KR102379145B1 - Composition for prevention, improvement or treatment of diabetes-mediated neurodegenerative diseases comprising a PICALM inhibitor and an mTORC1 inhibitor - Google Patents

Abstract

The present invention provides a method for preventing, improving or treating neurodegenerative diseases such as Alzheimer's disease by inhibiting PICALM and mTORC1-mediated early endosome disorders in a high-glucose environment by regulating PICALM and mTORC1, thereby inhibiting amyloid beta production. The present invention relates to a composition, by regulating PICALM and mTORC1, inhibiting PICALM and mTORC1-mediated early endosome disorders in a high glucose environment, and suppressing excessive production of amyloid beta, thereby preventing neurodegenerative diseases such as Alzheimer's disease early It can be prevented or effectively treated, and it can be widely used in various industrial fields such as the pharmaceutical industry and the bio industry.

Description

Composition for prevention, improvement or treatment of diabetes-mediated neurodegenerative diseases comprising a PICALM inhibitor and an mTORC1 inhibitor

The present invention relates to a composition for preventing, improving or treating a neurodegenerative disease comprising a PICALM inhibitor and an mTORC1 inhibitor. Specifically, the present invention regulates PICALM (phosphatidylinositol binding clathrin assembly protein) and mTORC1 (mammalian target of rapamycin complex 1, mammalian rapamycin complex 1) in a high glucose environment, PICALM and mTORC1 It relates to a composition for preventing, improving, or treating neurodegenerative diseases, such as Alzheimer's disease, by inhibiting mediated early endosome disorders, thereby inhibiting amyloid beta production.

Epidemiological and neuropathological studies suggest that diabetes (diabetes mellitus, DM) may substantially contribute to the pathogenesis or progression of Alzheimer's disease (AD) (Arnold, SE et al., Nat. Rev. Neurol. 14, 168-181, 2018; Biessels, GJ & Despa, F., Nat. Rev. Endocrinol. 14, 591-604, 2018; Rojas-Gutierrez, E. et al., Synapse, 71, e21990, 2017). Various in vitro and in vivo studies have provided evidence that DM is a factor that induces AD, and that the two diseases overlap in risk at the molecular level and in pathophysiological mechanisms (Moreno-Gonzalez, I. et al., Mol). Psychiatry, 22, 1327-1334, 2017; Mittal, K. et al., Sci. Rep. 6, 25589, 2016). Although there are many potential candidates (Moreno-Gonzalez, I. et al., Mol. Psychiatry, 22, 1327-1334, 2017; Mittal, K. et al., Sci. Rep. 6, 25589, 2016), DM The exact molecular mechanisms contributing to the development of this AD are still unknown. As a better understanding of the molecular mechanisms underlying this relationship, these findings may facilitate the development of effective therapeutic interventions to treat or modulate DM and, consequently, delay the onset or progression of AD. . Indeed, with respect to dysregulation of endosomal migration, a focus has been placed on amyloid beta (Aβ) production (Small, SA et al., Trends. Neurosci. 40, 592-602, 2017; Xu, W. et al. ., Traffic, 19, 253-262, 2018; Manucat-Tan, NB et al., Mol. Neurobiol. 56, 812-830, 2019). In particular, previous studies have reported that enlargement of early endosomes with Aβ immunoreactivity is the earliest pathological change in both diagnosed and undiagnosed AD patients (Cataldo, AM et al., Am (J. Pathol. 157, 277-286, 2000; Cataldo, AM et al., Neurobiol, Aging, 25, 1263-1272, 2004). Furthermore, hyperactivation of early endosomes is induced by a C-terminal fragment of APP cleaved by β-secretase (APP-CTFβ), considered a rate-limiting molecule of Αβ production (Kim, S. et al. al., Mol. Psychiatry, 21, 707-716, 2016). However, given that the amount of Aβ in the brain is already saturated before the onset of symptoms, early-stage AD diagnosis is important (Masters, CL et al., Nat. Rev. Dis. Primers, 1, 15056, 2015). However, it is necessary to investigate other factors contributing to endosomal dysfunction for disease prevention. It has been reported that DM up-regulates Aβ production by exacerbating endosomal dysfunction (Okabayashi, S. et al., PLoS One, 10, e0117362, 2015). Although the precise role and regulation of endosomes in Aβ production has not been elucidated, these studies suggest the possibility that perturbation of early endosomes may be a risk factor for early DM-induced AD pathology.

Hyperactivation of early endosomes is caused by various factors, such as upregulation of phagocytic genes, increased phagocytosis and hyperactivation of Rab5, a small GTPase indicative of early endosomes (Nixon, RA, FASEB J. 31, 2729). -2743, 2017). According to genome-wide association studies, phagocytic genes such as PICALM, BIN1, CD2AP, SORL1 and PLD3 are risk factors for AD through various, undefined, mechanisms associated with impaired trafficking (Kanatsu, K. & Tomita, T., Front. Biosci. (Landmark edition), 22, 180-192, 2017; Guimas Almeida, C., et al., Cell. Mol. Life Sci. 75, 2577-2589, 2018). Among them, phosphatidylinositol-binding clathrin assembly protein, which plays an important role in clathrin-mediated endocytosis (CME) (Xu, W. et al., Mol. Neurobiol. 52, 399-413, 2015) PICALM, which encodes (phosphatidylinositol binding clathrin assembly protein, PICALM), is a common risk factor for both AD and DM (Vacinova, G. et al., Mol. Biol. Rep. 44, 227-231, 2017; McFall, GP et al., Alzheimers Dement (Amst), 1, 395-402, 2015). However, the mechanisms by which DM regulates PICALM and possibly contributes to AD pathology through phagocytosis are not well understood. Therefore, studies on the role of PICALM in DM conditions should provide important clues in alleviating endosomal dysfunction. On the other hand, since endosomes are degraded through the autophagy pathway or direct fusion with intact lysosomes (Colacurcio, DJ et al, Free. Radic. Biol. Med., 114, 40-51, 2018), Injury results in dysregulation of early endosomes. In the past, phagocytic substances such as APP substrate and secretase were used in AD-induced dysfunctional autophagy vacuoles (Yu, WH et al., J. Cell Biol. 171, 87-98, 2005) and lysosomes (Yang, DS et al. , Brain, 137, 3300-3318, 2014) are known to be abundant. Moreover, this impairment functionally delays the turnover of endosomal migration, resulting in incomplete endosomal clearance (Lee, J. H. et al., Cell reports, 12, 1430-1444, 2015). Although DM damages the intracellular clearance system (Gonzalez, CD et al., Autophagy, 7, 2-11, 2014) or its overactivation (Moruno, F. et al., Cells, 1, 372-395, 2012) Although it is debated whether it induces an altered clearance system, it may affect the early endosomal degradation associated with memory loss.

In order to confirm that early endosomal expansion is a common risk factor for AD in various types of DM, we conducted a streptozotocin (STZ)-induced mouse model (Furman) for type 1 and type 2 DM, respectively. , BL, Curr. Protoc. Pharmacol. 70, 5-47, 2015) and a Zucker diabetic fatty (ZDF) mouse model (Srinivasan, K. & Ramarao, P., Indian. J. Med. Res. 125, 451-472, 2007) were used. To investigate the exact molecular mechanisms involved, the human neuroblastoma cell line, SK-N-MC, was used, and high glucose treatment was applied to generate DM. Using this experimental model, we hypothesized that DMdl is a metabolic risk factor for AD by upregulation of Aβ production through induction of early endosomal disturbance. To elucidate this, the role of expanded early endosomes in Aβ production and related specific regulatory mechanisms was investigated. In addition, the present inventors considered the effect of modulating early endosomes as a potential strategy for treatment for AD under DM conditions.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising a PICALM inhibitor and an mTORC1 inhibitor as active ingredients.

Another object of the present invention is to provide a food composition for preventing or improving neurodegenerative diseases comprising a PICALM inhibitor and an mTORC1 inhibitor as active ingredients.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising a PICALM inhibitor and an mTORC1 inhibitor as active ingredients.

The present invention also provides a food composition for preventing or improving neurodegenerative diseases comprising a PICALM inhibitor and an mTORC1 inhibitor as active ingredients.

