CN114685680A - Polypeptide and application thereof in improving learning and memory disorders of senile dementia - Google Patents
Polypeptide and application thereof in improving learning and memory disorders of senile dementia Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
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Abstract
The invention discloses an artificially synthesized small molecular polypeptide TAT-siP-Add1 and application of the polypeptide in improving learning and memory disorders in senile dementia. By utilizing the fusion protein polypeptide TAT-siP-Add1 of artificially synthesized TAT protein transduction structural domain and siP-Add1, TAT can carry siP-Add1 protein polypeptide to pass through blood-brain barrier to be taken by neurons, and the fusion protein polypeptide TAT-siP-Add1 is applied to an in-vivo mouse model, so that the biological effect of blocking the combination of Rock2 and Add1 protein can be effectively exerted, the phosphorylation level of Add1 is reduced, the stability of actin skeleton is enhanced, the normal physiological function of nerve synapse is maintained, and the synapse injury and the learning and memory disorder of AD mice are improved.
Description
Technical Field
The invention belongs to the field of medicine and pharmacy, particularly relates to micromolecule polypeptide and improvement and treatment of learning and memory disorder of senile dementia, and particularly relates to micromolecule polypeptide TAT-siP-Add1 and application thereof in preparation of a medicine for improving the learning and memory disorder of senile dementia.
Background
Alzheimer Disease (AD), also called senile dementia, has become a big killer threatening the health of the elderly all over the world, and is a high-incidence disease threatening the life and health of the elderly next to cardiovascular diseases. AD patients exhibit hypomnesis, impaired visuospatial function, and even dramatic changes in temperament and behavior in daily life. With the increasing of the disease condition of the patient, the patient gradually loses the self-care ability of daily life, which not only brings heavy economic burden to the society and families, but also has a great influence on the mood and the psychology of the patient and relatives around the patient. Therefore, at the present time when the aging problem of the population is increasingly highlighted, it is increasingly urgent to enhance the pathological basic research and drug development for AD.
The most typical clinical pathology of AD is senile plaques and neurofibrillary tangles found in postmortem brain autopsies of patients, and is also accompanied by massive neuronal loss, atrophy of hippocampus and cortex, etc. Based on the two pathological features of senile plaques and neurofibrillary tangles, an amyloid cascade hypothesis and a Tau protein hyperphosphorylation hypothesis are proposed, and a great deal of research has been conducted in the laboratory, and corresponding drugs have been clinically tested, but unfortunately, no specific drug has been effective for delaying or treating AD so far. Recent reports indicate that progressive cognitive function and memory decline has begun to occur in early stages of AD, while senile plaques and neurofibrillary tangles are often absent. Therefore, the intensive study on the pathological change mechanism of the AD in the early stage can bring new breakthrough to the treatment and the early diagnosis of the AD.
The neurosynaptic is a site where neurons are functionally interconnected, and is also a key point for information transmission and communication of neurons. Synapses are basic units in the brain, and synaptic activity can stimulate the maturation of mushroom dendritic spines and form new synapses, so that the synaptic strength can adapt to the change of internal and external environments, and further play an important role in learning and memory. Synapse loss and dysfunction mean that synapses are affected in terms of storage, processing, and transmission of information, leading to disruption of information flow throughout neural circuits and networks, and manifested as neurological dysfunction or dementia. Studies have shown that in AD brain, especially in early stages, it is possible to find interruptions in synaptic activity and loss of synapses. Several studies have shown a higher correlation between synaptic disorders characterized by loss of synapses and decreased synaptic activity and cognitive impairment in AD compared to senile plaques and neurofibrillary tangles. Therefore, treatment strategies for synaptic disorders in AD are already a major current direction.
