CN111253492B - Brain-penetrating polypeptide and application thereof in preparing medicine for preventing and treating senile dementia - Google Patents
Brain-penetrating polypeptide and application thereof in preparing medicine for preventing and treating senile dementia Download PDFInfo
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- CN111253492B CN111253492B CN201811456300.5A CN201811456300A CN111253492B CN 111253492 B CN111253492 B CN 111253492B CN 201811456300 A CN201811456300 A CN 201811456300A CN 111253492 B CN111253492 B CN 111253492B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- A—HUMAN NECESSITIES
- 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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- A—HUMAN NECESSITIES
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- A61K38/00—Medicinal preparations containing peptides
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- C07—ORGANIC CHEMISTRY
- 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
Abstract
The invention discloses a small molecular polypeptide TAT-siP-DC and application thereof in preparing a medicine for treating or preventing senile dementia, wherein a TAT protein transduction structural domain and a siP-DC fusion protein polypeptide TAT-siP-DC are artificially synthesized, TAT can carry siP-DC protein polypeptide to pass through blood-brain barrier to be absorbed by neurons, and the polypeptide is applied to an in-vivo senile dementia model to effectively play the biological effect of blocking the combination of beta-catenin and DAPK1, so that the accumulation/phosphorylation of the beta-catenin can be effectively reversed, presynaptic turgor damage shown by an animal of the senile dementia model can be improved, long-term enhanced inhibition and mode separation disorder can be improved, and the small molecular polypeptide TAT-siP-DC can be used for preparing the medicine for treating or preventing senile dementia.
Description
Technical Field
The invention belongs to the field of medicines, relates to a medicine for preventing and treating senile dementia, and particularly relates to application of small molecular polypeptide in preparing a medicine for preventing and treating senile dementia.
Background
Alzheimer's Disease (AD) is one of the most common neurodegenerative diseases in the elderly, accounting for about 2/3 of dementia cases worldwide. Clinically, AD is characterized by a series of cognitive disorders, including prominent episodic memory impairment that usually occurs at an early stage. Pathologically, there are two markers in the AD brain, senile plaques consisting of overproduced beta-amyloid (a β) protein and neurofibrillary tangles (NFTs) consisting of abnormally hyperphosphorylated tau protein. Over the past century, efforts have been made to explore the underlying mechanisms of memory deficits, and there is increasing evidence that overloaded a β in the brain may be the leading cause of AD memory impairment. Although numerous studies have found potential downstream signaling of a β mediated synaptic toxicity, the detailed mechanism by which a β induces episodic memory impairment is unclear.
One key feature of contextual memory is the ability to distinguish similar experiences, which are defined as strongly dependent on contextual discrimination of the hippocampal network, and are believed to be facilitated by pattern separation. Mode separation is a neural computation in which two similar input modes are made more orthogonal (dissimilar) as output modes. As described earlier, pattern separation is an indispensable step in information processing to avoid interference between stored information. It has been reported that early alzheimer patients have pattern-separated lesions. Meanwhile, disruption of the Dentate Gyrus (DG) -CA3 network of its hippocampus was found in a 12-month old Tg2576 mouse, a well-known mouse model of AD, which is critical to eliminating the overlapping pattern of information.
One of the main causes of senile dementia is memory decline due to choline deficiency. The commonly used medicines for slowing the senile dementia are as follows: (1) amide central stimulants: increase the synthesis of brain proteins and acetylcholine, such as piracetam. (2) Acetylcholinesterase inhibitors: inhibiting cholinesterase activity, and increasing the content of acetylcholine in brain, such as donepezil. (3) Calcium antagonists: selectively dilate cerebral vessels and improve memory and cognitive functions. (4) Other classes of drugs: for example, citicoline improves brain metabolism, and folium Ginkgo extract can improve brain function by scavenging oxygen free radicals. However, there is no specific therapeutic drug or method available, and the drug only can slightly relieve the symptoms of patients with middle and late-stage senile dementia or delay the progress of the disease course. Therefore, the development of new medicaments aiming at the pathological features of the senile dementia is particularly important.