The present invention regulates PICALM and mTORC1, suppresses PICALM and mTORC1-mediated early endosome disorders in a high glucose environment, and suppresses excessive production of amyloid beta, thereby preventing or effectively treating neurodegenerative diseases such as Alzheimer's disease at an early stage. It can be widely used in various industrial fields such as the pharmaceutical industry and the bio industry.

1 schematically shows a mechanism for upregulating the production of amyloid beta through early endosomal disturbance under high glucose conditions. High glucose concentrations increase PICALM, AP2A1 and CHC expression through ROS-stimulated Sp1 nuclear translocation, and PICALM promotes clathrin-mediated APP phagocytosis in lipid rafts, inducing early endosomal expansion, while high glucose concentrations It has been shown to impair endosomal clearance through AMPK/mTORC1- and ROS-mediated self-lysosomal dysregulation.
2-12 show that high glucose concentration upregulates Aβ production through early endosomal expansion. Figures 2 to 4 show samples obtained from ZLC and ZDF rat hippocampus. ^** p<0.01 vs. ZLC mice. 5 and 6 show C99, Rab5 and β-actin detected by Western blot. n = 4. 3 , Aβ 1-42 was measured by Aβ 1-42 specific ELISA assay. n=6. 4 and 6 show tissue slides for immunohistochemistry (IHC), immunostained with Rab5-specific antibody and counterstained with DAPI. Scale bar, 50 µm (magnification X200). n=5. 7 and 8 show the results of treating SK-N-MC cells with D-glucose (25 mM) or L-glucose (25 mM) for 24 hours or 48 hours, respectively. FIG. 7 shows C99 and Rab5 detected by Western blot. and β-actin (n=4), and FIG. 8 shows Aβ 1-42 derived from cell culture medium as measured by Aβ 1-42 specific ELISA assay (n=4). 9 shows the results of cells treated with high concentration of glucose (25 mM) for 24 hours, and immunoprecipitation of active Rab5 (GTP-binding Rab5) with a Rab5-specific antibody (upper panel). Expression of Rab5 and β-actin in whole lysates is shown in the middle and bottom panels. n=4. 10 and 11 show the results of immunoprecipitation of cells with Rab5- and C99-specific antibodies and counterstaining with DAPI. Scale bar, 8 μm (magnification X1,000). n=6. ^* p<0.05, ^** p<0.01 vs. control group. 12 shows cells transformed with NT siRNA or RAB5 siRNA for 12 hours before treatment with high concentration of glucose (25 mM) for 48 hours. Aβ 1-42 from cell culture medium was measured by Aβ 1-42 specific ELISA assay. n=3. ^** p<0.01 vs. NT siRNA transfection, ^## p<0.01 vs. High glucose concentration with NT siRNA transfection.
13 shows that there is no effect of high glucose concentration on the mRNA expression of Rab5 and its effector protein.
14-18 show that high glucose concentration promotes lipid raft-mediated APP phagocytosis, resulting in early endosomal expansion. 14 shows the results of biotin surface labeling and internalization analysis in SK-N-MC treated with high concentration of glucose (25 mM) for 24 hours. 0 and 15 min represent internalization time points. Western blot was performed on the whole lysate (bottom). APP and β-actin were detected. The proportion of internalized APP was measured by normalizing to total APP at 15 minutes in each condition. n=4. 15 is a result of treating cells with high concentration of glucose (25 mM) for 24 hours, and performing Western blot on the sucrose gradient-fractionated lysate. APP, CAV1 and FLOT1 were detected. n=3. 16 and 17 show the results of incubating cells with MβCD (1 mM) for 30 minutes before treatment with high concentration of glucose (25 mM) for 24 hours. 16 shows biotinylation of the cell surface and separation of the labeled protein with streptavidin beads. Surface APP, total APP and β-actin were detected by Western blot. n=4. 17 is a result of immunostaining the cells with an Rba5-specific antibody and counterstaining with DAPI. Scale bar, 8 µm (magnification X1,000). n=4. 18 shows the results of pretreatment with MβCD (1 mM) for 30 minutes before high-concentration glucose (25 mM) treatment for 48 hours, and measuring Aβ 1-42 derived from a cell culture medium by human Aβ 1-42 specific ELISA assay. . n=3. ^* p<0.05, ^** p<0.01 vs. Control, ^# p<0.05, ^## p<0.01 vs. high concentration of glucose.
19-25 show that high glucose concentration increases PICALM, AP2A1 and CHC expression through ROS-stimulated Sp1 nuclear translocation. 19 shows the results of processing SK-N-MC with high concentration of 25 mM glucose in a time-dependent manner, measuring intracellular ROS by DCF-DA staining, and then analyzing the results by flow cytometry. n=3. 20 and 21 show the results of incubating cells with NAC (4 mM) for 30 minutes before treatment with high concentration of glucose (25 mM) for 6 hours. 20 is a Western blot showing the detection results of Sp1, β-tubulin and lamin A/C in cytoplasmic and nuclear fraction samples. n=4. 21 is a result of immunostaining cells with Sp1-specific antibody and counterstaining with DAPI. Scale bar, 8 µm (magnification X1,000). n=4. 22 shows the results of pretreatment of cells with mithramycin A (25 mM) before treatment with high concentration of glucose (25 mM) for 12 hours. The mRNA expression levels of PICALM, AP2AP1 and CHC were analyzed by quantitative real-time PCR. n=4. 23 shows the results of incubating cells with mithramycin A (25 mM) before high-concentration glucose (25 mM) treatment for 24 hours. Western blots were performed for PICALM, AP2AP1, CHC and β-actin. n=4. ^* p<0.05, ^** p<0.01, ^*** p<0.001 vs. Control, ^# p<0.05, ^## p<0.01, ^### p<0.001 vs. high concentration of glucose. Figure 24 shows hippocampal samples obtained from ZLC and ZDF mice, Western blots were performed for PICALM, AP2AP1, CHC and β-actin. n=4. ^* p<0.05, ^** p<0.01 vs. ZLC mice. Figure 25 Western blots were performed for PICALM, AP2AP1, CHC and β-actin on hippocampal samples obtained from vehicle- and STZ-treated mice. n=4. ^* p<0.05, ^** p<0.01 vs. Vehicle-treated mice.
26 shows that PICALM is localized in lipid rafts in both normal and high glucose conditions. PICALM, CAV1 and FLOTI were detected. n=3.
