A SARM1 inhibitor for the treatment of Alzheimer's disease

Abstract

The present invention relates to a SARM1 inhibitor and its use in preparing a drug for treating Alzheimer's disease, wherein the SARM1 inhibitor is a SARM1 interference sequence SARM1-i-1shRNA, SARM1-i-2shRNA or SARM1-i-3shRNA. The SARM1 inhibitor of the present invention can significantly improve memory function and reduce Aβ deposition in brain tissue, and the inhibitor provides a practical basis for the research and development of drugs for treating Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to an application of a SARM1 inhibitor. Background Art

[0002] Alzheimer's disease (AD) is a chronic neurodegenerative disease that accounts for 60-80% of dementia patients. More than 50 million people suffer from the disease worldwide. The incidence rate rises from 5% to 10% at the age of 65-75, about 15% at the age of 85, and more than 50% at the age of 90 and above. As the most common cause of dementia, AD is a progressive neurodegenerative disease in the elderly characterized by memory loss, cognitive impairment, and abnormal behavior. Common initial symptoms of patients are forgetfulness and difficulty in daily work. The initial memory impairment is limited to short-term memory, manifested as a decrease in the ability to learn new information. As the disease progresses, AD patients will experience more severe memory loss, language disorders, visual and spatial disorders, and defects in coordination and fine motor control. It is worth noting that throughout the progression of AD, memory is the most severely affected aspect. Therefore, with such a high incidence and gradual deterioration, AD will undoubtedly bring huge economic losses and psychological trauma to mankind. As the most severely affected function in the onset and progression of AD, memory impairment should be the focus of our attention.

[0003] The main pathological features of AD are amyloid β deposition (Aβ), neurofibrillary tangles (NFTs), and neuronal loss. Among them, the accumulation of toxic amyloid β (Aβ) peptides leads to synaptic dysfunction, axonal degeneration, and ultimately neuronal death. Many recent studies have shown that increased inflammation increases the risk of AD, while long-term suppression of inflammation reduces and may delay its onset. Although inflammatory responses are thought to have a beneficial role in inhibiting the early stages of disease progression, chronic neuroinflammation and the production of related proinflammatory cytokines ultimately lead to neuronal death. Aβ can activate microglia and cause reactive proliferation of astrocytes, which then secrete a large number of inflammatory factors, such as TNF-α, IFN-γ, and produce neurotoxic substances, which can lead to neuronal dysfunction. A significant increase in the phosphorylation of NF-kB was observed in APP / PS1 mice, indicating that Aβ can also activate NF-kB. More importantly, the accumulation of Aβ and the accompanying inflammatory response ultimately lead to neuronal death and cognitive decline. Therefore, neuronal dysfunction and neuroinflammation may play a key role in the pathogenesis and progression of AD. However, the underlying mechanisms and consequences of the pathogenesis of AD and other conditions remain elusive.

[0004] Toll-like receptors are an important component of innate immunity, and SARM1 is an adaptor protein of the TIR domain of TLR downstream signals. It has a Toll / interleukin-1 receptor domain and is mainly expressed in the mammalian nervous system. It is widely distributed in the hippocampus, amygdala, cerebral cortex, midbrain and cerebellum. Its expression in neurons is higher than that in astrocytes and microglia. SARM1 mediates neuronal morphological changes by regulating axon growth and neuronal polarity. When hippocampal neurons are cultured in vitro, SARM1 also promotes axon death caused by Wallerian degeneration, and SARM1 deficiency has a protective effect on axon degeneration. In addition to participating in the regulation of neural development, SARM1 is also involved in the innate immunity of the brain and plays a prominent role in neurodegenerative diseases and mental illnesses. Therefore, it is speculated that the expression of SARM1 in neurons can actively mediate the inflammatory response of AD neurons.