TAT cell penetrating polypeptides (cell penetrating peptides) are a highly efficient transport vector discovered in recent years. TAT can penetrate cell membranes and nuclear membranes, and carries polypeptides, proteins, DNA molecules and the like to enter cytoplasm and nucleus by a receptor transport mode to exert corresponding biological effects. The current research shows that HIV-TAT can pass through all tissue cells and has no obvious toxic and side effects. TAT can bring the polypeptide connected with TAT into cells within minutes, and can cross blood brain barrier to enter neurons, and the polypeptide brought into cells retains the original biological activity, thereby playing a biological role.
Disclosure of Invention
The task of the invention is to provide a small molecular polypeptide which has the function of improving the learning and memory disorder of the senile dementia and can be used for preparing the medicine for treating the senile dementia.
The small molecular polypeptide provided by the invention is TAT-siP-Add1, the sequence of which is shown as SEQ ID NO.1 in a sequence table as follows: YGRKKRRQRRRKKKKFRTPSFLKKSKKK are provided.
The control of TAT-siP-Add1 is TAT-scramble, the sequence of which is shown as SEQ ID NO.2 in the sequence table as follows: YGRKKRRQRRRRPKKTKFFKKSKKLKKS is added.
TAT-siP-Add1 polypeptide and the control TAT-scramble polypeptide were prepared synthetically by the assignee of the present patent application, British Biotechnology, Inc.
Based on the recent research and TAT technology of the applicant, the applicant synthesizes a membrane-permeable small molecule polypeptide TAT-siP-Add1 consisting of an amino acid sequence which is in competitive binding with Add1 protein and Rock2 kinase, and effectively blocks the binding of Add1 and Rock2 by applying the polypeptide to an in-vivo AD mouse model, so that the phosphorylation level of the Add1 is reduced, the stability of an actin skeleton is enhanced, the synaptic injury and the learning and memory disorder of an AD mouse are improved, and a method is provided for improving the symptoms of the AD. The TAT cell-penetrating peptide (YGRKKRRQRRR) is connected with siP-Add1(KKKKFRTPSFLKKSKKK) to obtain the TAT-siP-Add1 polypeptide (SEQ ID NO.1: YGRKKRRQRRRKKKKFRTPSFLKKSKKK) with biological activity. The siP-Add1 polypeptide is transported into blood to cross blood brain barrier by utilizing the membrane penetrating function of TAT, and is taken up by brain nerve cells to exert the biological function of TAT.
The invention also aims to provide application of the small molecule polypeptide TAT-siP-Add1 in improvement of learning and memory disorders of senile dementia, and the small molecule polypeptide TAT-siP-Add1 is found to be capable of effectively blocking the combination of Add1 and Rock2 through intraperitoneal injection, so that the phosphorylation of Add1 is reduced, the stability of an actin skeleton is enhanced, the synaptic injury and the learning and memory disorders of AD mice are improved, and a method is provided for improving the symptoms of AD.
The inventor of the patent application finds that the mutual combination of Add1 and Rock2 kinase is reduced, and the learning and memory of AD mice can be obviously improved. Aiming at the discovery, the inventor connects TAT cell-penetrating peptide (YGRKKRRQRRR) with siP-Add1(KKKKFRTPSFLKKSKKK) to obtain a biologically active TAT-siP-Add1 polypeptide, and the TAT-siP-Add1 polypeptide enters blood to cross blood-brain barrier and is taken up by brain nerve cells to play a biological function by means of in vivo intraperitoneal injection.
The application of the small molecular polypeptide TAT-siP-Add1 in improving the learning and memory disorder of the senile dementia comprises the following application processes:
the APP/PS1 transgenic mouse is one of the most common AD model mice at present. APP/PS1 mice begin to develop cognitive impairment after 9 months of age, before which synaptic loss occurs. The method comprises the steps of selecting APP/PS1 and C57 mice with similar body weights and being 8 months old, and randomly dividing the mice into two groups, wherein 15mg/kg of TAT-siP-Add1 polypeptide solution is administered to the mice in an experimental group through intraperitoneal injection, 15mg/kg of TAT-scramble polypeptide solution is administered to the mice in a control group, the same volume of physiological saline for injection is administered to the mice in a C57 group through intraperitoneal injection, the physiological saline for injection is continuously injected for 14 days, the solutions are all prepared by diluting polypeptide powder with the physiological saline according to the specified concentration, and the solutions are used as the medicine when the medicine is prepared. After the Morris water maze experiment, APP/PS1 mice injected with TAT-scramble control solution have lower learning and memory abilities than C57 mice, and mice injected with TAT-siP-Add1 polypeptide solution have similar learning and memory abilities to C57 mice.