27 to 34 show that PICALM promotes clathrin-mediated APP phagocytosis under high glucose conditions. 27, 28, 30 and 31 show the results for SK-N-Mc transfected with NT siRNA or PICALM siRNA before high glucose treatment (25 mM) for 24 or 48 hours. 27 is a result of immunostaining cells with APP- and AP2A1-specific antibodies and counterstaining with DAPI. Scale bar, 8 µm (magnification X1,000). n=4. Figure 28 shows the results of immunostaining cells with APP- and CHC-specific antibodies and counterstaining with DAPI. Scale bar, 8 µm (magnification X1,000). n=4. 29 is a result of internalization analysis of cells treated with high concentration of glucose (25 mM) for 24 hours by immunostaining using Aβ- and PICALM-specific antibodies. 0 and 0 and 15 min represent internalization time points. Scale bar, 8 μm (magnification X1,000). 30 is a result of biotinylating the cell surface and separating the labeled protein with streptavidin beads. Surface APP, total APP and β-actin were detected by Western blot. n=4. 31 is a result of measuring Aβ 1-42 derived from a cell culture medium by an Aβ 1-42 specific ELISA assay. n=3. ^** p<0.01, ^*** p<0.001 vs. NT siRNA transfection, ^## p<0.01, ^### p<0.001 vs. High glucose concentration with NT siRNA transfection. 32 and 33 show the results of pre-treating cells with dynaso (25 μm) for 30 minutes before high-concentration glucose (25 mM) treatment for 24 hours. 32 is a result of biotinylating the cell surface and separating the labeled protein with streptavidin beads. Surface APP, total APP and β-actin were detected by Western blot. n=4. 33 is a result of immunostaining the cells with a Rab5-specific antibody and counterstaining with DAPI. Scale bar, 8 µm (magnification X1,000). n=5. 34 is a result of incubating cells with dynaso (25 μm) for 30 minutes before treatment with high concentration of glucose (25 mM) for 48 hours. These are the results of measuring Aβ 1-42 derived from a cell culture medium by an Aβ 1-42 specific ELISA assay. n=3. ^** p<0.01, ^*** p<0.001 vs. Control, ^# p<0.05, ^## p<0.01, ^### p<0.001 vs. high concentration of glucose.
35, 36 and 39-44 show that high glucose concentration impairs endosomal clearance through AMPK/mTORC1-mediated autophagy defects. Fig. 35 shows hippocampal samples obtained from ZLC and ZDF mice, and LC3, P62 and β-actin were detected by Western blot. n=4. ^** p<0.01, ^*** p<0.001 vs. ZLC mice. Fig. 36 shows hippocampal samples obtained from vehicle- and STZ-treated mice, and detection of LC3, P62 and β-actin by Western blot. n=3. ^** p<0.01 vs. Vehicle-treated mice.
37 and 38 show that high concentration of glucose impairs autophagy. ^* p<0.05 vs. Control, ^** p<0.01 vs control. FIG. 37 shows the results of processing SK-N-MC with high concentration of glucose (25 mM) in a time-dependent manner, and performing Western blots for LC3, P62 and β-actin. n=4. FIG. 38 shows the results of cells treated with D-glucose (25 mM) or L-glucose (25 mM) for 24 hours, and Western blotting was performed for LC3, P62 and β-actin. n=4.
Fig. 39 shows SK-N-MC treated with high concentration of glucose (25 mM) in a time-dependent manner, and p-AMPKα (Thr172), t-AMPKα, p-mTOR (Ser2448), t-mTOR and β-actin are treated by Western blot. was detected as n=4. 40, 41 and 43 show the results of incubating the cells with rapamycin (200 nM) for 30 minutes before treatment with high concentration of glucose (25 mM) for 24 hours. 40 is a result of Western blotting for LC3, P62 and β-actin. n=4. Figure 41 shows the results of immunostaining the cells with an LC3-specific antibody and staining with Lysotracker red and DAPI. Scale bar, 8 µm (magnification X1,000). Figure 42 shows the results of pretreatment of cells with PF-4708671 (10 μm) or rapamycin (200 nM) for 30 minutes before treatment with high concentration of glucose (25 mM) for 24 hours. Rab5 and β-actin were detected by Western blot. n=5. 43 is a result of immunostaining cells with Rab5-specific antibody and counterstaining with DAPI. Scale bar, 8 µm (magnification X1,000). n=4. 44 is a result of pretreatment of cells with rapamycin (200 nM) for 30 minutes before treatment with high concentration of glucose (25 mM) for 48 hours. These are the results of measuring Aβ 1-42 derived from a cell culture medium by an Aβ 1-42 specific ELISA assay. n=3. ^* p<0.05, ^** p<0.01, ^*** p<0.001 vs. Control, ^# p<0.05, ^## p<0.01 vs. high concentration of glucose.
45 and 47-56 show that ROS- and mTORC1-mediated lysosomal dysfunction exacerbate endosomal clearance and upregulate Aβ production. 45 and 55 show the results of treating SK-N-MC with high concentration of glucose (25 mM) for 24 hours. 45 shows the results of immunostaining cells with Rab5-specific antibody and staining with Lysosomal Tracker Red and DAPI.
Figure 46 shows that the high concentration of glucose does not have a significant change in the mRNA expression of the endolysosomal fusion-related protein. SK-N-MC was treated with high concentration of glucose (25 mM) in a time-dependent manner, and mRNA expression levels of ATG14, SNAP29, VTI1B, STX17 and VAMP7 were analyzed by quantitative real-time PCR. n=5.
FIG. 47 shows the results of high-concentration glucose (25 mM)-treated cells labeled with Lysosomal Tracker Red and analyzed in a time-dependent manner with a flow cytometer. n=3. ^*** p<0.001 vs. control group. Fig. 48 shows hippocampal samples obtained from ZLC and ZDF mice, and LAMP1 and β-actin were detected by Western blot. n=3. ^** p<0.01 vs. ZLC mice. Fig. 49 shows hippocampal samples obtained from vehicle- and STZ-treated mice, and LAMP1 and β-actin were detected by Western blot. n=3. ^*** p<0.001 vs. Vehicle-treated mice. 50 is a result of Western blotting on cells treated with 25 mM high-concentration glucose in a time-dependent manner. LAMP1 and β-actin were detected by Western blot. n=3. 51 and 53 show the results of pretreating cells with NAC (4 mM) for 30 minutes before treatment with high concentration of glucose (25 mM) for 24 hours. Figure 51 shows the detection of LAMP1 and β-actin by Western blot. n=4. 52 and 54 show the results of incubating cells with rapamycin (200 nM) for 30 minutes before treatment with high concentration of glucose (25 mM) for 24 hours. Figure 52 shows the detection of LAMP1 and β-actin by Western blot. n=4. 53 and 54 show the results of measuring intact lysosome and intracellular pH by lysosomal tracker red and BCECF-AM staining, respectively. n=3. 55 is a result of immunostaining the cells with a CTSB-specific antibody and staining with Lysosomal Tracker Red. Figure 56 is a result of pretreatment of cells with leupeptin (100 nM) for 30 minutes before treatment with high concentration of glucose (25 mM) for 48 hours. These are the results of measuring Aβ 1-42 derived from a cell culture medium by a human Aβ 1-42 specific ELISA assay. n=3. ^* p<0.05, ^** p<0.01, ^*** p<0.001 vs. Control, ^# p<0.05, ^## p<0.01 vs. high concentration of glucose.
Figures 57 to 59 and Figures 61 to 64 show that in the STZ-induced DM mouse model, endosomal expansion, Aβ increase, and cognitive impairment were restored by dynasore and rapamycin, respectively. 57 and 61 show results of immunostaining of hippocampal slides for IHC with Rab5-specific antibody and counterstaining with DAPI. Scale bar, 50 µm (magnification X200). n=4-5. 58 and 63 show the results of measuring hippocampal Aβ 1-42 concentrations by ELISA assay specific to rat and mouse Aβ 1-42. n=4-5. 59 and 64 are results of performing a Y-maze experiment on mice for evaluating cognitive function. n=4-5. Figure 60 shows that treatment with rapamycin attenuates STZ-induced phosphorylation of mTORC1. Western blots were performed for p-mTOR (Ser 2448), t-mTOR and β-actin. n=4. ^* p<0.05 vs. Vehicle-treated mice, ^# p<0.05 vs. STZ-treated mice. Figure 62 shows the results of performing Western blot on hippocampal Rab5 and β-actin. n=4. ^** p<0.01, ^*** p<0.001 vs. Vehicle-treated mice, ^# p<0.05, ^## p<0.01, ^### p<0.001 vs. STZ-treated mice.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art.

