Application of vasogenic BACE1 as treatment target of cerebral small vessel injury related diseases

A technology for cerebral small blood vessels and uses, applied in the field of vascular-derived BACE1 as a therapeutic target for cerebral small blood vessel diseases

Pending Publication Date: 2022-07-29
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] So far, no research results have confirmed that BACE1 can be used as a therapeutic target for diseases related to cerebral small vessel injury, and finding a drug that selectively regulates the BACE1 gene or protein is of great significance for the treatment of diseases related to cerebral small vessel injury

Method used

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  • Application of vasogenic BACE1 as treatment target of cerebral small vessel injury related diseases
  • Application of vasogenic BACE1 as treatment target of cerebral small vessel injury related diseases
  • Application of vasogenic BACE1 as treatment target of cerebral small vessel injury related diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1: BACE1 specific cleavage of Occludin.

[0076] 1. The experimental process of BACE1-specific cleavage of Occludin

[0077] In order to explore the relationship between human BACE1 and human Occludin, HEK293 cells stably transfected with HA-BACE1 and Flag-Occludin-Myc plasmids were constructed. terminal and C-terminal changes (see figure 1 ). At the same time, the HEK293 cells stably transfected with both HA-BACE1 and Flag-Occludin-Myc plasmids were transfected with different concentrations of HA-BACE1 plasmid and added with different concentrations of BACE1 inhibitor C3 were further detected by Western blot technology. Changes in the N-terminus and C-terminus of occludin (see figure 2 and 3 ). On the other hand, stable BACE1 knockdown (knockdown) and knockout (KO, knockout) cell lines were constructed using siRNA and CRISPR-Cas9 technology, respectively. technology to detect changes in the N- and C-termini of Occludin (see Figure 4 and 5 ). Furtherm...

Embodiment 2

[0135] Example 2: The site of cleavage of Occludin protein by BACE1.

[0136] 1. Experimental procedure

[0137] Construction of a 30-amino acid-linked biotin-polypeptide fragment in human Occludin

[0138] biotin-Occludin 81-110 :

[0139] biotin-ACVASTLAWDRGYGTSLLGGSVGYPYGGSG(Occludin 81-110 : SEQ ID NO: 6), the polypeptide fragment was incubated with BACE1 extracellularly and then analyzed by LC-MS / LM technology. The mass spectrometry results without BACE1 treatment detected two fragments biotin-Occludin 81-110 and Occludin 90-110 , the LC-MS / LM results of BACE1 treatment showed that biotin-Occludin 81-110 Peak reduction while Occludin 90-110 The increase of 4 times, while adding three fragments biotin-Occludin 81-87 , biotin-Occludin 81-88 , Occludin 88-110 and Occludin 89-110 . Therefore, it is speculated that the cleavage site of Occludin by BACE1 is in L 87 / A 88 , A 88 / W 89 and W 89 / D 90 . Further, construct Occludin L87G, A88G, W89G and D90G mutant...

Embodiment 3

[0175] Example 3: BACE1 cleaves endogenous Occludin in human umbilical vein endothelial cell (HUVEC) cells while promoting the degradation of other tight junction proteins.

[0176] 1. Experimental procedure

[0177] HUVEC cell lines can produce endogenous Occludin, Claudin-1, JAMA and ZO-1, so the inventors constructed HUVEC cell lines stably transfected with HA-BACE1, and used Western blot technology to detect the tight junction proteins Claudin-1, JAMA and ZO -1 changes, we found a significant decrease in the levels of all three claudins. Subsequently, using in vitro endothelial cell permeability assays, we found that BACE1 reduced the amount of tight junction proteins and also disrupted tight junction structures. Furthermore, using siRNA technology to construct a HUVEC cell line stably transfected with Occludin knockdown of BACE1, we found that the content of Claudin-1, JAMA and ZO-1 did not decrease in this cell line, confirming the cleavage of BACE1. Occludin protein d...

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Abstract

The invention relates to an application of vasogenic BACE1 as a treatment target of cerebral small vessel injury related diseases. In particular to application of a substance capable of reducing the expression level of a beta secretase (BACE1) gene and / or an enzyme activity inhibitor of BACE1 protein in preparation of a medicine for treating or relieving cerebrovascular diseases, and the cerebral small vascular injury related diseases are preferably Alzheimer's disease, amyloid cerebral small vascular disease and vascular dementia. The inventor finds that the increase of the BACE1 content can shear tight junction protein Occludin, so that the tight junction protein between vascular endothelial cells is degraded and lost, the structure and function of a blood brain barrier are damaged, and finally, the pathological phenotypes of small cerebral vessel injury related diseases such as cerebral microhemorrhage and cognitive dysfunction are caused; the inhibition on the BACE1 enzyme activity can improve the pathological phenotypes of the brain small vessel injury related diseases.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to the use of vascular-derived BACE1 as a therapeutic target for cerebral small vessel disease. Background technique [0002] Cerebrovascular disease has become the first major disease that seriously endangers human health with four high characteristics of high morbidity, high disability rate, high mortality rate and high recurrence rate. As an important part of cerebrovascular disease, cerebral small vessel disease has also attracted the attention of scientific researchers and medical workers A syndrome of clinical, cognitive, imaging and pathological manifestations caused by various lesions of perforating arteries, capillaries and venules. Diseases related to cerebral small vessel injury exist in a variety of clinical diseases, such as Alzheimer's disease, amyloid small vessel disease, and vascular dementia. [0003] The pathogenesis of cerebral small vessel injury-related d...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61K45/06A61K31/166A61K31/549A61K31/7105A61P25/28A61P9/10A61P25/00
CPCA61K45/00A61K45/06A61K31/166A61K31/549A61K31/7105A61P25/28A61P9/10A61P25/00A61K2300/00
Inventor申勇高峰周好月李人程忻
OwnerUNIV OF SCI & TECH OF CHINA