Compared with the prior art, the invention has the following characteristics: (1) the small molecular polypeptide TAT-siP-Add1 has high purity, is completely soluble, is suitable for intravenous injection and has no toxic or side effect. (2) The TAT-siP-Add1 polypeptide disclosed by the invention can carry siP-Add1 protein polypeptide, can pass through a blood brain barrier by blood and be taken by neurons, can be converted and applied to a nervous system, and can regulate and control synaptic functions, so that the TAT-siP-Add1 polypeptide has feasibility of practical operation. (3) The micromolecular polypeptide TAT-siP-Add1 can improve the learning and memory disorder of senile dementia and can be used for preparing a medicine for treating or preventing senile dementia. The pharmaceutical preparation for improving the learning and memory disorder of the low senile dementia can be prepared by combining pharmaceutically acceptable excipient or/and carrier.
Drawings
Description of abbreviations
FIG. 1 is a synthetic MS analysis diagram of a small molecule polypeptide TAT-siP-Add 1.
FIG. 2 is a graph of abnormal phosphorylation levels of Rock2 and Add1 in AD patients, wherein: FIG. 2A is an immunoblot of AD patient and normal human brain tissue. The brain tissue sample is quickly washed by PBS (phosphate buffer saline PH 7.4) which is pre-cooled to 0-4 ℃, then RIPA strong lysis buffer is added to ice for homogenate, the total protein of the tissue is extracted, and the protein content is measured for standby. The expression levels of Rock2, Add1-S726, Add1 and Actin proteins in brain tissues are respectively detected. Wherein, the Actin is an internal reference strip, which proves the consistency of the loading amount of the protein. By comparison, the content of Rock2 and phosphorylated Add1(Add1-S726) in the brains of AD patients was significantly higher than that of the control group, while Add1 was unchanged. Fig. 2B is a statistical chart. FIG. 2C is a graph of correlation analysis, illustrating a strong correlation between Rock2 and phosphorylation of Add 1-S726.
FIG. 3 immunoblot plot of Rock2 promoting phosphorylation of Add1-S72 sites in Neuro2a cells, wherein: FIG. 3A shows the transfection of empty plasmid overexpressing Rock2 or a control in Neuro2a cells, which were harvested 48 hours later and lysed using RIPA lysate to extract total protein for use in determining protein content. And respectively detecting the expression levels of Rock2, Add1-S726, Add1 and Actin in the lysis sample. Through comparison, the content of Rock2 in an experimental group for over-expressing Rock2 is obviously higher than that in a control group, which indicates that the plasmid is successfully over-expressed, and the phosphorylation level of Add1-S726 in the experimental group is also obviously higher than that in the control group, which indicates that Rock2 can actually promote the phosphorylation of Add1-S726, but Add1 is not changed. Fig. 3B is a statistical chart.