[0005] It can be seen that SARM1 may play an important role in the pathogenesis of AD. However, it is still unclear how SARM1 affects neuronal function and leads to the occurrence of AD. By inhibiting the expression of SARM1 gene in mice through SARM1 inhibitors, it is hoped that new research directions can be provided for the pathogenesis and treatment of AD. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings and deficiencies in the prior art and to provide a SARM1 inhibitor and its use in improving Alzheimer's disease neurology.

[0007] Inspired by the key role of SARM1 in the occurrence and development of AD, the inventors studied its effects on these processes. APP / PS1 double transgenic mice are classic Alzheimer's model mice, and the mice were divided into a control group (normal mice) and an AD group (APP / PS1 double transgenic mice). The results of animal behavior experiments showed that interfering with the SARM1 gene in the control group mice did not affect the depression and anxiety-like phenotypes of the mice, nor did it affect the memory function of the mice. However, the mice in the AD group had obvious memory impairment. In the AD group, the SARM1-siRNA1 mice showed a significant improvement in memory function compared with the SARM1-scramble mice. As we all know, Aβ deposition is a characteristic change of AD. Further studies have found that in the AD group, compared with the SARM1-scramble mice, the Aβ deposition in the brain tissue of the SARM1-siRNA1 mice was reduced at 9 months, which may be due to the fact that inflammatory cells or neurons play an important role in it. These results show that the expression of SARM1 plays a key regulatory role in improving memory function, and also provide new therapeutic targets and drug forms for Alzheimer's disease.

[0008] Based on the above findings, the technical solutions proposed by the present invention to solve the above technical problems are as follows.

[0009] A first aspect of the present invention provides a drug for treating Alzheimer's disease, wherein the drug comprises a SARM1 inhibitor.

[0010] A second aspect of the present invention provides a use of a SARM1 inhibitor in the preparation of a drug for treating Alzheimer's disease.

[0011] Preferably, the SARM1 inhibitor is a compound that reduces the expression of SARM1 nucleic acid or protein.

[0012] Preferably, the SARM1 inhibitor is a SARM1 interfering sequence.

[0013] Preferably, the SARM1 interference sequence is SARM1-i-1 shRNA, SARM1-i-2 shRNA or SARM1-i-3shRNA, the forward strand and reverse strand of the SARM1-i-1 shRNA sequence have at least 90% sequence homology with SEQ ID NO.1 and SEQ ID NO.2, respectively, the forward strand and reverse strand of the SARM1-i-2 shRNA sequence have at least 90% sequence homology with SEQ ID NO.3 and SEQID NO.4, respectively, and the forward strand and reverse strand of the SARM1-i-3 shRNA sequence have at least 90% sequence homology with SEQID NO.5 and SEQ ID NO.6, respectively.

[0014] More preferably, the forward strand and reverse strand of the SARM1-i-1 shRNA sequence have at least 95% sequence homology, or at least 98% sequence homology, or at least 99% sequence homology with SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

[0015] More preferably, the forward strand and reverse strand of the SARM1-i-2 shRNA sequence have at least 95% sequence homology, or at least 98% sequence homology, or at least 99% sequence homology with SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

[0016] More preferably, the forward strand and reverse strand of the SARM1-i-3 shRNA sequence have at least 95% sequence homology, or at least 98% sequence homology, or at least 99% sequence homology with SEQ ID NO. 5 and SEQ ID NO. 6, respectively.

[0017] Most preferably, the positive strand of the SARM1-i-1 shRNA sequence is: 5'-CCGGGCAGATCGGTTTCTCCAAGTACTCGAGTACTTGGAGAAACCGATCTGCTTTTTTG-3' (SEQ ID NO. 1);

[0018] The reverse strand of the SARM1-i-1 shRNA sequence is: 5'-AATTCAAAAAGCAGATCGGTTTCTCCAAGTACTCGAGTACTTGGAGAAACCGATCTGC-3' (SEQ ID NO. 2).

[0019] Most preferably, the positive strand of the SARM1-i-2 shRNA sequence is: 5'-CCGGGCAACTTTGTGTTGGTGCTATCTCGAGATAGCACCAACACAAAGTTGCTTTTTTG-3' (SEQ ID NO. 3);

[0020] The reverse strand of the SARM1-i-2 shRNA sequence is: 5'-AATTCAAAAAGCAACTTTGTGTTGGTGCTATCTCGAGATAGCACCAACACAAAGTTGC-3' (SEQ ID NO. 4).