Specific structural or functional descriptions presented in the embodiments of the present invention are only exemplified for the purpose of describing embodiments according to the concept of the present invention, and the embodiments according to the concept of the present invention may be implemented in various forms. In addition, it should not be construed as being limited to the embodiments described herein, and it should be understood to include all modifications, equivalents and substitutes included in the spirit and scope of the present invention.

In the present invention, the effect of high concentration of glucose on endosomes and related signaling pathways producing amyloid beta (Aβ) was investigated. We found that high glucose concentrations upregulate Aβ production through early endosomal expansion achieved by an increase in phosphatidylinositol binding clathrin assembly protein (PICALM)-mediated APP phagocytosis in lipid rafts. On the other hand, it was found that early endosomal expansion also induces dysfunction of AMPK/mTORC1 (mammalian target of rapamycin complex 1)-mediated autophagy and ROS- and mTORC1-mediated lysosomal dysfunction. In addition, it was found that increased Aβ production and cognitive deficits in the DM model were recovered by inhibition of early endosomal expansion. These results demonstrated that high glucose concentration induces early endosomal perturbation through PICALM-induced APP phagocytosis and ROS- and mTORC1-inhibiting endosomal clearance, which upregulates Aβ production.

The present invention has identified that high concentration of glucose induces amyloid beta production and cognitive impairment through induction of PICALM and mTORC1-mediated early endosomal disorders. In other words, the present invention demonstrated that high glucose upregulates the increase in PICALM-mediated APP phagocytosis and the production of Aβ through early endosomal perturbation induced by impairment of ROS- and mTORC1-mediated autolysosomal pathways. (Fig. 1). As shown in Figure 1, early endosomal disruption occurs by two mechanisms: increased influx of substances into early endosomes due to increased phagocytosis in the lipid raft of the PICALM-mediated Aβ precursor protein; and impairment of early endosomal clearance due to inhibition of mTORC1 and ROS-mediated cellular autophagy. Disorder of early endosomes due to the above mechanism promotes excessive production of Aβ, thereby causing cognitive impairment. Accordingly, the present invention relates to a technology for inhibiting early endosomal disorders through regulation of PICALM and mTORC1, and to a technology for preventing and inhibiting excessive production of Aβ in a diabetic environment. The present invention is the first study to identify early endosomal dysregulation and its precise mechanism in Aβ pathology under DM conditions. This means that it is possible to prevent and treat both diabetes and Alzheimer's disease by correcting early endosomal disorders while confirming that diabetes is an important factor inducing Alzheimer's disease. and to provide strong evidence that it is a promising strategy for managing the comorbidities of Alzheimer's disease.

The present invention in one aspect, PICALM (phosphatidylinositol binding clathrin assembly protein; phosphatidylinositol binding clathrin assembly protein) inhibitor and mTORC1 (mammalian target of rapamycin complex 1, mammalian target of rapamycin complex 1) neurodegenerative comprising an inhibitor as an active ingredient It relates to a pharmaceutical composition for preventing or treating a disease.

In another aspect, the present invention relates to a food composition for preventing or improving neurodegenerative diseases comprising a PICALM inhibitor and an mTORC1 inhibitor as active ingredients.

In the present invention, the PICALM inhibitor and the mTORC1 inhibitor may be characterized in that it inhibits the initial endosomal disorder caused by high glucose concentration.

In the present invention, the PICALM inhibitor and the mTORC1 inhibitor may be characterized in that the inhibition of amyloid beta production.

In the present invention, the PICALM inhibitor may be characterized in that it is mithramycin A, but is not limited thereto.

In the present invention, the mTORC1 inhibitor is at least one selected from the group consisting of rapamycin, deforolimus, everolimus, temsirolimus, torin1 and PP242. It may be characterized, but is not limited thereto.

In the present invention, the neurodegenerative disease is Alzheimer's disease, Huntington's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, Lou Gehrig's disease, frontotemporal dementia, cortical-basal ganglia degeneration, immune system dysfunction, brain ischemia and cerebral hemorrhage It may be characterized in that at least one selected from the group consisting of dementia, metabolic brain disease, and progressive supranuclear palsy, but is not limited thereto.

In the present invention, the term "prevention" refers to any action that inhibits or delays the onset of neurodegenerative diseases by administration of the pharmaceutical composition according to the present invention, and "treatment" refers to any action by administration of the pharmaceutical composition. It refers to any action that improves or beneficially changes the symptoms of a suspected or onset neurodegenerative disease.

The pharmaceutical composition for the prevention or treatment of neurodegenerative diseases of the present invention may further include an appropriate carrier, excipient or diluent commonly used in the preparation of the pharmaceutical composition. Specifically, the pharmaceutical composition is formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions, respectively, according to a conventional method. can be used In the present invention, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract and its fractions, for example, starch, calcium carbonate, It is prepared by mixing sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to simple diluents such as water and liquid paraffin, which are commonly used for suspensions, solutions, emulsions, and syrups. there is. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The content of the PICALM inhibitor and the mTORC1 inhibitor included in the pharmaceutical composition of the present invention is not particularly limited thereto, but is to be included in an amount of 0.0001 to 50% by weight, more preferably 0.01 to 20% by weight, respectively, based on the weight of the total composition. can

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, and the term "pharmaceutically effective amount" refers to an amount sufficient to treat or prevent a disease at a reasonable benefit/risk ratio applicable to medical treatment or prevention. Meaning, the effective dose level is the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the administration time of the pharmaceutical composition of the present invention used, the route of administration and the rate of excretion treatment The duration may be determined according to factors including drugs used in combination or concomitant with the composition of the present invention used and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. and may be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects.

The dosage of the pharmaceutical composition of the present invention may be, for example, 0.1 to 500 mg/kg body weight of the pharmaceutical composition of the present invention to mammals including humans per day. In addition, the frequency of administration of the pharmaceutical composition of the present invention is not particularly limited thereto, but may be administered once a day or administered several times by dividing the dose. The above dosage does not limit the scope of the present invention in any way.

In the present invention, the pharmaceutical composition may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

The contents of the PICALM inhibitor and the mTORC1 inhibitor included in the food composition of the present invention are not particularly limited thereto, but may be included in an amount of 0.0001 to 50% by weight, more preferably 0.01 to 20% by weight, respectively, based on the weight of the total composition. there is.

The food composition of the present invention may further contain nutrients, vitamins, electrolytes, sweeteners, colorants, organic acids, preservatives, and the like, and these components may be used independently or in combination.

Processed foods capable of preventing or improving neurodegenerative diseases can be prepared by using the food composition of the present invention, for example, confectionery, beverage, alcoholic beverage, fermented food, canned food, milk processed food, processed meat food or processed noodles. It can be manufactured as a health functional food in the form of food. In this case, the confectionery includes biscuits, pies, cakes, bread, candy, jelly, gum, cereals (including meal substitutes such as grain flakes), and the like. The beverage includes drinking water, carbonated beverage, functional ionized beverage, juice (eg, apple, pear, grape, aloe, tangerine, peach, carrot, tomato juice, etc.), sikhye, and the like. Alcoholic beverages include sake, whiskey, shochu, beer, Western liquor, fruit wine, and the like. Fermented foods include soy sauce, soybean paste, red pepper paste, and the like. Canned foods include canned seafood (eg, canned tuna, mackerel, saury, conch, etc.), canned livestock products (canned beef, pork, chicken, turkey, etc.), and canned agricultural products (canned corn, peaches, file apples, etc.). Milk products include cheese, butter, yogurt, and the like. Processed meat products include pork cutlet, beef cutlet, chicken cutlet, and sausage. Includes sweet and sour pork, nuggets, and breadcrumbs. Noodles such as sealed packaged raw noodles are included. In addition to this, the composition may be used in retort foods, soups, and the like.

In another aspect, the present invention comprises the step of administering a composition for the prevention or treatment of neurodegenerative diseases comprising the PICALM inhibitor and the mTORC1 inhibitor as an active ingredient in a pharmaceutically effective amount to an individual suffering from a neurodegenerative disease It relates to a treatment method for neurodegenerative diseases.

In the present invention, the term "individual" includes, without limitation, mammals including rats, livestock, humans, etc. that are likely to or have developed a neurodegenerative disease.

In the method of treating a neurodegenerative disease of the present invention, the administration route of the pharmaceutical composition may be administered through any general route as long as it can reach the target tissue. The pharmaceutical composition of the present invention is not particularly limited thereto, but routes such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, intranasal administration, intrapulmonary administration, rectal administration, etc. can be administered through However, during oral administration, since the extract or a fraction thereof may be denatured by gastric acid, the oral composition must be formulated to coat the active agent or to protect it from degradation in the stomach. In addition, the composition may be administered by any device capable of transporting the active agent to a target cell.

