. can In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant, and anti-coagulant, flavoring agent, emulsifier, solubilizer, dispersant, flavoring agent, antioxidant, and packaging agent. , pigments and preservatives, and the like may be further included.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspension solutions, emulsions, freeze-dried formulations, suppositories, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerol, gelatin, and the like may be used.

The composition of the present invention may be administered orally or parenterally, and may be formulated according to a method known in the art in the form of an injection for parenteral administration, intraperitoneal, rectal, intravenous, intramuscular, subcutaneous or intracerebrovascular injection. .

In the case of the injection, it must be sterilized and must be protected from contamination by microorganisms such as bacteria and fungi. Examples of suitable carriers for injections include, but are not limited to, water, ethanol, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.), mixtures thereof, and/or solvents or dispersion media containing vegetable oils. can More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) with triethanolamine or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose. etc. can be used. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, and thimerosal may be further included. Also, in most cases, the injection may further include an isotonic agent such as sugar or sodium chloride.

The composition of the present invention is administered in a pharmaceutically effective amount. A pharmaceutically effective amount means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level depends on the type of patient's disease, severity, activity of the drug, sensitivity to the drug, and administration time. , the route of administration and excretion rate, the duration of treatment, factors including concomitantly used drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. That is, the total effective amount of the composition of the present invention can be administered to the patient in a single dose, or it can be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. . Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

The present invention may also provide an information providing method for diagnosing diabetes-mediated neurodegenerative diseases, which includes checking the expression level of VPS26a (Vacuolar protein sorting-associated protein 26a) in a sample obtained from the subject.

A sample obtained from the subject may be serum, plasma, urine, saliva, cells or tissue.

According to a preferred embodiment of the present invention, the information providing method may further include a step of classifying as a diabetes-mediated neurodegenerative disease if the confirmed expression level of VPS26a is lower than that of a normal sample.

According to a preferred embodiment of the present invention, the method of providing information may be to check the levels of amyloid precursor protein (APP) and phosphorylated tau in addition to VPS26a.

According to a preferred embodiment of the present invention, if the level of the APP and phosphorylated tau is higher than that of the normal sample, a step of classifying as a diabetes-mediated neurodegenerative disease may be additionally included.

According to a preferred embodiment of the present invention, the diabetes-mediated neurodegenerative disease may be one or more selected from the group consisting of diabetes-mediated Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, stroke, and multiple sclerosis, , preferably diabetes-mediated Alzheimer's disease. In addition, the diabetes-mediated neurodegenerative disease may be an early diabetes-mediated neurodegenerative disease.

The present invention also includes i) treating a candidate drug to a sample obtained from a patient with diabetes-mediated neurodegenerative disease; and

ii) checking the expression level of VPS26a (Vacuolar protein sorting-associated protein 26a) in the sample treated with the drug;

It is possible to provide a drug screening method for the prevention, improvement or treatment of diabetes-mediated neurodegenerative diseases comprising a.

Samples obtained from patients with diabetes-mediated neurodegenerative diseases may be serum, plasma, urine, saliva, cells, or tissues.

According to a preferred embodiment of the present invention, the screening method includes: iv) classifying as a drug for preventing, improving or treating diabetes-mediated neurodegenerative diseases if the expression level of VPS26a identified is higher than before drug treatment; may be additionally included.

According to a preferred embodiment of the present invention, in step ii), levels of amyloid precursor protein (APP) and phosphorylated tau in addition to VPS26a may be checked together.

According to a preferred embodiment of the present invention, if the level of the APP and phosphorylated tau is reduced than before drug treatment, it may be classified as a drug for preventing, improving or treating diabetes-mediated neurodegenerative diseases.

According to a preferred embodiment of the present invention, the diabetes-mediated neurodegenerative disease may be one or more selected from the group consisting of diabetes-mediated Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, stroke, and multiple sclerosis, , preferably diabetes-mediated Alzheimer's disease. In addition, the diabetes-mediated neurodegenerative disease may be an early diabetes-mediated neurodegenerative disease.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for exemplifying the present invention, and it is obvious to those skilled in the art that the scope of the present invention is not construed as being limited by these examples.