[0021] Most preferably, the positive strand of the SARM1-i-3 shRNA sequence is: 5'-CCGGTATCAAGTGGTCCCACGAATACTCGAGTATTCGTGGGACCACTTGATATTTTTTG-3' (SEQ ID NO. 5);

[0022] The reverse strand of the SARM1-i-3 shRNA sequence is: 5'-CCGGTATCAAGTGGTCCCACGAATACTCGAGTATTCGTGGGACCACTTGATATTTTTTG-3' (SEQ ID NO. 6).

[0023] Preferably, the drug for treating Alzheimer's disease does not produce anxiety and depression-like behaviors.

[0024] The third aspect of the present invention provides a use of a SARM1 inhibitor in the preparation of a medicament for reducing Aβ deposition in brain tissue.

[0025] Preferably, the SARM1 inhibitor is a SARM1 interfering sequence.

[0026] Preferably, the SARM1 interference sequence is SARM1-i-1 shRNA, SARM1-i-2 shRNA or SARM1-i-3shRNA, the forward strand and reverse strand of the SARM1-i-1 shRNA sequence have at least 90% sequence homology with SEQ ID NO.1 and SEQ ID NO.2, respectively, the forward strand and reverse strand of the SARM1-i-2 shRNA sequence have at least 90% sequence homology with SEQ ID NO.3 and SEQID NO.4, respectively, and the forward strand and reverse strand of the SARM1-i-3 shRNA sequence have at least 90% sequence homology with SEQID NO.5 and SEQ ID NO.6, respectively.

[0027] More preferably, the forward strand and reverse strand of the SARM1-i-1 shRNA sequence are SEQ ID NO.1 and SEQ ID NO.2, respectively; the forward strand and reverse strand of the SARM1-i-2 shRNA sequence are SEQ ID NO.3 and SEQ ID NO.4, respectively; the forward strand and reverse strand of the SARM1-i-3 shRNA sequence are SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0028] A fourth aspect of the present invention provides a use of a SARM1 inhibitor in the preparation of a drug for improving memory function without causing anxiety and depression-like behaviors.

[0029] Preferably, the SARM1 inhibitor is a SARM1 interfering sequence.

[0030] Preferably, the SARM1 interference sequence is SARM1-i-1 shRNA, SARM1-i-2 shRNA or SARM1-i-3shRNA, the forward strand and reverse strand of the SARM1-i-1 shRNA sequence have at least 90% sequence homology with SEQ ID NO.1 and SEQ ID NO.2, respectively, the forward strand and reverse strand of the SARM1-i-2 shRNA sequence have at least 90% sequence homology with SEQ ID NO.3 and SEQID NO.4, respectively, and the forward strand and reverse strand of the SARM1-i-3 shRNA sequence have at least 90% sequence homology with SEQID NO.5 and SEQ ID NO.6, respectively.

[0031] More preferably, the forward strand and reverse strand of the SARM1-i-1 shRNA sequence are SEQ ID NO.1 and SEQ ID NO.2, respectively; the forward strand and reverse strand of the SARM1-i-2 shRNA sequence are SEQ ID NO.3 and SEQ ID NO.4, respectively; the forward strand and reverse strand of the SARM1-i-3 shRNA sequence are SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0032] Due to the particularity of the nucleotide sequence, any nucleic acid sequence or variant thereof containing SARM1-i-1, SARM1-i-2 and SARM1-i-3, as long as its fragment has a homology of more than 90% with the aforementioned nucleic acid sequence, or has the same function, is within the scope of protection of the present invention. Variants of this polynucleotide include substitution variants, deletion variants and insertion variants. For example, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of multiple nucleotides, but will not substantially change the function of the amino acid encoded by it.