FIG. 4 is a graph of immunoblots of TAT-siP-Add1 polypeptide inhibiting phosphorylation of Add1-S726 in HEK293T cells co-transfected with Rock2 and Add1 overexpression plasmids, wherein: FIG. 4A is a sequence layout of TAT-siP-Add1 polypeptide and a control polypeptide TAT-scramble. FIG. 4B shows the transfection of plasmids overexpressing Rock2 and Add1 in HEK293T cells, 48 hours later, treated with varying concentrations of TAT-siP-Add1 and TAT-scramblel polypeptides at 0-20 μ M in cell culture medium for 3 hours, followed by harvesting of the cells and lysis of the cells using RIPA lysate to extract total protein, which is then ready for use after determination of protein content. And respectively detecting the expression levels of Add1-S726, Add1 and Actin protein in the lysis sample. Through comparison, the phosphorylation levels of Add1 and Add1-S726 sites in the control group treated with different TAT-scramble polypeptides have no obvious change, while the phosphorylation level of Add1-S726 appears to be slightly reduced but has no statistical difference in the experimental group treated with TAT-siP-Add1 from 5 μ M, and the phosphorylation level of Add1-S726 is obviously reduced at 10-20 μ M and is most obviously reduced at 15 μ M, which indicates that TAT-siP-Add1 can indeed antagonize the phosphorylation promotion effect of Rock2 on Add1-S726 sites. Fig. 4C is a statistical chart.
FIG. 5 is a graph of immunoblotting of TAT-siP-Add1 polypeptide to inhibit phosphorylation of Add1-S726 in APP/PS1 mice, wherein: fig. 5A is an experimental flow chart. Randomly dividing APP/PS1 mice with similar body weight and age in months into two groups, injecting TAT-siP-Add1 solution into an experimental group in an intraperitoneal injection mode at a concentration of 15mg/kg every day after an adaptation period, injecting TAT-scramble solution into a control group in the same mode and concentration, continuously injecting for two weeks, anesthetizing the mice after 6 days, quickly cutting off heads, taking out brain tissues, placing the brain tissues in PBS (phosphate buffer solution) at 0-4 ℃, quickly separating bilateral hippocampus by using tweezers, homogenizing and extracting total protein of the hippocampus by using RIPA strong lysate, and determining the protein content for later use. FIG. 5B is a immunoblot of tissue from hippocampus of two groups of mice injected with TAT-siP-Add1 and TAT-scramblel polypeptides. The expression levels of Add1-S726, Add1 and Actin protein in hippocampal tissues were measured, respectively. Through comparison, the content of Add1-S726 in the hippocampal tissue of mice injected with TAT-siP-Add1 polypeptide is obviously lower than that of a control group, which shows that TAT-siP-Add1 polypeptide can effectively antagonize the phosphorylation promotion effect of Rock2 on Add1-S726 sites under in vivo conditions. Fig. 5C is a statistical chart.
FIG. 6 is a graph of the results of TAT-siP-Add1 polypeptide increasing APP/PS1 mouse hippocampal dendritic spine density, wherein: fig. 6A is an experimental flow chart. C57 mice (WT) and APP/PS1 mice (A/P) with similar body weight and age are selected and randomly divided into three groups, after an adaptation period, the APP/PS1 mice are injected with TAT-siP-Add1 or TAT-scramblel solution at the concentration of 15mg/kg by intraperitoneal injection every day, the WT mice are injected with physiological saline in the same way and dosage for two weeks continuously, and then experiments such as behavioral experiments of space memory capacity, Golgi staining and electrophysiological detection are carried out. Fig. 6B is a graph showing hippocampal dendritic spine density by golgi staining of three groups of mice. After three groups of mice were anesthetized, the thoracic cavity and septum were cut open, the heart was exposed, heart perfusion was performed with pre-cooled physiological saline until the liver of the mice became white, then the brain tissue was stripped off by decapitation, cut into small pieces, a golgi staining experiment was performed with a kit, then the brain tissue was sectioned, the neuronal dendritic spine morphology was observed with an optical microscope, and the number was counted. Golgi staining revealed that APP/PS1 mice injected with TAT-scramble polypeptide had significantly reduced hippocampal dendritic spine density and that the proportion of mature mushroom-type dendritic spines therein was also greatly reduced by half, compared to WT mice; compared with the prior art, APP/PS1 mice injected with TAT-siP-Add1 polypeptide have obviously increased density of hippocampal dendritic spines, and the proportion of mature mushroom dendritic spines is also increased. FIG. 6C is a statistical plot of the density of dendritic spines of hippocampus neurons in FIG. 6B. FIG. 6D is a statistical graph of the percentage of hippocampal mushroom dendritic spines in FIG. 6B.