Mouse model preparation

<1-1> Mouse preparation

Male ICR mice (9 weeks old) were purchased from OrientBio (Seongnam, Korea) and kept under standard conditions with appropriate temperature (20-25 °C) and humidity (less than 60%) with a 12-hour light/12-hour dark cycle. They had free access to autoclaved food and tap water. Animal handling, care and experiments were performed in accordance with the regulations and ethical approval of the Animal Care Committee of Seoul National University Animal Hospital (approval number: SNU-201013-7-1). Although sex is an important factor to consider when designing animal studies, it was reasonable to use male mice because the purpose of the study was to elucidate the effect of diabetes on retromers and the dysregulation of the associated APP processing and tau phosphorylation mechanisms. A total of 40 male mice were used in this experiment. Mice were randomly divided into equal-sized groups. Group size estimation was based on the guidelines established by the Seoul National University Animal Hospital Animal Care Committee (expected effect size: 15%, standard deviation: 6%, number of groups: 4, power (1-beta): 0.8, alpha: 0.05). The number of animals used was increased to account for the possibility of not obtaining a sufficient number of mice exhibiting the hyperglycemic phenotype.

<1-2> Preparation of diabetic mouse model

To investigate the effect of high glucose on retromer dysfunction, diabetes mellitus (DM) was induced in mice by injection of streptozotocin (STZ, Sigma Chemical Company; St. Louis, MO, USA). Mice were randomly administered an intraperitoneal injection (IP) of STZ (75 mg/kg) in 200 μl sodium citrate buffer (0.1 M, pH 4.5, vehicle) or 200 μl vehicle once daily for 3 consecutive days. Blood glucose was measured using a portable blood glucose meter (Roche, Mannheim, Germany), and body weight was measured before and after STZ injection. Mice with blood glucose levels above 300 mg/dl were considered severely diabetic. After obtaining enough diabetic mice, animals were randomly divided into 4 groups (n=6 for each group): vehicle, STZ, R33, and R33+STZ treated mice. 100 μl of R33 (12.5 mg/kg, dissolved in distilled water) and vehicle were administered intraperitoneally (IP). R33 was injected once daily for 8 weeks after DM induction. Body weight and blood glucose levels were measured at 9 and 18 weeks of age (Fig. 2A). After behavioral testing, mice were anesthetized with an intraperitoneal injection of alfaxalone (40 mg/kg) together with 10 mg/kg xylazine and euthanized for further biochemical analysis.

Downregulation of VPS26a by glucose

<2-1> Effect of glucose on retromer assembly

To demonstrate the effect of diabetes on retromer assembly, we first measured the levels of VPS35, VPS26a, VPS26b, VPS29, SORCS1, SORL1, and WASH Complex Subunit 1 (WASHC1) in the hippocampus of vehicle- or STZ-treated mice by Western blot. measured with

For neural stem cell (NSC) induction, iPSCs (Kangstem Biotech; Seoul, Korea) were cultured on recombinant human vitronectin (A14700, Thermo Fisher) coated plates using neural induction medium (A1647801, Thermo Fisher). After NSC induction, cells were re-cultured on poly-L-ornithine (Sigma, P3655)- and laminin- (23017, Thermo Fisher) coated dishes. NSCs were cultured for at least 10 days for neural differentiation in Neurobasal Medium (21103, Thermo Fisher) with 2% B27 serum-free supplement (17504, Thermo Fisher) and 2 mM GlutaMax-1 supplement (35050, Thermo Fisher). cultured. To promote neural differentiation, 0.5 mM dibutyryl-cAMP (D0627, Sigma) was added to the neural differentiation medium every day between 7 and 10 days after the start of differentiation. The results of characterization of iPSC-NDs using the neural markers NeuN and MAP2 are shown in FIG. 3 .

SH-SY5Ys (Korean Cell Line Bank; Seoul, Korea) were cultured in low glucose Dulbecco's essential _medium (DMEM; Hyclone, #SH30021FS), 10% FBS (Hyclone; Logan, UT, USA) and 1 % antibiotic-antifungal (Gibco; Grand Island, NY, USA) solution. After cells had grown to 70% confluency, the medium was replaced with DMEM containing 1% antibiotic-antifungal mixture for 24 hours prior to drug treatment. For high glucose conditions, cells were treated with 25 mM D-glucose (Sigma Chemical Company; St. Louis, MO, USA) or L-glucose (Sigma Chemical Company; St. Louis, MO, USA).

