WO2022126525A1 - Neurodegenerative disease marker prnp and application thereof - Google Patents

Abstract

Disclosed are a product for diagnosing and treating neurodegenerative diseases by using a prion protein encoding gene (Prnp) as a target, and a use therefor. Specifically, disclosed is a composition for the early assessment of a risk for Alzheimer's disease. The composition comprises a relevant reagent for detecting the level of the prion protein encoding gene in brain neuron synapses of a subject. Also disclosed is a use of the composition in assessing the risk of Alzheimer's disease. Also disclosed is a use of an inhibitor of the prion protein encoding gene in preparing a medicine for treating Alzheimer's disease.

Description

A neurodegenerative disease marker Prnp and its application technical field

The invention belongs to the field of biological detection, in particular to a neurodegenerative disease marker Prnp and its application.

Background technique

Alzheimer's disease (AD) is a relatively common chronic disease of the elderly, which causes memory impairment and eventually dementia. The quality of life of patients often declines sharply and they are unable to take care of themselves. There is no specific drug or cure for the disease, and the lengthy treatment and care of patients will impose a huge burden on the patient's family and society. According to the statistics of the AD Association in 2018, there are currently at least 50 million patients worldwide, and this number is expected to reach 152 million by 2050. As one of the countries with the largest population base in the world, China has now become the country with the largest number of Alzheimer's patients. AD has become an urgent medical and social problem to be solved.

Because the course of AD is an irreversible process. Therefore, the key to AD treatment is early diagnosis, intervening AD in the early stage of the disease and delaying the progression of the disease. But so far there is no precise method for early prediction or diagnosis of AD. The current diagnostic methods of AD are mainly combined diagnosis, including: neuropsychological assessment, cognitive impairment test; brain senile plaque and Tau protein PET scan; brain magnetic resonance (MRI) and cerebrospinal fluid (CSF) markers, β - Amyloid, phosphorylated tau protein detection, etc. [1]. However, because AD symptoms are not obvious in the early stage, when a definite diagnosis of the disease is made, most patients reach the late stage of the disease course, and most neurons die. Treatment or prevention can help patients stay in the stage of mild intellectual impairment (MCI) and slow down the deterioration, thereby ensuring the quality of life of patients and reducing social burden. Early clinical diagnosis and subsequent treatment of AD are the key points to solve AD symptoms [2-4]. At present, early molecular screening techniques for AD include positron emission tomography (PET) and cerebrospinal fluid Aβ molecular level detection. The former requires a certain dose of radioactive substances to be injected into the subject; cause surgical infection. The reliability of these diagnostic techniques for the early diagnosis of AD is also not stable, so it is difficult to be used for early AD screening. Therefore, the development of new biomarkers for early diagnosis of AD is one of the important directions for AD diagnosis and treatment in the future, and there is still a long way to go for new biomarkers to be used in clinical applications.

The APP/PSEN1 mouse model (hereinafter referred to as the AD mouse model) is one of the ideal animal models for simulating AD disease, and is widely used in the study of AD pathogenic mechanisms [5]. Numerous studies have confirmed that neuronal synaptic plasticity plays an important role in neurodegenerative diseases (eg AD, PD, HD, etc.) in living organisms. According to the difference of local translation protein expression in synaptic sites, the present invention screens out molecular biomarkers for AD early disease diagnosis, which can realize early detection, early intervention and early treatment, thereby delaying the course of disease and reducing mortality.