FIG. 7 is a graph of the results of TAT-siP-Add1 polypeptide increasing APP/PS1 mouse hippocampal dendritic complexity, wherein: fig. 7A is a golgi staining of the three groups of mice in fig. 6 showing hippocampal neuron morphology. Golgi staining shows that compared with WT mice, APP/PS1 mice injected with TAT-scramble polypeptide have obviously reduced number and branches of hippocampal neurons, and the complexity of the dendrites is reduced; compared with the mouse, the number and branches of hippocampal dendrites of the APP/PS1 mouse injected with the TAT-siP-Add1 polypeptide are obviously increased, and the dendrite complexity is increased, which shows that the injection of the TAT-siP-Add1 polypeptide can effectively improve the dendrite complexity of the APP/PS1 mouse neuron. Fig. 7B is a sholl analysis statistical chart of the hippocampal neuron dendritic branches in fig. 7A. Fig. 7C is a Dendron Complexity Index (DCI) statistical plot of the dendron branches of the hippocampal neuron in fig. 7A.
FIG. 8 is a statistical plot of the electrophysiological results of TAT-siP-Add1 polypeptides in improved mice, wherein: FIG. 8A is a LTP log. Three groups of mice in fig. 6 were decapitated after anesthesia, brain tissue was rapidly removed into frozen artificial cerebrospinal fluid (aCSF), mouse brain tissue was trimmed according to experimental requirements, and the tissue was cut into 300 μm thick tissue pieces with a vibrating microtome and incubated for 1h at room temperature. Appropriate stimulation and recording electrode locations were selected, ltp (long Term stimulation) baseline was stably recorded for 15-30 minutes, followed by induction with 2 bursts of high frequency stimulation (100Hz) at 10 s-sec intervals for 1 sec, and recording was continued for 1 hour. LTP electrophysiological recording shows that compared with WT mice, the LTP-induced amplitude of hippocampal neurons of APP/PS1 mice injected with TAT-scramble polypeptide is obviously reduced and can only reach 1.1 times; the amplitude of LTP induction of hippocampal neurons of an APP/PS1 mouse injected with TAT-siP-Add1 polypeptide is obviously enhanced compared with that of mice in a TAT-scramble group, and can reach 1.4 times, so that the electrophysiological activity of LTP of neurons of the APP/PS1 mouse can be effectively improved by injecting TAT-siP-Add1 polypeptide. FIG. 8B is a statistical chart of LTP results.
FIG. 9 is a statistical chart of the results of TAT-siP-Add1 polypeptide improving mouse learning and memory: three groups of mice in fig. 6 were subjected to behavioral experiments using the Morris water maze test system. The Morris water maze comprises a circular pool with the diameter of 120cm and the height of 60 cm; the diameter of the cylindrical organic glass platform is 10cm, and the height of the cylindrical organic glass platform is 40 cm. The water level in the pool is about 45cm high, the room temperature and the water temperature are kept at 22 +/-2 ℃, and the platform is placed in the center of a certain quadrant and is about 2cm below the water level. During training, the mouse is lightly put in water from any quadrant 1/2 with the arc head facing the pool wall, and the latency period (namely the time taken by the mouse to find a platform from entering the water) and the path of the mouse are used as indexes for measuring the learning memory and the test performance of the mouse. Each mouse is trained 3 times in 3 quadrants every day, the swimming time limit is 60s each time, namely the system automatically stops recording when a platform is not found in 60s, the latency period is recorded as 60s, a tester guides the mouse to stand, and the next training is carried out after the tester has a rest for 20 s. The training time was first 6 days, the time required for the mice to find the platform was recorded every day, and the latency required for the mice to find the platform was recorded. After resting for one day, the platform was removed, the residence time of