[Example]

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Example 1: Method

1-1: Experimental material

Cells derived from the human neuroblastoma cell line SK-N-MC were obtained from the Korean Cell Line Bank (Seoul, Korea). Fetal bovine serum (FBS) and antibiotics were purchased from Hyclone (Logan, UT, USA) and Gibco (Grand Island, NY, USA), respectively. β-actin (sc-47778), CAV1 (sc-53564), FLOT1 (sc-74566), CHC (sc-12734), PICALM (sc-271224), lamin A/C (sc-2068), LAMP1 (sc -20011), p-AMPKα (Thr 172) (sc-33524), BACE 1 (sc-33711), and cathepsin B (sc-365558) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). . Antibodies of β-tubulin (CSB-PA03874A0Rb) and AP2A1 (610502) were purchased from Cusabio (Wuhan, Hubei, China) and BD biosciences (San Jose, CA, USA), respectively. Antibodies of Rab5 (NB120-13253), P62 (NBP1-48320), and LC3 (NB100-2220) were purchased from Novus Biologicals (Centennial, CO, USA). Antibodies of p-mTOR (Ser 2448) (2971S), mTOR (2983 S) and AMPK (2532 S) were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies of APP (ab32136), Sp1 (ab227383), and Aβ (ab2539) were purchased from Abcam (Cambridge, England). The antibody of C99 (802801) was obtained from Biolegend (San Diego, CA, USA). MβCD, dynaso hydrate, rapamycin, NAC, DAPI, D-glucose, L-glucose, leupeptin and STZ were purchased from Sigma Chemical Company (St Louis, MO, USA). GM6001 and misramycin A were obtained from Cayman Chemical (Ann Arbor, MI, USA) and Tocris Bioscience (Bristol, UK), respectively. mRNA primers for PICALM, AP2A1, CHC, RAB5, EEA1, RABGEF1, RABEP1, APPL1, ATG14, SNAP29, VTI1B, STX17, VAMP7, and ACTB were purchased from Cosmo Genetech (Cosmo Genetech, Seoul, Korea). siRNAs for RAB5 and PICALM were obtained from Bioneer (Bioneer, Daejeon, Korea). NT siRNA was purchased from Dharmacon (Lafayette, CO, USA).

1-2: experimental animals

Male and female heterozygous (Leprfa/+) ZDF mice (rat) were obtained from Genetic Models Co. (Indianapolis, USA) and bred to obtain homozygous ZDF lean control (ZLC) and ZLC mice. . Rats were bred under standard environmental conditions set at an appropriate temperature (20-25° C.) and humidity (less than 60%) with a 12-hour light/12-hour dark cycle. Rats were given ad libitum access to an appropriate diet (Purina 5008, Purina Korea, Korea; recommended by Genetic Models Co.) and tap water. Animal handling and care was performed in accordance with the guidelines established by international law and policy (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No 85-23, 1985, revised 2011). The experimental protocol was approved by the Animal Experimental Ethics Committee of Seoul National University (approval number: SNU-140219-1). Male ICR mice (9 weeks old) were purchased from the Hallym Laboratory for Animal Research (Suwon, Korea) and bred under the existing environmental conditions (20-25°C, 60% humidity and 12 hours light/12 hours dark cycle). Mice were given free access to food and drinking water. Animal handling and management were performed and approved in accordance with the Animal Experimental Ethics Committee of Seoul National University (approval number: SNU-190122-1).

1-3: Animal Experimental Design

To investigate the effect of dysmorphic diabetes mellitus (DM) on early endosomal disturbance, male rats were divided into two groups: ZLC and ZDF groups (n=6 per group). For chronic heterozygous DM, mice were maintained until 33 weeks of age. Blood glucose and body weight were measured at 6 and 33 weeks with a portable glucose monitor (ACCU-CHEK® GO; 327 Roche, Mannheim, Germany) and a balance, respectively (Table 1).

Body weight and blood glucose in ZLC and ZDF rats ZLC ZDF Weight (g), 6 weeks 119±1.82 144.4±5.22 Weight (g), 33 weeks 394.2±9.05 355.2±6.77 Blood sugar (mg/dl), 6 weeks 102.8±5.38 153.3±15.85 Blood Sugar (mg/dl), Week 33 124.5±5.05 575.9±25.53

(Data is mean±SEM)

Rats were euthanized at 33 weeks of age. To investigate the effect of type 1 DM on endosomal dysregulation, STZ-induced DM mice were generated according to conventional methods (Furman, BL, Curr. Protoc. Pharmacol, 70, 5-47, 2015; Oh, JY). et al., Free. Radic. Biol. Med. 130, 328-342, 2019). Briefly, mice were randomly selected for intraperitoneal injection of STZ in 200 μl sodium citrate buffer (0.1M, pH 4.5). Blood glucose concentrations were measured every 2 days starting 72 hours after STZ injection with a portable glucose monitor (ACCU-CHEK® GO; 327 Roche, Mannheim, Germany). Mice with blood glucose concentrations exceeding 300 mg/dl were considered severe diabetes (Furman, B. L., Curr. Protoc. Pharmacol, 70, 5-47, 2015). Seven days after STZ injection, a sufficient number of diabetic mice were obtained. Dyansore (80 μM) (Hansen. C. et al., J. Clin. Invest. 121, 715-725, 2011) and 4 μl DMSO (1:1,000 with PBS) were injected into mice intraventricularly (ICV) (Kim, HY) et al., J. Vis. Exp. 109, e53308, 2016), and mice were randomly divided into 4 groups (n=6 in each group). vehicle; STZ; STZ + Dyansore; and Dyansore. Drugs were injected twice on days 5 and 21 after DM induction. Rapamycin (8.5 mg/kg) in 200 μl solution containing 99% corn oil (Sigma, #C8267) and 1% DMSO (Zhou, J. et al., J. Neurosci. 29, 1773-1783, 2009) Mice were injected IP, and mice were randomly divided into 4 groups (n=6 in each group). vehicle; STZ; STZ + rapamycin; and rapamycin. From the 5th day after DM induction, the drug was injected once a day for 5 consecutive days. Blood glucose concentration and body weight were measured at 9 and 18 weeks of age (Table 2).

Body weight and blood glucose concentration of experimental mice vehicle Dynasore STZ STZ
+ Dynasore Weight (g), 9 weeks 38.5±0.42 37.17±0.7 37.5±1.58 39±0.63 Weight (g), 18 weeks 42.33±1.45 44.83±0.87 34.83±1.62 42.6±1.20 Blood sugar (mg/dl), 9 weeks 116.3±4.64 137.7±7.08 120.3±15.02 135.7±7.24 Blood sugar (mg/dl), 18 weeks 126.7±6.29 131.8±4.64 497.7±20.49 439.6±35.16 vehicle rapamycin STZ STZ
+ Rapamycin Weight (g), 9 weeks 37.33±0.33 36.33±1.08 37±1.09 37.67±0.55 Weight (g), 18 weeks 44.83±1.24 43.5±0.92 40.67±1.47 46.67±1.145 Blood sugar (mg/dl), 9 weeks 126.7±7.37 116.5±7.85 118.2±9.26 118.5±5.38 Blood sugar (mg/dl), 18 weeks 127.3±8.78 120.8±6.39 484.7±5.94 503.7±10.56

(Data is mean±SEM)

At 18 weeks of age, mice were subjected to behavioral testing and were euthanized for further biochemical experiments.

1-4: cell culture

SK-N-MC was incubated with Dulbecco's essential medium (DMEM; Hyclone, #SH30021FS), 10% FBS and 1% antibiotic-antimycotic solution with low glucose at 37° C., 5% CO ₂ . After the cells had grown to 70% confluency, they were replaced with DMEM containing 2% Knockout™ serum replacement (SR; Gibco, #10828028) and 1% antibiotic-antifungal mixture for 12 hours prior to experimentation.

1-5: Y-maze Spontaneous Alternate Test

The Y-maze behavioral test is used to assess hippocampal-dependent cognitive impairment. Rodents instinctively prefer to challenge the new arms of the Y-maze. Prior to testing, animals were placed in the test room for 2 hours to minimize the effect of stress on behavior. Mice were placed on randomly selected arms of a Y-shaped maze obtained from Sam-Jung Company (Seoul, Korea). Each mouse was allowed to freely explore through the open field for 8 min. The total number of arm entries and sequence were recorded. Only the position in which all limbs were placed on the arm was considered complete. Percent shifts are the number of triads divided by the maximum shifts (total entries-2) X 100. When the animal exhibits a lower percentage of alternation, the animal indicates impaired memory function.