For immunohistochemistry (IHC), mice were completely anesthetized with a 4:1 mixture of alfaxalone and xylazine, transcardially perfused with PBS, and fixed with 4% paraformaldehyde (PFA). Brains were carefully removed and post-fixed in 4% PFA for 2 hours. Hemispheres were then dehydrated in 30% sucrose in PBS for 1–2 days. Coronal sections (thickness 40 μm) of the hippocampus were obtained by serial sectioning using a cryostat (Leica Biosystems, Nussloch, Germany). A free-floating method was performed to stain the hippocampus. Sections were blocked with 5% NGS for 1 hour at room temperature (RT) and incubated with primary antibodies (1:1,000 dilution) for 2 days at 4°C. Samples were washed 3 times with PBS and incubated with secondary antibody (1:300 dilution) for 2 hours at room temperature. Sections were visualized by Eclipse Ts2™ fluorescence microscope (Nikon, Tokyo, Japan). Image analysis was performed using Fiji software. Measurements of signal strength were performed applying the same threshold.

For immunofluorescence analysis, SH-SY5Ys were fixed with 4% PFA for 10 min. For cell permeability, cells were incubated with 0.2% PBST or 0.1% Triton X-100 (Sigma, T8787) for 10 minutes. Cells were blocked with 5% NGS for 1 hour and incubated with primary antibody (1:300 dilution) overnight at 4°C. Cells were then washed three times with PBS and incubated with Alexa Fluor 488- or 555- or 647-conjugated secondary antibodies (1:200 dilution) for 2 hours at room temperature. Immunostained cells were visualized with a super-resolution radial fluctuation (SRRF) imaging system (Andor Technology, Belfast, UK) and a confocal microscopy system (LSM 710, Carl Zeiss, Oberkochen, Germany). Fluorescence intensity quantification and measurement of Rab5+ endosome regions were performed using Fiji software. Co-localization analysis was performed by measuring manders coefficients using Fiji's JACop plugin.

For quantitative real-time polymerase chain reaction (qPCR), cellular RNA was obtained using an RNA extraction kit (TaKaRa, 9767) according to the manufacturer's instructions. Then, complementary DNA (cDNA) was prepared using reverse transcription-PCR premix (iNtRON Biotechnology, 25081). Reverse transcription was performed at 45°C for 1 hour followed by 95°C for 5 minutes. cDNA with two technical replicates was amplified with a mixture of mRNA primers (Bioneer; Daejeon, Korea) and TB™Green Premix Ex Taq™ by a Rotor-Gene 6000 real-time thermal cycling system (Corbett Research, NSW, Australia). qPCR was performed as follows: 95 °C for 10 min and 60 cycles of 95 °C for 10 sec, 55 °C for 15 sec, 72 °C for 20 sec for DNA polymerase activation. Double Δ analysis was performed for quantification of mRNA expression and data 3 was normalized to the ACTB gene.

Cells or tissues harvested for Western blot analysis and intracellular fractionation were dissociated with lysis buffer and protease and phosphatase inhibitor cocktail (100X) (Thermo Fisher, 78440). Then, the samples were homogenized with a vortexer and sonicator. Bichoninic acid (BCA) quantification assay (Thermo Fisher, 23227) using two technical replicates was performed for protein quantification. Identical samples (5-10 μg) with loading buffer were loaded onto an 8-12% SDS-polyacrylamide gel (SDS-PAGE) and transferred to a polyvinylindene fluoride (PVDF) membrane. Tris-buffered saline [TBST; 150 mM NaCl, 10 mM Tris-HCl (pH 7.6)] was used. Membrane blocking was performed with 5% skim milk (Gibco, 232100) for 1 hour and membranes were incubated with primary antibodies overnight at 4°C. Then, the membrane was washed three times and incubated for 2 hours at room temperature with a horseradish peroxidase (HRP)-conjugated secondary antibody. Blotting bands were detected with a chemiluminescent detection kit (Advansta Inc., K-12045-D50). Protein band quantification was performed with Image J software. Normalization of protein expression levels was performed relative to β-actin expression levels. The cytosolic and nuclear fractionated samples were separated using the EzSubcell™ subcellular fractionation kit (Atto, WSE-7421). Cytoplasmic and nuclear samples were obtained according to the manufacturer's instructions. α-Tubulin and lamin A/C were applied to normalize cytoplasmic and nuclear protein expression levels, respectively.