Prion protein (PrP) is a proven protein immersion factor, encoded by a single genomic gene, the prion protein-coding gene (Polymorphisms of the prion protein gene, Prnp) in animals. The C129 and C219 codon polymorphisms of Prnp gene are associated with the occurrence and development of neurodegenerative diseases. Studies have confirmed that the PrP ^C protein encoded by the Prnp gene has a high affinity for Aβ, and mediates the inhibition of long-term potentiation (LTP, which plays an important role in inducing synaptic toxicity [6,7]. Further research It was found that in AD mouse models, conditional deletion of Prnp can repair chemical synapse loss, neuronal activity, learning, memory impairment and behavioral cognitive ability [8,9]. In the detection of dementia patient samples, it was found that, Prion proteins caused by mutations in the Prnp gene often appear in hereditary family history, and are not uncommon in sporadic dementia [10]. Therefore, some researchers suggest that for patients with dementia, whether it is early-onset AD or AD-based The early diagnosis of prion disease, regardless of family history, should be considered, and genetic testing of Prnp is a simple and effective method.

Due to the insidious onset of AD, it is difficult for traditional detection methods to make judgments in the early stage of AD. Although some body fluid marker tests can theoretically be used for early screening, they have low sensitivity and poor controllability.

Although there have been reports of a correlation between Prnp and early AD, it is unknown whether Prnp can be used as a detection standard for AD.

1. Selkoe, D.J., Alzheimer disease and aducanumab: adjusting our approach. Nat Rev Neurol, 2019.15(7): p.365-366.

2. Mufson, E.J., et al., Mild cognitive impairment: pathology and mechanicss. Acta Neuropathol, 2012.123(1):p.13-30.

3. Long, J. M. and D. M. Holtzman, Alzheimer Disease: An Update on Pathobiology and Treatment Strategies. Cell, 2019.179(2): p.312-339.

4. Masters, C.L., et al., Alzheimer's disease. Nat Rev Dis Primers, 2015.1: p.15056.

5. Radde, R., et al., Abeta42-driven cerebral amyloidosis in transgenic mice reveals early and robust pathology. EMBO Rep, 2006.7(9):p.940-6.

6. Lauren, J., et al., Cellular prion protein mediates impairment of synaptic plasticity by amyloid-beta oligomers. Nature, 2009.457(7233): p.1128-32.

7. Zhang, J.E., et al., Polymorphisms of the prion protein gene(PRNP) in the Tibetan Mastiff.Anim Genet, 2009.40(6):p.1001-2.

8. Gimbel, D.A., et al., Memory impairment in transgenic Alzheimer mice requires cellular prion protein. J Neurosci, 2010.30(18): p.6367-74.

9.Salazar,S.V.,et al.,Conditional Deletion of Prnp Rescues Behavioral and Synaptic Deficits after Disease Onset in Transgenic Alzheimer's Disease.J Neurosci,2017.37(38):p.9207-9221.

10. Kovacs, G.G., et al., Genetic prion disease: the EUROCJD experience. Hum Genet, 2005.118(2): p.166-74.

SUMMARY OF THE INVENTION

One aspect of the present invention is to provide a composition for early assessment of Alzheimer's disease risk, the composition comprising a composition at the level of a prion protein-encoding gene in a subject's brain neuron synapses.

In the technical solution of the present invention, the level of the prion protein-coding gene refers to the composition of the mRNA level of the prion protein-coding gene.

In the technical solution of the present invention, the composition for detecting the mRNA level of the prion protein-encoding gene is selected from the combination of primers and or probes for detecting the NM_001278256.1 gene fragment.

In the technical scheme of the present invention, the composition for detecting the miRNA level of the prion protein-encoding gene is selected from the following primer compositions:

F:accagaacaacttcgtgcac SEQ ID No.1

R: ttctcccgtcgtaataggcc SEQ ID No. 2.

Another aspect of the present invention is to provide a kit comprising the above composition for detecting the level of a prion protein-encoding gene.

In the technical solution of the present invention, the kit further comprises reagents for transcriptome detection and/or reagents for quantitative reverse transcription PCR.

In the technical solution of the present invention, the kit further includes a detection composition for housekeeping genes.

In the technical solution of the present invention, the housekeeping gene in the kit is selected from GAPDH and or HPRT.