1-6: Immunohistochemistry

Mice and rats were completely anesthetized and perfused with PBS followed by 4% PFA in 0.1 M phosphate buffer (pH 7.4) by transcardiac perfusion. Brains were removed and post-fixed with 4% PFA. The brains were then placed in 30% sucrose in PBS for 1-2 days. Coronal sections (40 μm thick) were obtained by continuous cutting using a cryostat (Leica Biosystems, Nussloch, Germany). The free-floating hippocampus sections were carefully processed. Sections were blocked with 5% normal goat serum (NGS; Sigma-Aldrich, #566380) for 1 h at room temperature. Samples were incubated overnight at room temperature with primary antibody (1:1,000). Sections were washed three times with PBS and incubated with secondary antibody for 2 h (1:200 dilution). Immunostained slides were visualized with an Eclipse Ts2™ fluorescence microscope (Nikon, Tokyo, Japan). All IHC images were analyzed with Fiji software.

1-7: siRNA transfection for gene silencing

When SK-N-MC was grown to 60%, it was incubated for 2 hours with low glucose DMEM containing 25 nM of the indicated siRNA, transfection reagent TurboFect™ (Thermo Fisher, #R0531), and 2% SR. . Before the experiment, the medium was replaced with a medium containing 1% antibiotics. NT siRNA was used as a negative control.

1-8: Western blot analysis

The obtained cells or tissues were incubated with appropriate lysis buffer and protease and phosphatase inhibitor cocktail (100X) (Thermo Fisher, #78440). Then, homogenization was carried out on ice for 30 minutes using an ultrasonicator and a vortexer. The lysate was clarified by centrifugation (13,000×g, 4° C., 20 min). Protein concentration was measured using BCA (bichichoninic acid) quantitative analysis (Thermo Fisher, #23227). An equal amount of sample (5-10 μg) was loaded on an 8-12% SDS-polyacrylamide gel (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. tris-buffered saline (TBS) containing 0.2% Tween-20 [TBST; 150 mM NaCl, 10 mM Tris-HCl (pH 76)] was used to wash and incubate the membrane. The membrane was blocked with 5% skim milk (Gibco, #232100) for 1 hour. The blocked membrane was washed three times and incubated overnight at 4°C dptj with primary antibody (1:1,000 dilution). Then, the membrane was washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000 dilution) at room temperature for 2 hours. Blot bands were detected using a chemiluminescence detection kit (Advansta Inc, #K-12045-D50). Protein band quantification was performed using Image J software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). Protein expression levels were normalized to β-actin expression levels. The EzSubcell™ subcellular division kit (Atto, #WSE-7421) was applied to the preparation of cell fluid and nuclear division samples. Cell fluid and nuclear samples were obtained according to the manufacturer's instructions. Protein expression levels in cell fluid and nuclear samples were normalized to β-tubulin and lamin A/C expression levels, respectively.

1-9: sucrose density gradient classification method

Wash SK-N-MC with cold phosphate buffered saline (PBS; Hyclone, #SH30256) and 1 ml lysis buffer (10 mM EDTA, 500 mM Na in sterile water) with protease and phosphatase inhibitor cocktail (100X). ₂ CO ₃ , pH 11). The lysate was homogenized using an ultrasonicator (Branson Sonicator 250, Branson Ultrasonic Corp, Danbur, CT, USA), and incubated on ice while vortexing for 30 minutes. To form a 5-45% discontinuous gradient, 4 ml of 45% sucrose and 4 ml of 5% sucrose dissolved in MES-buffer [MBS: 25 mM MES (pH65), 0.15 M NaCl] were placed in an ultracentrifuge tube (Beckman Coulter, Fullerton, CA, USA). The same amount of protein was then adjusted to form 4 ml of 5% sucrose and added to the ultracentrifuge tube. The sample tube was centrifuged for 24 hours at 40,000 rpm in a SW41 rotor (Beckman Coulter). Resolved samples were obtained and analyzed by Western blot.

1-10: Immunocytochemical analysis

SK-N-MC was washed twice with PBS and fixed with 4% paraformaldehyde (PFA; Lugen Sci, Seoul, Korea, #LGB-1175) for 10 minutes. To permeate the cell membrane, cells were incubated with 0.2% TBST or 0.1% Triton X-100 (Sigma, T8787) for 10 minutes. Cells were blocked with 5% NGS for 40 min and incubated overnight at 4°C with primary antibody (1:100 dilution). Cells were then washed three times with PBS and incubated with Alexa Fluor™ 488 or 555-conjugated secondary antibody (1:200 dilution) at room temperature for 2 hours. Immunostained samples were visualized with a super-resolution radial fluctuations (SRRF) imaging system (Andor Technology, Belfast, UK) (Gustafsson, N et al., Nat. Commun. 7, 12471, 2016). Using Fiji software, fluorescence intensity was quantified and co-localization was analyzed by manders count.

1-11: Measurement of intracellular pH and reactive oxygen species (ROS)

cell-permeable pH-sensitive fluorescent probe BCECF-AM [2',7'-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester; Thermo Fisher, #B1150] and CM-H2DCFDA (Thermo Fisher, #C6821) were used to measure intracellular pH and ROS, respectively. After drug treatment, cells were incubated with 2 μM BCECF-AM or 10 μM DCFDA in media and maintained at 37° C. for 30 minutes. Then, the cells were washed three times with PBS. Signals of BCECF-AM- and DCFDA-stained cells were measured by flow cytometry (Beckman Coulter, Atlanta, USA).

1-12: Staining of intact lysosomes

Lysotracker deep red (Lysotracker deep red, Thermo Fisher, #L12492) was used for intact lysosomal staining. After drug treatment, cells were incubated with 2 μM Lysosomal Tracker Red in medium and maintained at 37° C. for 30 minutes. Then, the cells were washed three times with PBS. Lysosomal tracker-stained signals of cells were measured by flow cytometry (Beckman Coulter, Atlanta, USA), or cells were further tested by ICC.

1-13: Quantitative Real-Time Polymerase Chain Reaction (qPCR)

RNA samples were extracted using an RNA extraction kit (TaKaRa, #9767) according to the manufacturer's instructions. Then, 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 samples were amplified using a Rotor-Gene 6000 real-time thermal cycling system (Corbett Research, NSW, Australia) with the mRNA primers shown herein and TB™ Green Premix Ex Taq™ (TaKaRa, #RR420A). qPCR was performed as follows: DNA polymerase activation at 95 °C for 15 min and 60 cycles of 94 °C for 20 s, 55 °C for 20 s and 72 °C for 30 s. The specificity and identity of the amplified product was verified by melting curve analysis. Quantification of mRNA expression levels of target genes was performed by double delta Ct analysis, and data were normalized to those of the ACTB gene.

1-14: Determination of Aβ in in vitro and in vivo samples

In vitro, a culture medium of SK-N-MC was obtained and stored at -70°C with a phosphatase inhibitor cocktail (100X). In vivo, hippocampal samples (200 μg) from equal amounts of sacrificed animals were prepared with the appropriate lysis buffer and BCA assay. According to the manufacturer's instructions, the optical density (OD) of Aβ(1-42) in culture medium and animal samples was measured with human Aβ(1-42) ELISA kit (Thermo Fisher, #KHB3544) and mouse Aβ(1-42), respectively. It was obtained using an ELISA kit (Thermo Fisher, #KMB3441). OD values were converted to concentrations using a standard curve.

1-15: GTP-Rab5 activation assay

Measurement of Rab5 activation was performed using the Rab5 activation assay kit (Neweast Bioscience, #83701). All procedures were performed according to the manufacturer's instructions. Briefly, cells were incubated with lysis buffer containing the provided anti-active Rab5 monoclonal antibody. The bound active Rab5 was then pulled down from the lysate using A/G agarose. The precipitated active Rab5 was detected by Western blot using a Rab5-specific polyclonal antibody. The ratio of expression levels of active Rab5 and total Rab5 was analyzed.

1-16: APP internalization analysis

SK-N-MC was washed with cold PBS and incubated with Aβ antibody (1:100 dilution) at 4° C. for 1 hour to label surface APP. Cells were washed twice with cold PBS and incubated at 37° C. for 0 and 15 min to allow internalization. Internalization was stopped by rapid cooling in ice. Cells were fixed at room temperature with 4% PFA for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 5% NGS for 40 min. Cells were incubated overnight at 4°C with primary antibody (1:100 dilution). Cells were then washed three times with PBS and incubated with Alexa Fluor™ 488 or 555-conjugated secondary antibody (1:200 dilution) at room temperature for 2 hours. Immunostained samples were visualized with an SRRF imaging system (Andor Technology, Belfast, UK).