As a result of expression level analysis, VPS26a was downregulated in the hippocampus of diabetic mice, iPSC-ND and SH-SY5Y treated with hyperglycemia. That is, only the protein level of VPS26a among retromeric components was decreased in STZ-induced diabetic mice compared to vehicle-treated mice (FIG. 4a). STZ-induced diabetic mice also had lower staining intensity of hippocampal VPS26a than vehicle-treated mice (FIG. 4B). In vitro experiments, iPSC-NDs exposed to high glucose showed down-regulation of VPS26a (Fig. 4c, Fig. 5a). In SH-SY5Y, high glucose also decreased the mRNA and protein expression levels of VPS26a after 12 h (Fig. 4d). Only D-glucose treatment, but not L-glucose treatment, reduced VPS26a expression in cells, indicating that osmotic effects were not involved in protein downregulation (FIG. 5B).

<2-2> Effect of glucose on retromer function

Since recruitment of retromeric complexes to endosomes is essential for cargo recognition core (CRC)-mediated cargo transport, we investigated the recruitment of VPS35-bound cargo directly to early endosomes to investigate the effect of high glucose on retromeric function. Measured.

As a result, compared to normal glucose-treated cells, high glucose treatment reduced the degree of colocalization between Rab5 and VPS35, early endosome markers, in both iPSC-ND (Fig. 4e) and SH-SY5Y (Fig. 5c). These results suggest that high glucose downregulates the expression of VPS26a causing retromeric dysfunction.

Mechanism of VPS26a downregulation by glucose

Given that diabetes-mediated genetic repression mainly occurs by epigenetic downregulation, we focused on changes in the expression of epigenetic regulators to investigate how high glucose downregulates VPS26a. First, mRNA of DNA methyltransferases, which are enzymes that methylate DNA, such as DNMT1, DNMT3A, and DNMT3B, and ten-eleven translocation methylcytosine dioxygenases, which are enzymes that demethylate DNA, such as TET1, TET2, and TET3 Changes in expression levels were investigated.

Dot blot analysis was performed to detect DNA methylation levels in SH-SY5Ys. Genomic DNA (gDNA) was extracted using a genomic DNA extraction kit (Bioneer, K-3032). 100 ng of gDNA was denatured at 95°C for 10 minutes and then neutralized on ice for 10 minutes. gDNA was loaded onto a N+ nylon membrane (Amersham, RPN203B). Membranes were dried and incubated at 80° C. for 2 hours. Membranes were blocked with 5% skim milk for 1 hour and incubated with anti-5-mC antibody overnight at 4°C. Membranes were washed three times with TBST and then incubated with HRP-conjugated secondary antibodies for 2 hours at room temperature. DNA was detected with a chemiluminescence detection kit (Advansta Inc., K-12045-D50). Quantification of dot blot intensities was performed by Image J software.

Conversion of gDNA for methylation analysis as methylation-specific PCR (MSP) was performed by bisulfite treatment using the EZ DNA Methylation-Lightning Kit and following the manufacturer's instructions (Zymo Research, D5030). The methylation status of the VPS26A gene in SH-SY5Ys was determined by MSP. Methylation status was assessed as the relative methylation level compared to the unmethylated form.

As a result, it was confirmed that high glucose downregulated VPS26a through ROS/NF-κ-mediated promoter hypermethylation. That is, only the mRNA expression of DNMT1 was increased by high glucose, and the protein expression level of DNMT1 also increased in a time-dependent manner upon high glucose treatment (Fig. 7a). STZ-induced diabetic mice had higher hippocampal DNMT1 levels compared to vehicle-treated mice (FIG. 6A).