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 brings the polypeptide connected with TAT into cells within minutes, crosses the blood brain barrier and enters neurons, and the polypeptide brought into the cells retains the original biological activity, thereby playing a biological role.
Disclosure of Invention
The invention aims to provide a brain-penetrating small-molecule polypeptide which has the effect of treating and preventing senile dementia.
The technical scheme for realizing the invention is as follows:
the amino acid sequence of the brain-penetrating small molecule polypeptide provided by the invention is shown in SEQ ID NO. 1.
SEQ ID NO.1：M YGRKKRRQRRRAHQDTQRRTSMGGTQQQFVE。
The TAT cell-penetrating peptide (YGRKKRRQRR) is connected with siP-DC (RAHQDTQRTSMGGTQQQFVE) to obtain the TAT-siP-DC polypeptide (SEQ ID NO.1: M YGRKKRRQRRRAHQDTQRRTSMGGTQQQFVE) with biological activity, and the TAT cell-penetrating function is utilized to convey the siP-DC polypeptide into blood to penetrate through a blood brain barrier and be taken up by brain nerve cells so as to play the biological function.
The brain-penetrating small molecule polypeptide TAT-siP-DC (SEQ ID NO:1: MYGRKKRRQRRRAHQDTQRRTSMGGTQQQFVE) provided by the invention has the effect of treating or preventing senile dementia, and is found to be capable of effectively improving the mode separation disorder shown by senile dementia model animals by intravenous injection, so that a molecular target is provided for further developing a medicine for clinically treating senile dementia.
Based on the recent research and TAT technology of the applicant, the applicant synthesizes a membrane-permeable small molecule polypeptide TAT-siP-DC which is composed of an amino acid sequence competitively bound with beta-catenin (beta-catenin) to Death-related protein kinase 1 (DAPK 1), and the biological effect of blocking the binding of the beta-catenin and the DAPK1 is effectively exerted by applying the membrane-permeable small molecule polypeptide TAT-siP-DC to an in-vivo senile dementia transgenic animal model, so that the phosphorylation of the beta-catenin is weakened, the presynaptic function is restored, the pattern separation (pattern separation) obstacle shown by the senile dementia animal model is improved, and a molecular target is provided for further developing a medicament for clinically treating the senile dementia.
The inventor of the patent application finds in research that the beta-catenin and the DAPK1 are combined with each other in an animal model of senile dementia to mediate phosphorylation and aggregation of the beta-catenin, so that presynaptic function of moss fibers (mossy fiber, MF) -CA3 of the hippocampus is damaged, and pattern separation disorder is caused. Blocking the combination of beta-catenin and DAPK1 can effectively improve the symptoms. In response to this finding, applicants linked TAT-penetrating peptide (YGRKKRRQRR) to siP-DC (RAHQDTQRTSMGGTQQQFVE) to obtain a biologically active TAT-siP-DC polypeptide, which was then injected in vivo intravenously to allow TAT-siP-DC polypeptide to enter the blood and cross the blood-brain barrier and be taken up by brain neurons to exert its biological function.
The amino acid sequence related to the invention comprises:
(1) a small molecule polypeptide TAT-siP-DC with brain penetration property has the following sequence:
M YGRKKRRQRRRAHQDTQRRTSMGGTQQQFVE。
(2) the control of TAT-siP-DC is TAT-scramblel, the sequence of which is:
M YGRKKRRQRRATHRQRQSFQGETQDQMVGTR。
TAT-siP-DC polypeptide and the reference TAT-scramblel are synthesized and purified from the C end to the N end of the sequence by Qianzhou Biotechnology, Inc.