In the technical scheme of the present invention, the detection primer composition of described housekeeping gene is:

The primer composition for detecting GAPDH is:

F: TCAACAGCAACTCCCACTCTTCCA SEQ ID No.3

R:ACCCTGTTGCTGTAGCCGTATTCA SEQ ID No.4;

The primer composition for detecting HPRT is:

F: GGAGTCCTGTTGATGTTGCCAGTA SEQ ID No.5

R: GGGACGCAGCAACTGACATTTCTA SEQ ID No. 6.

Another aspect of the present invention is to provide the use of the above-mentioned composition or kit for detecting RNA level in the preparation of a reagent for detecting Alzheimer's disease risk assessment.

In the technical solution of the present invention, the Alzheimer's disease risk assessment is an early Alzheimer's disease risk assessment.

In the technical solution of the present invention, the early risk assessment of Alzheimer's disease is an assessment performed before the occurrence of amyloid in the subject.

Yet another aspect of the present invention provides a method for predicting Alzheimer's disease risk, the method comprising the steps of:

1) Obtain the subject's brain synapses;

2) Detecting the expression level of the prion protein-encoding gene in the brain synapse.

In the technical solution of the present invention, the method for predicting the risk of Alzheimer's disease further comprises the step of comparing the expression level of the prion protein-encoding gene in the brain synapse of the subject with a negative control or with a normal value range .

In the technical solution of the present invention, the detection method in the step 2) is selected from using transcriptome detection and or quantitative reverse transcription PCR.

In the technical solution of the present invention, the above-mentioned composition or kit of the present invention is used in the step 2).

In the technical solution of the present invention, when the expression level of the prion protein-encoding gene in the brain synapse is higher than the negative control or the normal value range, it is considered to be at risk of Alzheimer's disease.

In the technical solution of the present invention, the method for predicting the risk of Alzheimer's disease is a method for predicting the risk of Alzheimer's disease at an early stage, and the early stage means that amyloid does not appear in the brain of the subject.

Another aspect of the present invention provides a drug for reducing the risk of Alzheimer's disease, the drug comprising an inhibitor of a gene encoding a prion protein.

In the technical solution of the present invention, the inhibitor of the prion protein encoding gene is used to inhibit the transcription, translation and expression of the prion protein encoding gene.

Another aspect of the present invention provides the use of an inhibitor of a gene encoding a prion protein in the preparation of a medicament for treating Alzheimer's disease.

Another aspect of the present invention provides the use of an inhibitor of a prion protein-encoding gene in the preparation of a medicament for repairing neuronal synapses and or increasing the density or volume of neuronal dendritic spines.

In the technical scheme of the present invention, the inhibitor of the prion protein encoding gene is selected from the shRNA, siRNA, dsRNA, miRNA, antisense nucleic acid, siRNA targeting sequence or iRNA targeting sequence of the prion protein encoding gene; or can express or Form the construct of described shRNA, siRNA, dsRNA, miRNA, antisense nucleic acid, described siRNA sequence is shown as SEQ ID No.13 or SEQ ID No.14.

Another aspect of the present invention provides the use of neuronal cells with up-regulated prion protein-encoding genes in the preparation of drugs for screening and repairing neuronal synapses and/or dendritic spines.

beneficial effect

1) The present invention discovers the Prnp gene. Compared with traditional detection methods, gene diagnosis is more timely, specific, convenient and sensitive.

2) Early diagnosis of AD can be achieved, thereby providing guidance for early intervention in AD, thereby delaying AD symptoms and prolonging the life of patients.

Description of drawings

Figure 1 is a volcano plot of neurodegenerative disease-related genes obtained by RNA-seq analysis of brain tissue synapses. The volcano plot reveals the differential gene distribution between littermate wild-type and AD mice in the SD group. Figure A shows the same 3-month-old Gene differences between litters of wild-type and AD mice; panel B shows the gene differences between 6-month-old littermates of wild-type and AD mice.