Since ROS is known to stimulate NF-κ, which can upregulate DNMT1 expression, we found that the high glucose-stimulated nuclear translocation of NF-κ was mediated by NAC (ROS scavenger, Sigma Chemical Company; St. Louis, MO, USA). We investigated whether it could be blocked by (Fig. 6b, S5c). The mRNA and protein expression levels of DNMT1, which were increased by high glucose, were also decreased by Bay 11-7082 (NF-κ inhibitor, Sigma Chemical Company; St. Louis, MO, USA) (Fig. 6c, S5d). These results suggest that the ROS-stimulated NF-κ pathway upregulates DNMT1 expression under high glucose conditions.

To elucidate the relationship between high glucose-mediated DNMT1 and VPS26a downregulation, we first determined the global DNA methylation status by measuring the intensity of 5-methylcytosine (5-mC) staining in cells exposed to high glucose. The high glucose-mediated increase in 5-mC staining intensity was reduced by DNMT1 silencing (Fig. 7d, S5f).

For siRNA transfection for gene silencing, when SH-SY5Ys have grown to 60%, the medium is low-glucose DMEM without serum and antibiotics containing 25 nM of the indicated siRNA, transfection reagent TurboFect™Fisher, R0531) for 12 hours. has been replaced with Cells were cultured in medium containing 1% antibiotics before the experiment. NT siRNA (Dharmacon; Lafayette, CO, USA) was treated as a negative control.

We further determined the specific methylation status of the VPS26A promoter, which contains two CpG sites at -118 and -346. As shown in FIG. 6D , cells transfected with NT siRNA in high glucose increased the level of VPS26A promoter methylation at -118 and -346 compared to cells transfected with NT siRNA in normal glucose conditions. Interestingly, DNMT1 silencing reduced high glucose-mediated hypermethylation of the VPS26A promoter. In addition, the downregulated mRNA and protein expression levels of VPS26a in high glucose were reversed by DNMT1 silencing (Fig. 6e, Fig. 7f). These data indicate that DNMT1, upregulated by high glucose, suppresses VPS26a expression through hypermethylation of the VPS26A promoter.

Increased APP and p-tau levels by glucose-induced retromer dysfunction

To investigate the effect of retromer dysfunction on high glucose-mediated amyloidogenesis and tau phosphorylation, cells were treated with R33 (5 μM) for 30 min before high glucose treatment (25 mM) for 24 h.

For Aβ measurement, cell culture fluid was collected in vitro and centrifuged at 1,000g for 20 minutes. Phosphatase inhibitor cocktail (100X) was added to the supernatant to inhibit proteolysis. In vivo, the same amount of hippocampal sample (100 μg) was prepared in lysis buffer and BCA assay. According to the manufacturer's instructions, the optical density (OD) of Aβ was obtained in culture media and tissue samples using a human AβELISA kit (Thermo Fisher, KHB3544) and a mouse & rat AβELISA kit (Thermo Fisher, KMB3441), respectively. Absorbance at 450 nm was converted into concentration and analyzed using a standard curve.

As a result, R33, a pharmacological chaperone, blocked increased Aβ production and tau phosphorylation by high glucose, stabilizing and restoring retromer function. That is, treatment with R33 restored high glucose-mediated downregulation of VPS26a (Fig. 8a). Retromers restored by R33 treatment reduced the size of early endosomes, an early pathological feature of AD, in both iPSC-NDs (Fig. 8b) and SH-SY5Y (Fig. 8c) exposed to high glucose. Given that high glucose regulates APP processing in abnormal early endosomes, R33 may normalize APP processing. Increased APP, the C-terminal fragment of the amyloid β (Aβ) protein precursor (APP-CTFβC99) and Aβ were observed in both iPSC-ND (Fig. 8d) and SH-SY5Y (Fig. 9A, B) under high glucose conditions. Recovered by R33 under Retromers may be involved in tau phosphorylation in diabetes because degradation of phosphorylated tau is regulated by retromer-mediated cathepsin D (CTSD) maturation. We confirmed that R33 treatment downregulated tau phosphorylation at Ser396 and Thr181 in both iPSC-ND (Fig. 8e) and SH-SY5Y (Fig. 9C) exposed to high glucose. These confirmations suggest that retromeric dysfunction is involved in APP processing and tau phosphorylation under high glucose conditions.