the mouse in the target quadrant was calculated for 60s and the number of crossings of the mouse at the location of the platform was analyzed to evaluate its memory. Wherein: FIG. 9A is a graph of the movement traces of three groups of mice on day 8 of the water maze experiment; fig. 9B is a graph of the latency statistics of the training phase 6 days prior to the three groups of mouse water maze experiments. In the learning phase of days 4-6, the incubation period for APP/PS1 mice injected with TAT-scramble polypeptide solution to reach the platform was prolonged; in contrast, APP/PS1 mice given TAT-siP-Add1 polypeptide solution injections had significantly reduced latency to reach the platform; FIG. 9C is a graph of the latency statistics of the 8 th day testing phase of the three groups of mouse water maze experiments. In the 8 th day of detection, APP/PS1 mice injected with TAT-scramble polypeptide solution had prolonged latency to reach the platform; in contrast, APP/PS1 mice given TAT-siP-Add1 polypeptide solution injections had significantly reduced latency to reach the platform; FIG. 9D is a graph of the statistics of the number of platform crossings at the 8 th day detection stage of the three groups of mouse water maze experiments. In the 8 th day of detection, APP/PS1 mice injected with TAT-scramble polypeptide solution crossed the platform significantly less frequently; compared with the TAT-siP-Add1 polypeptide solution, APP/PS1 mice injected with the TAT-siP-Add1 polypeptide solution have obviously improved times of crossing the platform. Therefore, the TAT-siP-Add1 polypeptide injection can effectively improve the learning and memory functions of APP/PS1 mice.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example 1:
experimental Material
According to the invention, mice are randomly selected and subjected to intraperitoneal injection of TAT-siP-Add1 solution, so that the combination of Add1 and Rock2 can be blocked, the phosphorylation of Add1 is reduced, the stability of an actin skeleton is enhanced, and the synaptic injury and learning and memory disorder of AD mice are improved.
The specific operation steps are as follows:
firstly, TAT-siP-Add1 polypeptide is applied to improvement of senile dementia.
Subject: male APP/PS1 mice (purchased from Jackson Laboratory), C57/BL mice 6-8 months old (purchased from Beijing Wittingerihua Laboratory animals Co., Ltd.), SPF grade, body weight 20-25 g, normal environment feeding.
The research method comprises the following steps: firstly, detecting the space memory of the mouse: intraperitoneal injection and water maze; ② quantifying the content of the mouse hippocampal-related protein: immunoblotting; ③ TAT-siP-Add1 polypeptide antagonizes the S726 site of Rock2 phosphorylating Add 1: immunoblotting; mouse hippocampal LTP: electrophysiology; detecting the dendritic trees and the dendritic spines of the mouse hippocampal neurons: golgi staining, sholl analysis.
The experimental results are as follows: see fig. 1-9, where the notations of a and # are as follows:
p <0.05, compared to Con/WT group;
p <0.01, compared to Con/WT group;
p <0.001, compared to Con/WT group;
# P <0.05, as compared to TAT-scramble (Scr) group;
# P <0.01, compared to TAT-scramble (Scr) group;
# P <0.001, compared to TAT-scramble (Scr) group.
TAT-siP-Add1 has a sequence shown in SEQ ID NO.1, is artificially synthesized by Jiangsu Qiangmao Biotechnology Co., Ltd, and is shown in FIG. 1 by MS analysis, and the analysis result shows that the amino acid sequence of the synthesized polypeptide is consistent with the SEQ ID NO. 1. The synthesized TAT-siP-Add1 polypeptide has the purity of 98.67%, 10mg of one polypeptide is white powder, can be completely dissolved in water, and is sealed and stored at-20 ℃ in a dark place. Before use, the preparation is diluted by normal saline for injection according to a specified concentration and is used as it is. The TAT-scramble sequence is shown in SEQ ID NO.2 and was also synthesized by this company.