Figure 2 is a heat map of neurodegenerative disease-related gene expression obtained by RNA-seq analysis of brain tissue synapses. In the transcriptome sequencing results, the differential expression results of genes related to AD disease. Among them, those marked with grid fills represent up-regulation, and those that are not marked represent down-regulation. Different gray levels represent different degrees of difference, and gray levels that are approximately darker represent higher degrees of difference.

Figure 3 shows the data classification analysis. Picture A shows the genes with FDR (false discovery rate) < 0.01, in which genes with expression changes greater than 2 were found. These genes were classified, and 12 genes with significant differences were enriched in energy metabolism-related pathways; it could be found that they were significantly associated with AD disease signaling pathways, of which the core gene was Prnp.

In the figure, 1 is the binding of amyloid beta protein, 2 is the negative regulation of dendritic spines, 3 is the binding of copper ions, 4 is the regulation of age-related behavioral decline, 5 is the regulation of the entry of calcium ions through the cell membrane, and 6 is the Protein binding, 7 is the detoxification of copper ions, 8 is the protection of glial cells, 9 is myelin sheath, and 10 is the regulation of chemical synapses.

Figure 4. Distribution of Prnp in mouse cortex and synaptosome (SD) after validation by qPCR. Differential expression of Prnp between wild-type and AD-type fractions. Picture A shows the expression of Prnp in the cortex and hippocampus of 3-month-old mice; Picture B shows the expression of Prnp in SD components of 3-month-old and 6-month-old mice.

Figure 5 shows the calcium transduction of shPrnp in primary neurons. shPrnp can restore the density of dendritic spines and synaptosomes in primary neurons of rat hippocampus at 14 days.

Detailed ways

In order to make the above objects, features and advantages of the present invention more clearly understood, the specific embodiments of the present invention will be described in detail below, but should not be construed as limiting the scope of the present invention.

Materials, Equipment and Methods

The AD mouse strain used came from the Jackson Laboratory, and was bred and raised in the SFP animal room of the Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences. The operation was in line with animal ethics and experimental standards; the anatomy and other related surgical instruments were purchased from Ruiward; the ultracentrifuge and The supporting rotor and centrifuge tube were purchased from Beckman; the gradient centrifuge was from Thermo Fisher Scientific; the TRIzol ^TM Reagent was from Invitrogen; the RNA-seq service and the preliminary data processing service were provided by Novogene; phosphate buffered saline (PBS), etc. Produced by Gibco Company; SYBR Green and corresponding qPCR detection instruments are from Thermo Fisher Scientific Company.

Example 1 Tissue sample collection

The 3-month-old (3M) and 6-month-old (6M) littermates of wild-type (WT) and transgenic AD mice (AD) were anesthetized with isoflurane (gas), and then quickly decapitated with scissors after menstruation. , place the head on ice, and rapidly dissociate the cerebral cortex and isolate the cerebral cortex and hippocampus of the mouse. The isolated tissue was washed twice in DPBS containing 4 U/mL of protease inhibitor and RNase inhibitor to obtain a cerebral cortex tissue sample.

Example 2 Isolation and purification of brain synapses (SD)

a) The removed brain tissue was placed on ice, the pia mater and vascular tissue were removed in a tissue homogenate balance solution containing protease inhibitors and RNase inhibitors, and 2 mL of cerebral cortex was taken and homogenized in the same tissue homogenate balance solution. , filter the tissue homogenate with a 40 μm tissue filter to remove large tissue fragments;

b) The filtered tissue homogenate was centrifuged horizontally for 10 minutes at 4°C at a speed of 10,000 g, and the precipitate was collected and subjected to the following gradient centrifugation to separate brain nerve synapses;

c) The pellet was then suspended in 35% OptiPrep tissue homogenate equilibration solution, followed by 9%, 12.5%, 15%, 25% and 35% OptiPrep gradient centrifuge tubes at 4°C at 10000g Gradient centrifugation was performed for 24 minutes by centrifugal force, and the suspension between the 9%-12.5% interface was carefully collected, which was the suspension containing brain synaptosomes;

d) The collected brain synaptosome suspension was centrifuged again at a speed of 6000g under the condition of 4°C for 30min, and the obtained precipitate was the brain nerve synapse (SD).