[0033] Beneficial effects of the present invention:

[0034] The present invention unexpectedly discovered that SARM1 is mainly expressed in neurons, and interfering with the SARM1 gene in neural stem cells of Alzheimer's disease model mice can significantly improve the memory function of mice and reduce the Aβ deposition in the brain tissue of mice. The development of substances that reduce SARM1 expression is helpful to improve memory function and intervene in the occurrence and development of Alzheimer's disease. Based on the above findings, the present invention provides a SARM1 inhibitor and its use in the preparation of a drug for treating Alzheimer's disease, providing a practical basis for the development of new drugs for treating Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the result of the open field test after the 3-month-old control group and AD group mice were treated with SARM1 inhibitors;

[0036] Figure 2 These are the results of the elevated plus maze test of 3-month-old control and AD group mice after using SARM1 inhibitors;

[0037] Figure 3 These are the results of the sucrose preference test of 3-month-old control and AD group mice after administration of SARM1 inhibitors;

[0038] Figure 4 This is the result of the tail suspension test in the 3-month-old control group and AD group mice after using SARM1 inhibitor;

[0039] Figure 5This is the result of Morris water maze test of 3-month-old control group and AD group mice after using SARM1 inhibitor;

[0040] Figure 6 This is the result of the open field test after the 8-month-old control group and AD group mice were treated with SARM1 inhibitors;

[0041] Figure 7 These are the results of the elevated plus maze test of 8-month-old control and AD group mice after administration of SARM1 inhibitors;

[0042] Figure 8 A is the result of sucrose preference test of 8-month-old control group and AD group mice after using SARM1 inhibitor;

[0043] Figure 8 B and Fig. 9 A is the results of the tail suspension test in the 8-month-old control group and AD group mice after using SARM1 inhibitors;

[0044] Fig. 9 B is the results of the forced swimming test of 8-month-old control group and AD group mice after using SARM1 inhibitor;

[0045] Fig.10 These are the results of the Y-maze test of 8-month-old control and AD group mice after using SARM1 inhibitors;

[0046] Fig.11 These are the results of the novel object recognition test in the 8-month-old control and AD group mice after using the SARM1 inhibitor;

[0047] Fig.12 This is the result of Morris water maze test of 8-month-old control group and AD group mice after using SARM1 inhibitor;

[0048] Fig.13 This is the result of immunofluorescence staining of SARM1, Iba1, and Aβ deposition in the brain tissues of 9-month-old control group and AD group mice after using SARM1 inhibitor. DETAILED DESCRIPTION

[0049] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0050] Experimental Example 1: Effects of SARM1 gene on memory function, depression and anxiety in mice

[0051] 1. Test methods

[0052] Animal behavioral experiments confirmed the importance of the SARM1 gene in improving memory function in Alzheimer's disease. Specific SARM1 inhibitors can improve the memory function of AD mice, but do not affect the anxiety and depression-like behaviors of mice.

[0053] In order to study the possibility and importance of SARM1 in Alzheimer's disease, highly specific SARM1 antibodies were used to test the effects of the SARM1 gene on memory function, depression and anxiety in mice through animal behavioral experiments.

[0054] 1.1. Construction of SARM1 gene interference model mice

[0055] First, construct SARM1-i-1 shRNA that specifically recognizes SARM1. After large-scale data analysis and experimental verification, it does not target any known human, mouse, or rat genes. After 100 μL of competent cells were spotted and placed on ice, the constructed SARM1-i-1 shRNA sequence (the positive and reverse strands of the SARM1-i-1 shRNA sequence are SEQ ID NO.1 and SEQ ID NO.2, respectively) was added; after being placed on ice for 30 minutes, it was placed in a 42°C environment for 90 seconds; then it was placed on ice for 5 minutes, and finally 1 ml of incubated LB medium was added and shaken at 37°C and 250 rpm for 1 hour, and half of the plate was spread (Amp+) and kept at 37°C overnight. The plate was divided into several small grids, and different single colonies on the plate were streaked in the small grids. Pick a single colony, amplify it, and extract the plasmid.