1-17: Cell surface biotinylation assay and internalization assay

In order to prevent the protein from falling on the cell surface spontaneously, GM6001 (MMP inhibitor; 20 μM) was pretreated for 30 min before drug treatment (Kanatsu, K. et al., Nat. Commun. 5, 3386, 2014). Surface protein isolation was performed using a cell surface protein isolation kit (Biovision, #K295-10). All procedures were performed according to the manufacturer's instructions. Briefly, cells were washed with cold PBS and labeled with Sulfo-NHS-SS-Biotin, a non-membrane-permeable, thiol-degradable, biotinylated reagent. For internalization assays, biotinylated cells were incubated at 37° C. for 0 and 15 min to allow internalization. Internalization was stopped by rapid cooling in ice. In order to remove biotin remaining on the cell surface, it was incubated with MesNA (50 mM 2-mercaptoethanesulfonic acid in 50 mM Tris-HCl, 100 mM NaCl, 25 mM CaCl ₂ , and pH 87) three times at 4° C. for 20 minutes. After quenching the biotin reagent with 0.1 M glycine, the cells were lysed, and the labeled surface protein was isolated using streptavidin beads. Then, the cells were incubated with a DTT (dithiothreitol) solution, and the attached beads were released. Western blot was performed for the biotin-labeled protein.

1-18: Statistical Analysis

Quantitative data were expressed as mean ± standard error of the mean (S.E.M) and analyzed by Prism (GraphPad software, USA). ANOVA was used to analyze the differences between the experimental groups. In some cases, the Bonferroni-Dunn test was used for multiple comparisons. Results with p values <0.05 were considered statistically significant.

Example 2: Results

2-1: Upregulation of Aβ production through early endosomal expansion by high glucose concentration

To elucidate the effect of DM on Aβ-producing early endosomes, first, hippocampal C99 (APP-CTFβ) and Rab5 were measured by Western blotting. Compared to the level of ZLC mice, both proteins were increased in ADF mice (Fig. 2). In addition, ZDF mice had higher Aβ levels than ZLC mice (Fig. 3). Early endosomes were also enlarged and had a higher staining intensity in the hippocampus of ZDF mice than in ZLC mice (Fig. 4). Moreover, STZ-induced type 1 DM mice increased C99 and Rab5 compared to vehicle-treated mice ( FIG. 5 ). In addition, the early endosomes of the hippocampus of the STZ model had changes similar to those of the ZDF mice ( FIG. 6 ). In in vitro experiments involving treatment of D- or L-glucose to cells, only D-glucose increased C99 and Rab5 ( FIG. 7 ). Likewise, the level of Aβ in the cell culture medium was increased only in the D-glucose-treated-sample ( FIG. 8 ), indicating that the osmotic effect was not related to amyloidogenesis. In addition to the increase in Rab5 protein expression, high glucose concentration also increases early endosomal activation, which was measured by confirming the level of the GTP-Rab5 form ( FIG. 9 ). In order to confirm that high glucose concentration increases Aβ production in early endosomes, immunocytochemical staining was used with Rab5, beta-secretase 1 (BACE1) and C99 antibodies. As a result, high glucose concentration induces early endosomal expansion and increases co-localization with C99 and BACE1, reflecting an increase in Aβ processing in expanded early endosomes ( FIGS. 10 and 11 ). These results were confirmed by RAB5 siRNA transfection, which is in contrast to the increase in Aβ in the cell culture medium induced by high glucose concentration (Fig. 12). These findings suggest that early endosomal expansion is an important feature for the increase in Aβ under DM conditions.

2-2: Induction of early endosomal expansion by increased APP uptake in lipid rafts by high glucose concentration

Many factors contribute to early endosomal expansion, such as increased endocytosis, upregulation of the expression level of Rab5 and activation of Rab5 (Nixon, R. A. FASEB J. 31, 2729-2743, 2017). As shown in Figure 9, except for the increase in Rab5 protein expression, other factors may activate the early endosome. To investigate how high glucose concentration induces early endosomal expansion, we measured the mRNA expression of Rab5 and its effector protein, which is related to the activation of Rab5. However, no valid change could be identified ( FIG. 13 ). Next, it was hypothesized that high glucose concentration increases APP endocytosis causing early endosomal expansion. It was confirmed that high concentration of glucose increased APP intracellular uptake (FIG. 14). To investigate where APP endocytosis occurs, based on a previous study that high glucose concentrations promote lipid raft reorganization (Lee, HJ et al., Sci. Rep. 6, 36746, 2016), Focused. High glucose concentrations markedly migrate APP to the lipid raft fraction, as indicated by caveolin-1 (CAV-1) and flotilin-1 (FLOT1) (Fig. 15). Moreover, the expression level of surface APP in cells treated with high glucose and MβCD (lipid raft disruptor) was higher than in cells treated with high glucose ( FIG. 16 ). These results indicate that high glucose concentration induces lipid-raft-mediated APP endocytosis. Moreover, pretreatment with MβCD reduced high-dose glucose-induced early endosomal expansion, suggesting that endocytosis of APP in lipid rafts contributes to early endosomal expansion (Fig. 17). Thereafter, the increase in Aβ induced by high glucose concentration was blocked by disruption of the lipid raft ( FIG. 18 ). These results suggest that the initial endosomal expansion is mediated by APP endocytosis in lipid rafts under high glucose conditions.

2-3: Increase in expression of phagocytic protein by promoting Sp1 nuclear translocation by high concentration of glucose-induced ROS

How high glucose concentration promotes APP endocytosis was investigated, and it was hypothesized that high glucose concentration regulates cellular uptake-related protein. Based on the fact that ROS is the main cause of pathological conditions under high glucose conditions (Giacco, F & Brownlee, M. Circ. Res. 107, 1058-1070, 2010), ROS generation and related pathological mechanisms were investigated. The high concentration of glucose significantly increased the amount of ROS after 24 hours (FIG. 19). To investigate how high glucose-induced ROS modulates phagocytic proteins, we focused on the role of ROS in regulating cell signaling and gene expression through changes in kinase activation (Schieber, M. & Chandel, N.S.). Curr. Biol. 24, R453-462, 2014). Among the kinases regulated by ROS, Sp1 was found to be involved in phagocytic protein expression. Indeed, high glucose-stimulated Sp1 nuclear translocation was blocked by pretreatment with N-acetylcysteine (NAC; ROS scavenger) (Figure 20). Similarly, immunocytochemistry using Sp1 antibody also confirmed ROS-dependent Sp1 nuclear translocation under high glucose concentration (FIG. 21). As shown in FIGS. 22 and 23 , mRNA and protein expression levels of PICALM, adapter-associated protein complex 2 alpha 1 (AP2A1), and clathrin heavy chain (CHC) were increased by high concentration of glucose, but mithramycin A (Sp1) inhibitor) was inhibited. Moreover, compared to the levels in ZLC mice, ZDF mice had high expression levels of the hippocampal phagocytic proteins PICALM, AP2A1 and CHC ( FIG. 24 ). STZ-treated mice also had increased hippocampal PICALM, AP2A1 and CHC compared to vehicle-treated mice ( FIG. 25 ). These results suggest that high glucose concentration upregulates the expression of phagocytic proteins by ROS-stimulated Sp1 activation.