Example 3 RNA extraction

Total RNA was extracted from brain tissue (cortex and hippocampus) and synapses by Trizon method. For detailed methods, please refer to the instructions for use of TRIzol ^TM Reagent from Invitrogen as follows:

a) Preparation reagents: chloroform, isopropanol, 75℅ ethanol, RNase-free water or 0.5℅ SDS (all solutions should be prepared with DEPC-treated water).

b) Operation steps:

i. Homogenization treatment: Grind the tissue or cells in liquid nitrogen, add 1 mL of TRIzol per 50-100 mg of tissue, and perform homogenization treatment with a homogenizer. The sample volume should not exceed 10℅ of the TRIzol volume.

ii. Place the homogenized sample at room temperature (15-30° C.) for 5 minutes to completely separate the nucleic acid-protein complexes.

iii. Add 0.2 mL of chloroform for every 1 mL of TRIzol used, shake vigorously for 15 seconds, and leave at room temperature for 3 minutes.

iv. Centrifuge at 10000×g for 15 minutes at 2-8°C. The sample is divided into three layers: the bottom layer is the yellow organic phase, the upper layer is the colorless aqueous phase and an intermediate layer. The RNA is mainly in the aqueous phase, and the volume of the aqueous phase is about 60℅ of the TRIzol reagent used.

v. Transfer the aqueous phase to a new tube, if you want to separate DNA and protein, you can keep the organic phase, see below for further operations. RNA in the aqueous phase was precipitated with isopropanol. Add 0.5 mL of isopropanol for every 1 mL of TRIzol used, and leave at room temperature for 10 minutes.

vi. Centrifuge at 10000×g at 2～8℃ for 10 minutes, no RNA precipitate can be seen before centrifugation, and gelatinous precipitate appears on the side and bottom of the tube after centrifugation. Remove the supernatant.

vii. Wash RNA pellet with 75℅ ethanol. Add at least 1 mL of 75℅ ethanol for every 1 mL of TRIzol used. Centrifuge at 2-8°C at no more than 7500 × g for 5 minutes, and discard the supernatant.

viii. Dry at room temperature or vacuum dry the RNA precipitate for about 5 to 10 minutes. Do not vacuum centrifuge to dry, as excessive drying will greatly reduce the solubility of RNA. Add 25-200 μL of RNase-free water or 0.5℅ SDS, pipette several times with a pipette tip, and place at 55-60°C for 10 minutes to dissolve the RNA. Do not use SDS solution if RNA is used for digestion. RNA can also be dissolved in 100℅ of deionized formamide and stored at -70°C.

Example 4 cDNA synthesis, transcriptome sequencing and quantitative PCR detection

The synthesis of cDNA was completed with the reverse transcription kit K1632 of ThermoFisher Company. The RNAs from different sources obtained in Example 3 were reverse transcribed into cDNAs, and the synthesized cDNAs were subjected to quantitative RT-PCR detection and transcriptome sequencing. The housekeeping genes used were GAPDH and HPRT.