[0056] The obtained SARM1-i-1 shRNA plasmid (250 μg / kg) was injected intramuscularly to construct SARM1 gene interference mice (from Shanghai Southern Model Biotechnology Co., Ltd.), and the APP / PS1 double transgenic mice (from Nanjing Southern Model Animal Center, a classic AD model mouse) were also treated in the same way to generate four types of mice, including APP / PS1; SARM1-scramble, APP / PS1; SARM1-siRNA1, SARM1-scramble and SARM1-siRNA1 mice. All mice were based on the C57BL / 6 background.

[0057] 1.2 Behavioral experiments on model mice

[0058] The four types of mice were subjected to the following animal behavior tests:

[0059] (1) Open field test: The mice were placed in the open field test room 30 minutes before the start of the test. After 30 minutes, the mice were placed in the center of the open field box and allowed to freely explore the environment for 5 minutes under appropriate lighting conditions. Panlab video behavioral analysis software SMART was used to measure and record the time spent in the center area.

[0060] (2) Elevated plus maze test: The mouse was placed in the center of the elevated plus maze with its head facing the open arm and allowed to explore freely for 5 min. All behaviors were recorded by video surveillance. The time spent in the open and closed arms was measured using Panlab video behavioral analysis software SMART.

[0061] (3) Sucrose preference test: It is divided into two stages. The first stage is the adaptation stage: the mouse is placed in a cage for 48 hours. Two bottles are placed, one of which contains water and the other contains 1% sucrose. After 24 hours, the positions of the two bottles need to be changed to avoid position preference. The second stage is the test stage: after the adaptation, the mouse is fasted and deprived of water for 24 hours. After the fasting and water deprivation, water and feed are given normally, and the water and sucrose consumption is measured after 24 hours. Sucrose preference index = sugar water consumption / (sugar water consumption + pure water consumption).

[0062] (4) Tail suspension test: Before the start, the mice were brought into the test environment and allowed to adapt for one hour. After adaptation, the posterior 1 / 3 of the tail of the mice was fixed with tape and hung on a bracket with the head 15 cm away from the table. The immobility time of the mice within the last 4 minutes of the 6-minute period was recorded. The Panlab video behavioral analysis software SMART was used to count the immobility time of the mice in the last four minutes.

[0063] (5) Morris water maze test: It is divided into two stages. The first stage is acquisition training: the mouse is placed in the water with its head facing the pool wall, and the time (in seconds) for the animal to find the underwater platform is recorded. The mouse is placed in one of the four starting positions: east, west, south, and north. In the first few training sessions, if the mouse takes more than 60 seconds to find the underwater platform, the animal is guided to the platform and allowed to stay on the platform for 10 seconds before being wiped dry. Each animal is trained 4 times a day, with a 30-min interval between two training sessions, and the training lasts for 4 consecutive days. The second stage is exploration training: the exploration training is carried out on the second day after the last acquisition training. In this stage, the platform is removed and the mouse begins a 60-second exploration. The animal is placed in the water from the opposite side of the original platform quadrant. The time the animal spends in the target quadrant (the quadrant where the platform was originally placed) and the number of times it enters the quadrant are recorded as a test indicator of spatial memory.

[0064] (6) Forced swimming test: Put 1.6L of water in a 2L glass beaker, and control the water temperature at about 24°C. Place the mouse in the beaker and swim for 6 minutes. Record the immobility time of the mouse in the last 4 minutes of the 6 minutes.

[0065] (7) Y-maze test: It consists of three identical arms, with an angle of 120 degrees between each two arms. The inner arms and the bottom of the maze are painted black. The mouse is placed at the end of any arm of the Y-maze and allowed to explore freely for 10 minutes. The camera system records the behavioral changes of the animal for 10 minutes and the order in which the mouse enters each arm within 10 minutes. Alternation is defined as consecutive entry into three arms, such as (1, 2, 3 or 1, 3, 2). The maximum alternation is the sum of the number of arm entries minus 2. The spontaneous alternation behavior score = total number of alternations / maximum number of alternations*100%.