2-4: Regulation of clathrin-mediated APP endocytosis, upregulating Aβ production by PICALM

PICALM is considered a common risk factor for both AD and DM (Vacinova, G. et al., Mol. Biol. Rep. 44, 227-231, 2017; McFall, GP et al., Alzheimers Dement (Amst); 1, 395-402, 2015), which promotes clathrin-mediated endocytosis, employing CHC and AP-2 complexes (Xu, W. et al., Mol. Neurobiol. 52, 399-413, 2015). ), the role of PICALM on endocytosis under high glucose conditions and AD pathology was studied. First, changes in the intracellular localization of PICALM were investigated. PICALM was localized in lipid rafts in both normal and high glucose conditions ( FIG. 26 ). To elucidate the function of PICALM, loss-of-function experiments were designed. Using immunocytochemical staining with APP, CHC and AP2A1 antibodies, the level of co-localization of AP2AP1 and CHC of APP under high glucose in cells transfected with non-target siRNA (NT) was treated with high glucose. It was confirmed that the PICALM siRNA was higher than that of the transfected cells ( FIGS. 27 and 28 ). Moreover, high glucose concentration induced APP co-localization with PICALM during endocytosis ( FIG. 29 ). Likewise, the expression level of surface APP in cells transfected with PICALM siRNA under high glucose concentration was higher than in NT siRNA transfected cells treated with high glucose concentration ( FIG. 30 ). These results indicate that PICALM upregulated by high glucose concentration induces clathrin-mediated APP endocytosis. In addition, the amount of medium Aβ in cells transfected with high glucose-treated PICALM siRNA was lower than in high-concentration glucose-treated NT siRNA-transfected cells ( FIG. 31 ). To confirm the effect of clathrin-mediated APP uptake induced by high glucose concentration, dynasore (dynasore, a dynamin inhibitor) was used. The expression level of surface APP in the high glucose and dynaso-treated samples was higher than in the high glucose-only-treated samples ( FIG. 32 ). Moreover, dynasomal treatment reversed high glucose-induced early endosomal expansion, suggesting that clathrin-mediated APP endocytosis contributes to early endosomal expansion ( FIG. 33 ). The increase in Aβ under high glucose conditions was then blocked by inhibition of clathrin-mediated endocytosis ( FIG. 34 ). These results demonstrate that increased PICALM promotes clathrin-mediated APP endocytosis, resulting in Aβ production.

2-5: Inhibition of early endosomal clearance at high glucose concentration by AMPK/mTORC1-mediated autophagy defect

It was hypothesized that blockade of the degraded trafficking pathway also causes early endosomal expansion under high glucose conditions. Compared to the levels in ZLC mice, ZDF mice had higher expression levels of hippocampal P62 and LC3 ( FIG. 35 ). Moreover, STZ-treated mice had increased P62 and decreased LC3II/LC3I ratio compared to vehicle-treated mice ( FIG. 36 ). In the in vitro model, high glucose concentration decreased the LC3II/LC3I ratio after 12 h and increased P62 ( FIG. 37 ). Compared with L-glucose, only D-glucose decreased the LC3II/LC3I ratio and increased P62 (FIG. 38). This indicates that autophagy dysfunction was induced under DM conditions. Since the mammalian target of rapamycin complex 1 (mTORC1) is the most important regulator of autophagy, it was hypothesized that it would be a factor involved in the downregulation of autophagy under high glucose conditions. It was demonstrated that high glucose levels downregulate AMPKα phosphorylation at phosphorylated threonine 172 by increasing the AMP:ADP ratio in a time-dependent manner and phosphorylates its downstream molecule, mTORC1, at serine 2448 ( FIG. 39 ). The decreased LC3II/LC3I ratio and increased P62 induced by high glucose concentration were reversed by pretreatment with rapamycin (mTOR inhibitor) ( FIG. 40 ). Moreover, the reduction of co-localization with LC3 of lysosomes under high glucose conditions was also restored by pretreatment with rapamycin ( FIG. 41 ). To investigate the relationship between mTORC1-mediated autophagy impairment, and the S6K1-mediated translation pathway regulated by mTORC1 with increased Rab5 protein levels, PF-4708671 (S6K1 inhibitor) and rapamycin were used. The increased Rab5 protein expression level under high glucose concentration was decreased by pretreatment with rapamycin, but not by pretreatment with PF-4708671, indicating that the increase in Rab5 is dependent on defective autophagy (Fig. 42). Indeed, it was confirmed that ramaphycin attenuated the increase of Rab5 signal induced by high glucose concentration (FIG. 43). In addition, the increase in Aβ under high glucose conditions was blocked by pretreatment with rapamycin (FIG. 44). These results revealed that high glucose concentrations impair early endosomal clearance through AMPK/mTORC1-mediated autophagy defects.

2-6: Upregulation of Aβ production through impairment of endosomal clearance by lysosomal dysfunction induced by ROS and mTORC1

To further investigate endosomal degraded migration, the co-localization of Rab5 and lysosomes was tested. High glucose concentration increased the co-localization of Rab5 with lysosomes ( FIG. 45 ). In order to confirm the cause of the increase in endolysosomal fusion, screening was performed for changes in the mRNA expression level of endolysosomal fusion-related genes, but no effective findings were found ( FIG. 46 ). Therefore, it was hypothesized that the reason for the increase in fusion was lysosomal dysfunction, which impairs the degradation of enlarged endosomes. High glucose concentration decreased the intensity of the lysosomal tracker after 12 hours, which reached its worst at 36 hours ( FIG. 47 ). Hippocampal lysosomal-associated membrane protein 1 (LAMP1) was increased in ZDF mice and STZ-treated mice compared to controls ( FIGS. 48 and 49 ). In the in vitro model, LAMP1 was also increased after 12 h under high glucose conditions ( FIG. 50 ). To account for lysosomal dysfunction under DM conditions, lysosomal dysfunction is associated with ROS-mediated lysosomal membrane permeabilization (Serrano-Puebia. A. & Boya, P., Ann. NY Acd. Sci. 1371, 30-44, 2016) and Reference was made to the literature described as being caused by impairment of lysosomal quality control (Papadopoulos. C. & Meyer, H., Curr. Bio. 27, R1330-R1341, 2017). It was confirmed that pretreatment with NAC and rapamycin reversed the LAMP1 expression level increased by high glucose concentration ( FIGS. 51 and 52 ). Furthermore, attenuation of signals of both the lysosomal tracker and intracellular pH indicator BCECF-AM by high concentration of glucose was restored by pretreatment with NAC and rapamycin, respectively ( FIGS. 53 and 54 ). In addition to the impairment of early endosomal degradation, Aβ degradation-related lysosomal dysfunction was investigated. Since the extralysosomal localization of cathepsin B (CTSB), an Aβ-degrading lysosomal enzyme, induces impairment of enzymatic activity, changes in its intracellular localization were investigated (Mueller-Steiner, S. et al., Neuron, 51, 703-714, 2006). Co-localization of lysosomes with CTSB was reduced by high glucose concentration ( FIG. 55 ). Moreover, pretreatment with leupeptin (an inhibitor of serine and thiol protease) further increased the amount of Aβ upregulated by high glucose concentration ( FIG. 56 ). These results suggest that ROS- and mTORC1-mediated lysosomal dysfunction are important in blocking endosomal clearance and increasing Aβ under high glucose conditions.

2-7: Expansion of early endosomes, increase in Aβ and cognitive impairment, restored by dynaso and rapamycin in STZ-induced DM mouse model

Changes in proteins characteristic of cognitive impairment and their clinical symptoms were investigated in STZ-treated mice. In histological data, the signal intensity and magnitude of the hippocampal early endosomes of STZ-treated mice were greater than that of vehicle-treated mice and STZ + dynasore-treated mice ( FIG. 57 ). Together with enlarged early endosomes, STZ increased hippocampal Aβ levels, which were inhibited by dynasomal treatment ( FIG. 58 ). Furthermore, the Y-maze results indicated that STZ-treated mice exhibited cognitive impairment, whereas STZ-treated mice with dynaso treatment exhibited restoration of cognition ( FIG. 59 ). On the other hand, in STZ-treated mice, phosphorylation of mTORC1 at serine 2448 was increased, which was downregulated by treatment with rapamycin ( FIG. 60 ). In histological data, the signal intensity and magnitude of hippocampal early endosomes of STZ-treated mice were greater than that of vehicle-treated mice and STZ+rapamycin-treated mice ( FIG. 61 ). The increased protein expression level of hippocampal Rab5 in STZ-treated mice was reversed in STZ+rapamycin-treated mice ( FIG. 62 ). In addition, STZ increased hippocampal Aβ levels and induced cognitive impairment, which was restored by rapamycin treatment ( FIGS. 63 and 64 ). These results demonstrate that DM induces cognitive impairment and Aβ increase through endocytosis- and autophagy-mediated early endosomal dysregulation.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereby. something to do. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

Claims (12)

delete A method of inhibiting mTOR-mediated early endosomal dysfunction and the production of amyloid beta due to high glucose concentration by treating isolated brain neurons cultured in a high glucose environment in vitro with rapamycin.
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