The amplified sequence of the Prnp gene is NM_001278256.1;

Prnp gene amplification primer is F:accagaacaacttcgtgcac SEQ ID No.1

R:ttctcccgtcgtaataggcc SEQ ID No.2

The amplified sequence of GAPDH gene is NM_008084.3;

GAPDH gene amplification primer is F: TCAACAGCAACTCCCACTCTTCCA SEQ ID No.3

R:ACCCTGTTGCTGTAGCCGTATTCA SEQ ID No.4

The amplified sequence of HPRT gene is NM_013556.2;

HPRT gene amplification primer is F: GGAGTCCTGTTGATGTTGCCAGTA SEQ ID No.5

R:GGGACGCAGCAACTGACATTTCTA SEQ ID No.6

The cDNA derived from brain neuron synapse samples was amplified and sequenced by RNA-seq, and the data was analyzed by bioinformatics, including volcano map, heat map, and gene enrichment analysis (GO analysis). In the GO analysis, the differences between wild-type and AD-type mice were analyzed and compared, the genes with FDR (false discovery rate)<0.01 were screened, and the gene enrichment analysis was performed on the genes whose relative expression difference was more than 2 times. There are 12 genes. Significantly different genes were enriched in pathways related to energy metabolism and were significantly associated with neurodegenerative diseases.

qRT-PCR was used to verify the difference of Prnp gene expression between wild-type mice and AD mice at different time periods with RNA derived from brain neuron synapse samples, cerebral cortex tissue and hippocampus tissue.

GraphPad Prism software was used for data processing, and T test was used for statistical methods, and P<0.05 was considered statistically significant.

Example 6 Calcium phosphate transfection experiment of primary neurons

Using AD mouse brain tissue, primary culture of neurons in vitro was performed.

For calcium phosphate transfection experiments, the slides with neurons cultured on the inner surface of the 24-well plate were placed in equilibrated 1.5 mL DMEM, and put into a 7.5% CO ₂ incubator to starve for 1 hour. During the preparation of plasmid and calcium transfer reagent, the required amount of plasmid and 5 μL of 2M CaCl ₂ were added to water to form a 50 μL system. 50 μL of 2×HBS (pH=7.05) was added dropwise to the CaCl ₂ /plasmid mixture while vortexing, and allowed to stand in a dark environment for about 20 minutes to form a transfection solution. Add the transfection solution dropwise evenly to the glass slides in the wells, put them into a 7.5% CO ₂ incubator and let stand for about 15 minutes (depending on the size of calcium phosphate crystals). After washing twice with 1 mL of DMEM, the slides were placed in the neuron maintenance medium, returned to the 5% CO ₂ incubator, and the medium was half-changed after 1 hour.

Among them, the experiment was divided into 4 groups, and the plasmids were selected from pSUPER vector plasmid (blank group), pSUPER vector plasmid transfected with scr-Prnp (negative control), SUPER vector plasmid transfected with shPrnp#1p and transfected with shPrnp#2. pSUPER vector plasmids, respectively.

The plasmid construction method is to use shRNA primers to co-incubate with the pSUPER vector plasmid to obtain a plasmid that generates siRNA in the transfected cells.

The primers used for each group of plasmids are as follows:

shPrnp 1#:

F:GATCTCCcctgtgatcctcctcatctTTCAAGAGAagatgaggaggatcacaggTTTTTGGAAC SEQ ID No.7

R:TCGAGTTCCAAAAAcctgtgatcctcctcatctTCTCTTGAAagatgaggaggatcacaggGGASEQ ID No.8

shPrnp 2#

F:GATCTCCtcctcatctccttcctcatTTCAAGAGAatgaggaaggagatgaggaTTTTTGGAACSEQ ID No.9

R:TCGAGTTCCAAAAAtcctcatctccttcctcatTCTCTTGAAatgaggaaggagatgaggaGGA SEQ ID No.10

scr-Prnp.

F:GATCTCCgttcctgccctctctactaTTCAAGAGAtagtagagagggcaggaacTTTTTGGAAC SEQ ID No.11

R:TCGAGTTCCAAAAAgttcctgccctctctactaTCTCTTGAAtagtagagagggcaggaacGGASEQ ID No.12

The siRNA targeting sequences obtained from shPrnp 1# and shPrnp 2# obtained by sequencing are:

RRNP siRNA 1#: CCTGTGATCCTCCTCATCT SEQ ID No.13

PRNP siRNA 2#:TCCTCATCTCCTTCCTCAT SEQ ID No.14

Result analysis

The RNA-seq detection is aimed at the analysis of brain neuron synapses (SD). The experimental results are shown in Figures 1-3.