The experimental studies conducted by the present invention include:
(1) application research of TAT-siP-DC in senile dementia animal model
APP/PS1 transgenic mice are a recognized animal model for senile dementia. Model separation experiments, long-term enhancement and moss fiber knot (MFB) staining detect early complex hippocampal-related memory capacity of transgenic mice. 5-month-old APP/PS1 transgenic mice are injected with 10 mug/mg TAT-siP-DC solution once per vein, injected once every three days, and after 1 month of continuous injection, animals are subjected to mode separation experiments, electrophysiological experiments or sacrifice, and brain tissues are taken. The results show that the compound can effectively improve the pattern separation disorder, long-term potentiation (LTP) inhibition, MFB injury and beta-catenin accumulation/phosphorylation shown by APP/PS1 mice which are model animals of senile dementia. Further proves that TAT-siP-DC has exact therapeutic effect on senile dementia models.
(2) Application research of TAT-siP-DC in senile dementia cell model
A β oligomer (5 μ M, 24h) treatment was given as a model of senile dementia cells on N2a cells and mouse primary hippocampal neurons. Immunoblotting was used to detect changes in beta-catenin accumulation/phosphorylation, and whole-cell patch clamp was used to detect presynaptic function.
Experimental data:
according to the invention, through injecting TAT-siP-DC polypeptide into 5-month-old APP/PS1 mice and giving siP-DC treatment to an in vitro senile dementia cell model, the phosphorylation and aggregation of beta-catenin, the presynaptic function of MF-CA3 and pattern separation disorder in senile dementia model animals can be improved.
The specific operation steps are as follows:
1. test object
Male APP/PS1 mice (purchased from Jax laboratories), SPF grade, weight 25 ~ 32 g, 20, conventional environment feeding. Grouping experiments: (ii) normal group (wild type control mice); ② a model group (APP/PS1 mice are injected with TAT-scrambles); ③ treatment group (APP/PS1 mice injected with TAT-siP-DC); fourthly, a control group (wild type mice are injected with TAT-siP-DC); each group had 10.
N2a cells and mouse primary hippocampal neurons were treated by administering a β oligomer as a model of senile dementia cells, on which siP-DC treatment was administered as a treatment group.
2. Preparation of experimental animal model
Normal group: 5-month old wild-type control mice, untreated.
Model group: 5-month-old APP/PS1 mice were given TAT-scramble polypeptide by tail vein single injection at a dose of 10 μ g/mg.
(iii) treatment group: 5-month-old APP/PS1 mice were given TAT-siP-DC polypeptide by single injection into the tail vein at a dose of 10 μ g/mg.
Fourthly, comparison group: 5-month old wild-type control mice were given TAT-siP-DC polypeptide by single injection into the tail vein at a dose of 10. mu.g/mg.
3. Research method
Polypeptide interference binding: detecting the combination condition of DAPK1 and beta-catenin by co-immunoprecipitation under the conditions of polypeptide existence and polypeptide nonexistence;
second, early complex hippocampal-related memory in mice: training and detecting mode separation;
③ mouse electrophysiology: enhancing electrophysiological recording over a long period of time;
MFB form: performing immunofluorescence statistics;
beta-catenin stability: immunoblotting to detect aggregation and phosphorylation;
sixthly, presynaptic function: whole-cell patch clamp detection of micro excitatory postsynaptic current (mepscs);
activity of DAPK 1: immunoblots detect the expression of itself and its substrates.
4. Results of the experiment
An artificial synthetic chromatogram of small molecular polypeptide siP-DC. See fig. 1.
(1) The co-immunoprecipitation verifies the effectiveness of the polypeptide: see fig. 2. Performing co-immunoprecipitation by using DAPK1 antibody, wherein the beta-catenin combined with DAPK1 is not changed when no polypeptide is added; after addition of the polypeptide, the binding of beta-catenin to DAPK1 was significantly reduced. Co-immunoprecipitation with β -catenin antibodies further demonstrated that TAT-siP-DC can interfere with DAPK1 and; binding of beta-catenin. At the same time, the polypeptides we synthesized were also shown to be effective.
(2) Training and detection results of pattern separation: see fig. 3. There was no difference in stiffness performance between the training phase and the initial testing phase. In the identification stage, the identification rate of the model group is obviously reduced compared with that of the normal group; the discrimination rate was clearly restored in the siP-DC-administered treatment group compared with the model group.