[0066] (8) New object recognition test: The first stage is the adaptation period. On the first day, the mice move freely in the test device (without objects) for 5 minutes. On the second and third days, the mice move freely for 15 minutes. The second stage is the familiarization period. On the fourth day, two identical objects are placed in the device (make sure the objects have no odor and cannot be pushed). The objects are 10 cm away from the two side walls. The mice are placed in the device with their backs facing the objects at an equal distance from the objects. A camera and software are used to record the time the mice spend exploring each object (the exploration of the object is counted as the mouth or nose touching the object and the object is within about 2-3 cm). The number, time and distance of the animal's exploration of each object are measured within 10 minutes. The third stage is the test period. One hour after the completion of the second stage is used as the time interval for testing memory. One of the two identical objects is replaced with a different object and placed in the device. The mice are placed in the device with their backs facing the object at an equal distance from the object for 5 minutes. The time the mice spend exploring the two target objects is measured. If the memory function of the test mice is normal, they will spend more time exploring the novel object than exploring the familiar object. If exploration is identical for all objects, this behavior indicates that the mouse has a memory impairment.

[0067] 2. Test results

[0068] (1) Open field test and elevated plus maze test

[0069] Anxiety is one of the most common comorbidities in AD, so the open field and elevated plus maze tests were performed on 3-month-old mice. The results showed that compared with the control group SARM1-scramble mice, the other three groups of mice did not spend a significant difference in the time spent in the central area of ​​the open field test. In addition, there was no difference in the movement distance of the four groups of mice in the open field, indicating that they did not have obvious anxiety-like behavior ( Figure 1To make the phenomenon more clear, the elevated plus maze test was further used, and the results showed that there was no significant difference in the movement distance of the other three groups of mice compared with the control group SARM1-scramble mice ( Figure 2 These results suggest that at 3 months of age, inhibition of SARM1 gene expression does not affect anxiety-like behavior in mice.

[0070] The 8-month-old mice were subsequently tested for anxiety-like behavior-related phenotypes. The results of the open field test showed that the percentage of time spent in the central area by the other three groups of mice was not significantly different compared with the control group SARM1-scramble mice ( Figure 6 A-6B). In addition, compared with the SARM1-scramble mice in the control group, the distance covered by the AD group mice in the open field was reduced. It is speculated that inhibition of the SARM1 gene in the AD model may affect the motor function of mice to a certain extent ( Figure 6 C-6D). To make the phenomenon more clear, the elevated plus maze test was further used, and it was found that compared with the control group SARM1-scramble mice, the other three groups of mice did not show anxiety-like behavior ( Figure 7 A), but also reduced the distance traveled in the elevated cross-country test ( Figure 7 B) These results indicate that mice did not display overt anxiety-like behaviors at 8 months of age.

[0071] (2) Sucrose preference test and tail suspension test

[0072] In addition to anxiety, the most common comorbidity of AD is depression-like behavior, so the four groups of mice were subjected to sucrose preference test and tail suspension test. The results of the sucrose preference test showed that compared with the control group SARM1-scramble mice, the other three groups of mice had no significant difference in the preference index for sucrose, indicating that there was no obvious depression-like behavior ( Figure 3 ).

[0073] To make the phenomenon more certain, a tail suspension test was also performed, and the results showed that there was no significant difference in the percentage of immobility time of the other three groups of mice compared with the control group SARM1-scramble mice ( Figure 4 ). These results indicate that at 3 months of age, inhibiting SARM1 gene expression does not affect the depressive-like behavior of mice.

[0074] The depressive-like behavior of 8-month-old mice was further measured. The results of the sucrose preference test showed that compared with the SARM1-scramble mice in the control group, the preference index for sucrose in the AD group SARM1-scramble mice decreased, indicating the presence of obvious depressive-like behavior, but the SARM1 gene interference mice in the AD group did not have depressive-like behavior ( Figure 8A).