Among them, through the analysis of the volcano map (Figure 1) and the heat map (Figure 2), it was found that the comparison between the littermate wild-type group and the AD group in the SD of 3-month-old mice and 6-month-old mice showed that Prnp in AD mice expression was significantly increased. Comparing the expression difference between 3-month-old and 6-month-old AD mice, it can be seen that the differential expression becomes more obvious with the increase of time.

After gene function enrichment analysis (GO analysis) of genes with significant gene expression in the transcriptome, it was found that Prnp gene functions were mainly enriched in: Aβ recognition and binding, dendritic spine regulation, metal ion recognition and transport, age-related neurodegeneration Sexual disease regulation, etc. (Figure 3). Therefore, Prnp can serve as a candidate biomarker for early diagnosis of neurodegenerative diseases.

The qRT-PCR results are shown in Figure 4. The experimental results show (Figure 4A) that there is no significant difference in the expression of Prnp in the cerebral cortex of 3-month-old wild-type mice and AD mice. The comparison of hippocampal tissues of 3-month-old wild-type mice and AD mice, and the comparison of cerebral cortex and hippocampal tissues of 6-month-old wild-type mice and AD mice showed that the expression of Prnp in AD mice was relatively high. The expression level in wild-type mice was significantly reduced. For brain synapses, the results showed (Fig. 4B) that the expression of Prnp in synapses of 3-month-old AD mice was significantly higher than that of wild-type mice, while 6-month-old mice did not show this difference.

From the above experimental results, different detection methods and different detection sources will affect the early diagnosis of Alzheimer's. From the comprehensive RNA-seq results and qPCR results, the two can be used for the early detection of neuronal dendrites. The results showed the same trend that Prnp expression was significantly increased in neuronal dendrites early in Alzheimer's development. For the AD mouse model, amyloid plaques have not yet appeared in the brains of 3-month-old mice, while amyloid plaques have appeared in the brains of 6-month-old mice. The results of qPCR and RNA-seq showed that the expression of Prnp was significantly different before the appearance of amyloid plaques. It shows that the expression of Prnp in neuronal synapses can be used as a marker for early Alzheimer's disease for early screening and detection of Alzheimer's disease.

The experimental results of calcium phosphate transfection of primary neurons are shown in Figure 5. The experimental results show that shPrnp calcium transfection was performed on neurons on day 14, and it was found that knocking down the expression of Prnp in neurons on day 14 can repair dendrites in neurons. Density and size of spines and synapses, etc. (Figure 5). Dendritic spines are protruding structures on the dendritic trunk of a neuron that serve as the main structure of the postsynaptic component in the synaptic structure. Dendritic spines are the most direct anatomical structures for synaptic connections and transmission, and the dynamic changes in their formation and degradation are widely regarded as hallmarks of synaptic plasticity. Studies have shown that the length, number and density of dendritic spines and synapses in AD mice are significantly less than those in wild-type mice, and the reduction in the length and density of dendritic spines and synapses occurs before the appearance of amyloid plaques. Features occur early in Alzheimer's. The experimental results of the present invention confirm that the increased expression of Prnp gene can cause synapse loss and decrease in the volume of dendritic spines, and the volume and number of dendritic spines and synapses can be increased by knocking down the Prnp gene. From the results of calcium phosphate transfection experiments, it can be seen that the Prnp gene is highly expressed in early Alzheimer's brain neurons and can be used as a marker for early Alzheimer's, which is consistent with the results of qPCR and RNA-seq.