(3) Electrophysiological recording results: see fig. 4. The MED64 recording system measures LTP in the MF-CA3 pathway, with the LTP in the model group being suppressed compared to the normal group; the LTP inhibition was significantly improved in the siP-DC-administered treatment group compared to the model group.
(4) Statistical results of hippocampal MFB morphology: see fig. 5. Compared with the normal group, the MFB density of the model group is obviously reduced, the main bouton area is reduced, and the number of presynaptic filamentous pseudo feet is reduced; the siP-DC-administered treatment group exhibited improved MFB density, size and complexity of lesions compared to the model group.
(5) Immunoblotting results for in vivo beta-catenin stability: see fig. 6. Compared with the normal group, the accumulation/phosphorylation of the beta-catenin of the model group is obviously increased; compared with the model group, the beta-catenin accumulation/phosphorylation of the treatment group which is given with siP-DC is obviously reduced.
(6) Immunoblotting results for ex vivo beta-catenin stability: see fig. 7. Compared with the control group, the phosphorylation of beta-catenin accumulation/PS 552 site of the A beta treatment group is obviously increased, and is improved after siP-DC is given.
(7) The detection result of the whole-cell patch clamp is as follows: see fig. 8. Compared with the control group, the frequency of mEPSC in the A beta treatment group is obviously reduced, and the presynaptic damage is recovered after siP-DC administration.
(8) Immunoblotting results for DAPK1 activity: see fig. 9. Compared to the model group, DAPK1 itself and its substrate P-MLC were unchanged in the siP-DC-administered treatment group, indicating that siP-DC treatment did not alter DAPK1 activity.
5. Conclusion and usage recommendations:
the above results show that a β may impair MF-CA3 presynaptic function by promoting β -catenin accumulation/phosphorylation, siP-DC treatment may ameliorate pattern segregation defects, LTP inhibition, MFB injury, and β -catenin accumulation/phosphorylation in AD mice by interfering with DAPK1 binding to β -catenin.
Our research shows that the polypeptide siP-DC reduces phosphorylation and accumulation of beta-catenin by blocking interaction between DAPK1 and beta-catenin for a long time, saves functions of MF-CA3 loop, and improves the capability of AD mouse mode separation. Our studies provide a new epigenetic mechanism for the early stage characteristic cognitive deficits in AD for the first time. The outstanding advantages of the invention also include:
(1) the brain penetrating small molecular polypeptide TAT-siP-DC provided by the invention is easy to induce expression purity, is completely soluble, is suitable for intravenous injection, and has no toxic or side effect.
(2) The TAT-siP-DC polypeptide disclosed by the invention can carry siP-DC protein polypeptide to pass through blood brain barrier to be taken by neurons, and can be converted and applied to nervous system diseases such as senile dementia, so that the TAT-siP-DC polypeptide has feasibility of practical operation.
Drawings
FIG. 1 is an artificial chromatogram of a small molecule polypeptide TAT-siP-DC.
FIG. 2 is a graph showing the co-immunoprecipitation result of the polypeptide TAT-siP-DC interfering the combination of DAPK1 and beta-catenin.
FIG. 3 is a statistical plot of the results of TAT-siP-DC improving APP/PS1 mouse pattern isolation. FIG. 3(A) is a statistical graph of the percent stiffness during the training phase on days 1-3, FIG. 3(B) is a statistical graph of the percent stiffness during the initial testing phase on days 4-5, and FIG. 3(C) is a statistical graph of the discrimination rate during the discrimination phase on days 6-17.
FIG. 4 is a graph of the results of TAT-siP-DC improving long-term enhancement in APP/PS1 mice.
FIG. 5 is a statistical chart of the results of TAT-siP-DC in improving MFB injury in APP/PS1 mice. Graph A is a statistical graph of MFB density, graph B is a statistical graph of the size of a bouton region in the center of MFB, and graph C is a statistical graph of the number of presynaptic pseudopodia.
FIG. 6 is a diagram showing the result of the immunoblotting of TAT-siP-DC for improving the aggregation and phosphorylation of β -catenin in APP/PS1 mice. The result of western blotting is shown in FIG. A, and the result is shown in FIG. B.