[0075] (3) Morris water maze test

[0076] Since AD ​​model mice have memory impairment, in order to verify whether the inhibition of SARM1 gene will affect the memory function of mice, the memory-related phenotypes of AD mice were tested. The results of the Morris water maze test showed that in the 3-month-old AD group mice, there was no significant difference in the latency of mice with SARM1 gene inhibition compared with SARM1-scramble ( Figure 5 ).

[0077] The results of the Morris water maze test on 8-month-old mice showed that compared with the memory impairment of SARM1-scramble mice in the AD group, the memory function of SARM1 gene interference mice was improved ( Fig.12 ).

[0078] (4) Tail suspension test and forced swim test

[0079] In order to confirm the phenomenon, a tail suspension test was also performed ( Figure 8 B and 9A) and forced swim test ( Fig. 9 B), the results showed that there was no significant statistical difference in the percentage of immobility in the other three groups of mice compared with the control group SARM1-scramble mice. The above results indicate that at 8 months of age, mice did not show obvious depressive-like behavior.

[0080] (5) Y-maze test

[0081] The results of the Y-maze test on the memory function of 3-month-old mice showed that there was no significant difference in the percentage of spontaneous alternations in the other three groups compared with the control group SARM1-scramble mice. The above test results indicate that the mice did not have memory impairment at 3 months of age.

[0082] The results of the Y-maze test on the memory function of 8-month-old mice showed that compared with the SARM1-scramble mice in the control group, the spontaneous alternation rate of the AD group mice in the Y-maze test was reduced, indicating that the 8-month-old AD model mice had obvious memory impairment, and compared with the SARM1-scramble mice in the AD group, the memory impairment of the SARM1 gene interference mice in the AD group was alleviated, indicating that SARM1 can affect the memory function of AD model mice ( Fig.10 ).

[0083] (6) Novel object recognition test

[0084] The results of the novel object recognition test showed that compared with the phenomenon that the SARM1-scramble mice in the control group preferred novel objects, the SARM1-scramble mice in the AD group had a reduced preference for novel objects, but the preference for novel objects by the SARM1 gene interference mice in the AD group did not change significantly ( Fig.11 ).

[0085] In summary, inhibiting SARM1 at 3 months of age did not affect anxiety, depression-like behaviors and memory function in mice. AD model mice at 8 months of age showed obvious memory impairment, and SARM1 played a vital role in AD model mice. Inhibiting the SARM1 gene could alleviate the memory impairment of AD model mice.

[0086] Referring to the method of Experimental Example 1, SARM1-i-2 shRNA plasmid and SARM1-i-3 shRNA plasmid (250 μg / kg) were respectively injected intramuscularly to construct mice with SARM1 gene interference, and the above experiment of Experimental Example 1 was carried out. The experimental results also verified that inhibition of SARM1 gene can alleviate memory impairment in AD model mice.

[0087] Experimental Example 2: Effect of SARM1 interference on Aβ deposition in brain tissue of AD model mice

[0088] Immunofluorescence test showed that in AD model mice, the Aβ deposition in brain tissue of mice injected with SARM1-i-1 shRNA plasmid (the construction method was the same as that of Experimental Example 1) was reduced. The expression of SARM1 in neurons may be involved in the inflammatory response of AD neurons.

[0089] 1. Test methods

[0090] The Aβ deposition in the brain of AD model mice was stained. The brain tissue of mice was stained with immunofluorescence using 6E10, a biomarker of Aβ plaques, and Iba1, a biomarker of microglia.