Abnormal phosphorylation levels of Rock2 and Add1 in AD patients: see fig. 2. After the AD patient and normal human brain tissue samples are quickly washed by PBS (phosphate buffer solution) with the temperature of precooling to 0-4 ℃, RIPA strong lysate is added to ice for homogenate, the total protein of the tissue is extracted, and the expression quantification of the protein is carried out. We found that the brain contents of Rock2 and Add1-S726 in AD patients are obviously higher than those in the control group, and the two contents have stronger correlation.
Rock2 in Neuro2a cells promoted phosphorylation of Add1-S72 site: see fig. 3. An empty plasmid overexpressing Rock2 or a control was transfected into Neuro2a cells, and 48 hours later, cellular proteins were collected and quantified for protein expression. Immunochromatographic analysis revealed that Rock2 indeed promotes phosphorylation at the Add1-S726 site.
TAT-siP-Add1 polypeptide inhibits Add1-S726 phosphorylation in HEK293T cells: see fig. 4. Plasmids overexpressing Rock2 and Add1 were transfected into HEK293T cells and 48 hours later, treated with different concentrations of TAT-siP-Add1 and TAT-scramble polypeptides for 3 hours, followed by harvesting of the cells. Immunoblot detection shows that TAT-siP-Add1 with the concentration of 15 mu M can effectively antagonize the phosphorylation promotion effect of Rock2 on Add1-S726 sites. TAT-scramble polypeptides do not have this effect.
TAT-siP-Add1 polypeptide inhibits Add1-S726 phosphorylation in APP/PS1 mice: see fig. 5. Randomly dividing APP/PS1 mice with similar body weight and month age into two groups, injecting TAT-siP-Add1 or TAT-scramblel polypeptide solution into the two groups of mice in an intraperitoneal injection mode at a concentration of 15mg/kg every day, continuously injecting for two weeks, anesthetizing and decapitating the mice after 6 days, quickly separating bilateral hippocampus, and homogenizing and extracting total protein of the hippocampus by using RIPA strong lysate. Immunoblotting shows that TAT-siP-Add1 polypeptide can effectively antagonize phosphorylation promotion of Add1-S726 site by Rock2 under in vivo conditions.
TAT-siP-Add1 polypeptide increased APP/PS1 mouse hippocampal dendritic spine density: see fig. 6. C57 mice (WT) and APP/PS1 mice (A/P) of similar body weight and age were selected and randomized into three groups, and after the adaptation period, APP/PS1 mice were injected daily by intraperitoneal injection with TAT-siP-Add1 or TAT-scramblel solution at a concentration of 15mg/kg, and WT mice were injected with physiological saline in the same manner and dose for two weeks, followed by Golgi staining. Golgi staining shows that the density of hippocampal dendritic spines and the proportion of mushroom type dendritic spines of APP/PS1 mice injected with TAT-scramble polypeptide are also greatly reduced; while mice injected with APP/PS1 having TAT-siP-Add1 polypeptide significantly improved dendritic spine density and a reduction in the proportion of mushroom-type dendritic spines.
TAT-siP-Add1 polypeptide increases the hippocampal dendritic complexity of APP/PS1 mice: see fig. 7. Hippocampal neuronal morphology was analyzed after golgi staining of three groups of mice in fig. 6. As a result, APP/PS1 mice injected with TAT-scramble polypeptide have reduced complexity of hippocampal dendrites; compared with the mouse neuron dendritic complexity, the mouse neuron dendritic complexity of the mouse hippocampal injected with the TAT-siP-Add1 polypeptide is obviously improved, and the TAT-siP-Add1 polypeptide can effectively improve the dendritic complexity of the APP/PS1 mouse neuron.