Regulation of APP processing of retromeric complexes

To elucidate how the retromeric complex regulates APP processing, we measured APP localization to early endosomes and the trans-Golgi apparatus.

After SH-SY5Y reached 60% confluency, cells were incubated for 24 hours with a mixture of plasmid DNA, low glucose DMEM and Lipofectamine 3000 (Thermo Fisher). Cells were cultured in serum-free medium containing 1% antibiotics prior to experiments. The pcDNA3.1-eGFP plasmid (Koma Biotech; Seoul, Korea) was used as a negative control.

As a result, it was confirmed that the upregulation of VPS26a reversed the retention of APP in the early endosome by high glucose, which reduced Aβ. That is, in iPSC-NDs, high glucose increased the colocalization of APP and the early endosome marker EEA1, which was reversed by R33 (Fig. 10a). Colocalization of APP and the trans-Golgi marker TGN38 was reduced at high glucose and restored by R33 treatment (Fig. 10b).

To focus on the effect of VPS26a in high glucose, we compared SH-SY5Y with pcDNA3.1-eGFP or pcDNA3.1-VPS26A-eGFP (Koma Biotech; Seoul, Korea) VPS26A was overexpressed by transfection.

Colocalization of APP and EEA1 in cells treated with high glucose with control pcDNA3.1-eGFP transfection was reduced by VPS26a overexpression (Fig. 10c). Colocalization of APP and TGN38 in high glucose treated cells with VPS26a overexpression was higher than in high glucose treated cells with control pcDNA3.1-eGFP transfection (FIG. 10D). VPS26a overexpression downregulated high glucose-mediated increases in APP, C99 and Aβ levels (Fig. 10e). These results suggest that high glucose-mediated downregulation of VPS26a impairs APP transport from early endosomes to the trans Golgi, thereby altering APP processing.

Downregulation of VPS26a inhibits p-tau degradation

Phosphorylation of tau can be regulated by the kinases p35/25-CDK5 and GSK3β or protein phosphatase 2 (PP2A), but there was no significant change in the levels of these proteins under high glucose conditions (FIG. 11). Given that maturation of CTSD is regulated by retromer-mediated CI-MPR recycling, and that VPS35 interacts with CI-MPR to transfer from early endosomes to the trans-Golgi apparatus, we first investigated the activation of early endosomes and trans-Golgi. The localization of CI-MPR was measured.

Lysotracker deep red (Thermo Fisher, #L12492), which has a high affinity for acidic organelles, was used to stain intact lysosomes. Cells were incubated with 2 μM Lysotracker red in medium and maintained at 37° C. for 30 minutes. Cells were then washed 3 times with PBS. Signals of Lysotracker stained cells were visualized by a confocal microscopy system (LSM 710, Carl Zeiss, Oberkochen, Germany).

Cathepsin D (CTSD) activity was measured using a cathepsin D activity fluorescence assay kit (abcam, K143-100). The assay was performed according to the manufacturer's protocol. Briefly, harvested cells were lysed in an appropriate lysis buffer for 10 min on ice and centrifuged for 5 min at maximum speed. Equal amounts of lysate were loaded into a 96-well plate with reaction buffer and substrate and then incubated at 37°C for 2 hours. Fluorescence intensities were read with a fluorometer equipped with 328 nm excitation/460 nm emission filters.