[0091] First, the frozen sections were dried at room temperature for 5-10 minutes and then placed in a 55°C oven for 45 minutes to make the tissue stick more firmly; then the sections were washed with PBS buffer for 5 minutes, repeated 3 times to remove the OCT on the sections; 0.3% Triton was used for perforation for 30 minutes; then 5% BSA was used for blocking for 45-60 minutes (the blocking time was adjusted according to the room temperature); the tissue sections were marked with a water-blocking pen, and fresh primary antibodies were prepared with 5% BSA and incubated at 4°C overnight to recover the primary antibodies; primary antibody diluent: mouse anti-Aβ (Covance 6E10, BioLegend, concentration 1:500), goat anti-Iba1 (ab5076, Abcam, concentration 1:500), rabbit anti-Iba1 (ab178847, Abcam, concentration 1:500); wash with PBS for 5 minutes, repeat 3 times, mark the tissue section again with a water-blocking pen, incubate with fresh 5% BSA prepared fluorescent secondary antibody of the corresponding species at room temperature for 60 minutes (secondary antibody dilution concentration 1:1000), wash with PBS again after incubation; finally, add an appropriate amount of anti-fluorescence quencher containing DAPI to seal the slide, then seal the edge with nail polish to prevent the coverslip from slipping and drying, and take pictures on a Nikon fluorescence microscope and immunofluorescence confocal microscope after drying.

[0092] 2. Test results

[0093] The results showed that Aβ deposition was present in the brain tissues of both SARM1-scramble and SARM1-siRNA1 mice in the 9-month-old AD group, but Aβ deposition was reduced in mice after SARM1 interference ( Fig.13 AB). Thioflavin S staining showed the Aβ deposition in the two groups of mice under the AD model. It can be seen that the Aβ deposition in the hippocampus and cortex of SARM1-inhibited mice was reduced ( Fig.13 C). Microglia have phagocytic and clearance effects on Aβ plaques. Double immunofluorescence staining of 6E10 and Iba1 was used to show the presence of Aβ plaques and microglia in the cortex ( Fig.13 D) and hippocampus ( Fig.13 F) and statistical analysis of Aβ plaques ( Fig.13 EG). The results showed that compared with the AD group SARM1-scramble, the deposition of Aβ plaques in SARM1-siRNA1 mice was reduced, and the plaques were surrounded by the aggregation of microglia.

[0094] SARM1-i-2 shRNA plasmid and SARM1-i-3 shRNA plasmid (250 μg / kg) were respectively injected intramuscularly to construct SARM1 gene interference mice, and the above experiment of Experimental Example 2 was carried out. The experimental results also verified that inhibition of SARM1 gene can reduce Aβ deposition in mouse brain tissue.

[0095] Since the current Alzheimer's disease drugs are ineffective, gene regulation methods are used to inhibit the expression of SARM1 at the transcriptional level and combined with other treatment methods to achieve better treatment effects for Alzheimer's disease.

[0096] The present invention can also be applied to the diagnosis and drug development targets of other systemic diseases. Any diagnosis by detecting the expression of SARM1, and any treatment of diseases by reducing or inhibiting the expression of SARM1, are within the scope of protection of the present invention.

[0097] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A drug for treating Alzheimer's disease, characterized in that: The drug includes a SARM1 inhibitor, which is SARM1-i-1 shRNA, SARM1-i-2 shRNA or SARM1-i-3 shRNA, the positive chain and reverse chain of the SARM1-i-1 shRNA sequence are SEQ ID NO.1 and SEQ ID NO.2 respectively; the positive chain and reverse chain of the SARM1-i-2 shRNA sequence are SEQ ID NO.3 and SEQ ID NO.4 respectively; the positive chain and reverse chain of the SARM1-i-3 shRNA sequence are SEQ ID NO.5 and SEQ ID NO.6 respectively.

2. Use of a SARM1 inhibitor in the preparation of a drug for treating Alzheimer's disease, characterized in that: The SARM1 inhibitor is SARM1-i-1 shRNA, SARM1-i-2 shRNA or SARM1-i-3 shRNA, the positive chain and reverse chain of the SARM1-i-1 shRNA sequence are SEQ ID NO.1 and SEQ ID NO.2 respectively; the positive chain and reverse chain of the SARM1-i-2 shRNA sequence are SEQ ID NO.3 and SEQ ID NO.4 respectively; the positive chain and reverse chain of the SARM1-i-3 shRNA sequence are SEQ ID NO.5 and SEQ ID NO.6 respectively.

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