TAT-siP-Add1 polypeptide improves the electrophysiology of mice: see fig. 8. Three groups of mice in fig. 6 were decapitated after anesthesia, and brain tissue was rapidly removed into frozen artificial cerebrospinal fluid (aCSF) and sectioned with a vibrating microtome. LTP was stably recorded (baseline 15-30 minutes, followed by induction with high frequency stimulation and recording continued for 1 hour) by selecting appropriate stimulation sites and stimulation intensities, and the magnitude of LTP induction was found to be significantly enhanced in APP/PS1 mouse hippocampal neurons injected with TAT-siP-Add1 polypeptide compared to TAT-scramble group mice, indicating that TAT-siP-Add1 polypeptide injection was effective in improving LTP electrophysiological activity in APP/PS1 mouse neurons.
Learning and memory ability of mice: see fig. 9. Three groups of mice in fig. 6 were subjected to behavioral experiments using the Morris water maze test system. As a result, it was found that in the learning phase of days 4-6, the incubation period until reaching the platform was prolonged in APP/PS1 mice injected with TAT-scramble polypeptide solution; in contrast, mice injected with TAT-siP-Add1 polypeptide solution had significantly reduced latency to reach the platform.
In the detection of the 8 th day, the incubation period of the APP/PS1 mouse injected with TAT-scramble polypeptide solution to reach the platform is prolonged, the frequency of crossing the platform is reduced, and the learning and memory ability is poor; compared with the prior art, the incubation period of the APP/PS1 mice injected with the TAT-siP-Add1 polypeptide solution to reach the platform is obviously reduced, the frequency of crossing the platform is obviously improved, and the learning and memory ability is enhanced.
The results show that the density, the dendritic complexity and the electrophysiology of the neuron dendritic spines of the APP/PS1 mouse can be obviously improved by injecting TAT-siP-Add1 polypeptide solution into the abdominal cavity. Immunoblot detection shows that TAT-siP-Add1 polypeptide can obviously inhibit phosphorylation of Add1-S726 site of APP/PS1 mouse. In the Morris water maze, APP/PS1 mice injected with TAT-siP-Add1 polypeptide solution have obviously shortened latency, obviously increased frequency of platform crossing and enhanced learning and memory ability. The experiments prove that the TAT-siP-Add1 polypeptide can antagonize the promotion effect of Rock2 on Add1-S726 phosphorylation, so that the neuron dendritic spine density and dendritic complexity are increased, the electrophysiology is enhanced, and the learning and memory of mice are improved.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
The following is a sequence listing of amino acid sequences to which the present patent application relates, wherein: the sequence 1 is a small molecule polypeptide TAT-siP-Add1 provided by the invention; sequence 2 is a reference TAT-scramble polypeptide; the sequence 3 is TAT cell-penetrating peptide; the sequence 4 is siP-Add1 polypeptide.
Sequence listing
<110> university of science and technology in Huazhong
<120> polypeptide and application thereof in improving learning and memory disorders of senile dementia
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 28
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Lys Lys Lys Lys Phe
1 5 10 15
Arg Thr Pro Ser Phe Leu Lys Lys Ser Lys Lys Lys
20 25
<210> 2
<211> 28
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Arg Pro Lys Lys Thr
1 5 10 15
Lys Phe Phe Lys Lys Ser Lys Lys Leu Lys Lys Ser
20 25
<210> 3
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
1 5 10
<210> 4
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Lys Lys Lys Lys Phe Arg Thr Pro Ser Phe Leu Lys Lys Ser Lys Lys
1 5 10 15
Lys
Claims (5)
1. An artificially synthesized polypeptide, which is characterized in that the amino acid sequence of the polypeptide is shown as SEQ ID NO.1 in a sequence table.
2. A drug for improving learning and memory disorders of senile dementia, which is the polypeptide of claim 1.
3. A pharmaceutical preparation for improving learning and memory disorders due to low senile dementia, comprising the polypeptide of claim 1 and a pharmaceutically acceptable excipient or/and a carrier.
4. Use of the polypeptide of claim 1 in the manufacture of a medicament for the prevention or treatment of senile dementia.
5. The use of the polypeptide of claim 1 in the preparation of a medicament for improving learning and memory disorders due to senile dementia.
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