As a result, it was confirmed that the up-regulation of VPS26a reversed the retention of CI-MPR in early endosomes by high glucose and induced the increased CTSD activity-mediated p-Tau degradation. That is, in iPSC-NDs, high glucose increased the colocalization of CI-MPR and EEA1, which was reversed by R33 pretreatment (Fig. 12a). Colocalization of CI-MPR and TGN38 was reduced in cells treated with high glucose and restored in cells pretreated with R33 (FIG. 12B). In addition, cells in high glucose conditions had lower colocalization of lysosomes and CTSD than cells in normal glucose conditions restored by R33 pretreatment (FIG. 12c). In SH-SY5Y transfected with pcDNA3.1-eGFP or pcDNA3.1-VPS26a-eGFP, the colocalization of CI-MPR and EEA1, which was increased by high glucose, was reduced by VPS26a overexpression (FIG. 12D). Cells transfected with pcDNA3.1-VPS26a-eGFP in high glucose had higher colocalization of CI-MPR and TGN38 than cells transfected with pcDNA3.1-eGFP in high glucose (FIG. 12E). Lysosome and CTSD colocalization was reduced by high glucose with pcDNA3.1-eGFP and restored by pcDNA3.1-VPS26a-eGFP transfection (Fig. 12f). Although high glucose-mediated upregulation of pro-CTSD and downregulation of mature CTSD were reversed by pcDNA3.1-VPS26a-eGFP transfection, there was no significant change in CI-MPR expression levels in all groups, and no significant change was observed by high glucose. Reduced CTSD activity was restored by VPS26a overexpression (FIG. 12G). The levels of phosphorylated tau at Ser396 and Thr181, which were increased by high glucose, were reduced by VPS26a overexpression, which was blocked by pretreatment with the CTSD inhibitor Pepstatin A (Fig. 12h). These results suggest that VPS26a, which is downregulated at high glucose, inhibits CTSD maturation by blocking CI-MPR recycling between early endosomes and trans-Golgi, resulting in defects in the degradation of phosphorylated tau.

Induction of AD-like pathology of retromeric dysfunction

R33 restored amyloidogenesis, tau phosphorylation, synaptic deficits, astrocyte hyperactivation and cognitive impairment in diabetic mice. That is, the level of VPS26a was reduced and restored in the hippocampus of STZ-diabetic mice by R33 treatment (FIG. 13a). The hippocampal Rab5 signal intensity of STZ mice was higher than that of vehicle-injected and STZ+R33-injected mice (FIG. 13B). Diabetic mice showed upregulated levels of APP, C99, Aβ phosphorylated tau and pro-CTSD, and downregulated levels of mature-CTSD, all of which were restored by R33 treatment (Fig. 13c). These results indicate that the diabetes-associated Alzheimer's disease phenotype is mediated by retromeric dysfunction.

In addition to the AD phenotype, we investigated changes in synaptic integrity and astrocyte hyperactivation.

The Y-maze behavioral test is used to assess hippocampal-dependent spatial learning and memory. Mice instinctively prefer to explore the new arm of the Y-maze. Prior to testing, animals were housed for 2 hours in the testing room to minimize stress. Mice were placed on randomly selected arms of a Y-maze apparatus obtained from GaonBio (Yongin, Korea). Each mouse was allowed to freely explore the maze for 8 min. The total number of arm entries and sequences was recorded. Entry was considered complete only when all four limbs were placed on the arm. Percentage alternation is the number of triads divided by the maximum number of alternations (total items-2) × 100. Animals exhibit impaired spatial learning and memory when they exhibit lower alternation percentages.

The novel object recognition test (NOR) is now a commonly used mouse behavioral test to assess object working memory and depends on the mouse's innate preference for novelty. Mice were housed in an outdoor box for 5 min in the testing room for habituation. After 24 h, mice were free to explore for 10 min in the same open field using two similar objects during the first session. After 4 h, one of the two objects was replaced with a new one and then the mice were allowed to freely explore for 10 min in the open field for the second session. Discrimination index, which is the most frequently used value for cognitive evaluation, was calculated by dividing the search time for a new object minus the search time for a familiar object by the total search time. Novelty preference was calculated as the time spent exploring the novel object as a percentage of the total exploration time. An increase in discrimination index indicates an improvement in cognitive memory.