In addition, the results of calcium phosphate transfection experiments in primary neurons also revealed the use of the Prnp gene as a target for early therapy. Knockdown of Prnp can restore the density and size of dendritic spines and synapses in neurons. The dendritic spines and synapses in neurons have been shown to be associated with changes in synapses or dendritic spine morphology associated with Alzheimer's disease, and are important factors in neurodegenerative diseases. The invention inhibits the expression of Prnp gene through the interference of RRNP siRNA 1# and 2#, so that the volume and number of dendritic spines and synapses in neurons become larger, and the reduction of Prnp expression can increase synaptic plasticity and improve dendritic spines. Density and thickness, and then used for neuron repair and slow down the process of neurodegenerative diseases, improve or treat neurodegenerative diseases. Therefore, the experimental results show that the Prnp gene expression inhibitor can restore the density and thickness of dendritic spines and synapses, which in turn predicts the improvement and therapeutic effect of neurodegenerative diseases.

Claims (10)

A composition for early assessment of Alzheimer's disease risk, characterized in that the composition comprises a composition for detecting the level of a prion protein-encoding gene in a subject's brain neuron synapses; Preferably, the composition comprises a combination of primers and or probes selected from the group consisting of detecting NM_001278256.1 gene fragments; More preferably, the composition comprises the following primer composition: F:accagaacaacttcgtgcac SEQ ID No.1 R: ttctcccgtcgtaataggcc SEQ ID No. 2. A test kit, characterized in that, the test kit comprises the composition of claim 1; Preferably, the kit further comprises reagents for transcriptome detection and/or reagents for quantitative reverse transcription PCR. Use of the composition of claim 1 or the kit of claim 2 in the preparation of a reagent for detecting Alzheimer's disease risk assessment; Preferably, the Alzheimer's disease risk assessment is an early Alzheimer's disease risk assessment. A drug for reducing the risk of Alzheimer's disease, characterized in that the drug comprises an inhibitor of a gene encoding a prion protein; Preferably, the inhibitor of the prion protein-coding gene is selected from the shRNA of the prion protein-coding gene, siRNA, dsRNA, miRNA, antisense nucleic acid, siRNA targeting sequence or iRNA targeting sequence; or can express or form the shRNA, siRNA, dsRNA, miRNA, antisense nucleic acid constructs. Use of a prion-encoding gene inhibitor in the preparation of a medicament for treating Alzheimer's disease; Preferably, the inhibitor of the prion protein-coding gene is selected from the shRNA of the prion protein-coding gene, siRNA, dsRNA, miRNA, antisense nucleic acid, siRNA targeting sequence or iRNA targeting sequence; or can express or form the shRNA, siRNA, dsRNA, miRNA, antisense nucleic acid constructs. Use of an inhibitor of a prion-encoding gene in the preparation of a medicament for repairing neuronal synapses and or increasing the density or volume of neuronal dendritic spines; Preferably, the inhibitor of the prion protein-coding gene is selected from the shRNA of the prion protein-coding gene, siRNA, dsRNA, miRNA, antisense nucleic acid, siRNA targeting sequence or iRNA targeting sequence; or can express or form the shRNA, siRNA, dsRNA, miRNA, antisense nucleic acid constructs. Use of neuronal cells with up-regulated prion protein-encoding genes in the preparation of reagents for screening and repairing neuronal synapses and/or dendritic spines. A method for predicting the risk of Alzheimer's disease, characterized in that the method comprises the following steps: 1) Obtain the subject's brain synapses; 2) detecting the expression level of the prion protein-encoding gene in the brain synapse; Optionally, the method for predicting the risk of Alzheimer's disease further comprises, 3) the step of comparing the expression level of the prion protein-encoding gene in the brain synapse of the subject with a negative control or with a normal value range. The method according to claim 8, wherein the detection method in step 2) is selected from using transcriptome detection and or quantitative reverse transcription PCR. The method according to claim 8, wherein the method for detecting in step 2) adopts the composition according to claim 1 or the kit according to claim 2.

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