In STZ mice, PSD95 (post-synaptic marker) levels decreased but hippocampal synaptophysin (pre-synaptic marker) levels did not decrease and GFAP (astrocytic marker) levels increased, all of which were reversed by R33 treatment (FIGS. 13D-7H). ). The results of the Y-maze showed a decrease in the total number of arm items in both STZ-injected rats and STZ+R33-injected rats, but R33 increased spontaneous alternation, which was reduced by diabetes (Fig. 13h). As a result of the NOR test, mice injected with STZ showed a decrease in the discrimination index and novelty preference recovered by R33 injection (FIG. 13h). The results of the Y-maze and NOR indicate that R33 restored spatial and object working memory impaired by diabetes. This suggests that Aβp-tau, synaptic deficits, hyperactivation of astrocytes, and cognitive impairment in diabetic mice were induced by retromer dysfunction.

[Statistical analysis]

Sample size n represents the number of biologically independent replicates used for statistical analysis. Outliers were identified according to the exclusion criterion beyond the external fence (Q1-3*IQ or Q3+3*IQ) in the box plot configuration and no outliers were observed. All data were obtained for normality using the Shapiro-Wilk test and analyzed by parametric statistics. The heteroscedasticity of the sample was confirmed by Spearman's test, and logarithmic transformation was performed if the variance did not satisfy homogeneity. After data conversion, the unit of the variable was the percentage agreement control value. Imaging and animal studies were evaluated in a blinded manner. A two-tailed unpaired Student's t-test was performed to compare the mean of the treatment group and the mean of the control group. One-way ANOVA (Dunnett's multiple comparison test and two-tailed) or two-way ANOVA (Tukey's multiple comparison test and two-tailed) were used to compare differences between groups. Quantitative data are expressed as mean ± SD and analyzed with Prism 8 software (Graphpad, CA, USA). A probability level (p) < 0.05 was considered statistically significant.

Claims (12)

A pharmaceutical composition for preventing, improving or treating diabetes-mediated neurodegenerative diseases, including a retromer stabilizer.
The method of claim 1, wherein the retromer is VPS35 (Vacuolar protein sorting ortholog 35), VPS26a (Vacuolar protein sorting-associated protein 26a), VPS26b (Vacuolar protein sorting 26 homolog B), VPS29 (Vacuolar protein sorting 29), SORCS1 (Sortilin A pharmaceutical composition, characterized in that at least one selected from the group consisting of Related VPS10 Domain Containing Receptor 1), SORL1 (Sortilin Related Receptor 1) and WASHC1 (WASH Complex Subunit 1).
The pharmaceutical composition according to claim 1, wherein the retromer stabilizer is at least one selected from the group consisting of R33 and R55.
The pharmaceutical composition according to claim 1, wherein the diabetes-mediated neurodegenerative disease is at least one selected from the group consisting of diabetes-mediated Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, stroke, and multiple sclerosis. .
An information providing method for diagnosing diabetes-mediated neurodegenerative diseases comprising checking the expression level of VPS26a (Vacuolar protein sorting-associated protein 26a) in a sample obtained from the subject.
The information providing method according to claim 5, further comprising classifying as a diabetes-mediated neurodegenerative disease if the confirmed expression level of VPS26a is lower than that of the normal sample.
The information providing method according to claim 5, wherein, in addition to VPS26a, the levels of APP (amyloid precursor protein) and phosphorylated tau are also checked.
The method of claim 7, further comprising classifying the APP and phosphorylated tau as a diabetes-mediated neurodegenerative disease if the level of the APP and phosphorylated tau is higher than that of the normal sample.
i) treating a sample obtained from a patient with diabetes-mediated neurodegenerative disease with a candidate drug; and
ii) checking the expression level of VPS26a (Vacuolar protein sorting-associated protein 26a) in the sample treated with the drug;
A drug screening method for preventing, improving or treating diabetes-mediated neurodegenerative diseases comprising a.
10. The method of claim 9, wherein the screening method comprises: iv) classifying as a drug for preventing, improving or treating diabetes-mediated neurodegenerative diseases if the expression level of the identified VPS26a is higher than before drug treatment; A screening method characterized in that it further comprises.
The screening method according to claim 9, wherein, in step ii), the levels of APP (amyloid precursor protein) and phosphorylated tau are checked together in addition to VPS26a.
The screening method according to claim 11, wherein if the level of APP and phosphorylated tau is reduced compared to before drug treatment, the screening method can be classified as a drug for preventing, improving or treating diabetes-mediated neurodegenerative diseases.

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