FIG. 7 is a diagram showing the result of immunoblotting that TAT-siP-DC improves the aggregation and phosphorylation of beta-catenin induced by A beta. The result of western blotting is shown in FIG. A, and the result is shown in FIG. B.
FIG. 8 is a graph of the results of TAT-siP-DC improving the decrease in mEPSC frequency induced by A β.
FIG. 9 is a graph of the immunoblot results of TAT-siP-DC not affecting DAPK1 activity. The result of western blotting is shown in FIG. A, and the result is shown in FIG. B.
In each figure, P <0.05, P <0.01, P <0.001 was compared to the normal group; # P <0.01, # # P <0.001 compared to the model group; ns indicates no significant difference.
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
Artificial synthesis of TAT-siP-DC
TAT-siP-DC has a sequence shown in SEQ ID NO.1 and is artificially synthesized by Shanghai Qianzhiya Biotechnology Co., Ltd, the synthesis report is shown below, and the chromatogram is shown in FIG. 1.
TAT-siP-DC Artificial Synthesis HPLC report
The synthesized TAT-siP-DC polypeptide has the purity of 97.14 percent, each TAT-siP-DC polypeptide is 1mg, is white powder, is completely soluble in water, and is stored at-20 ℃ in a sealed and lightproof manner. Before use, the preparation is diluted by normal saline for injection according to a specified concentration and is used as it is.
Example 2
The polypeptide TAT-siP-DC interferes with the combination of DAPK1 and beta-catenin. See fig. 2.
The polypeptide was synthesized by Shanghai Qiaozhi. Co-immunoprecipitation with DAPK1 antibody showed that the binding of DAPK1 and β -catenin was unchanged without administration of the polypeptide; in the presence of the polypeptide, the binding of DAPK1 and beta-catenin is interfered. Co-immunoprecipitation using β -catenin antibodies further demonstrated that TAT-siP-DC can interfere with the binding of DAPK1 to β -catenin. At the same time, the polypeptides that we synthesized have also proven effective.
Example 3
Application of TAT-siP-DC in senile dementia animal model
(1) TAT-siP-DC ameliorated pattern isolation disorders in APP/PS1 mice. See fig. 3.
Pattern segregation is a more complex hippocampal-associated memory that is impaired by established patterns in early AD patients. The contextual fear house includes a shock generator (AniLab, which can provide a 0.1 to 1.0mA shock) and is associated with a computer. The sound-proof box is equipped with lights and a small fan (for ventilation and background sound). In addition, there are voltmeters and sonographers to detect the intensity of the stimulus. The difference between the chamber A and the chamber B we used is that the bottom of the chamber A was placed with a 100% alcohol solution of benzaldehyde (0.25% strength), the side walls of the chamber B were angled inward at 60 degrees, and the chamber A was wiped with a 5% sodium hydroxide solution which was odorless and the chamber B was wiped with a harsh 1% acetic acid solution. Days 1-3 were the training phase, and each group of mice received the same conditioning training in chamber A, i.e., 192s later, a single foot shock (2 s; 0.65mA), and was removed from the chamber 1 minute after termination of the foot shock; day 4 and day 5 are the initial testing stages, each mouse was placed in chamber a and chamber B for testing (8 min; no shock) respectively, and recorded every 8 s; day 6 to day 17 (which began the day after the second test described above) were the identification phase, mice were placed in the a and B compartments each day, following the baababbaab design in order, such that on day 7, day 8, day 10, day 13, day 15 and day 16, all animals were placed first in the a compartment and then in the B compartment. For the remaining days, the two chambers were reversed in order. Throughout this period, all animals received a single foot shock in chamber a, and not chamber B. The first 192s in each chamber were recorded every day, every 8 s. The recording relies on the rigor behavior, i.e. immobility except breathing, and the recording score is converted into a rigor rate, and the discrimination rate in the discrimination stage is: chamber A/(chamber A + chamber B).
We found that all mice completed the tasks required for the training phase from day 1 to day 3 and completed the initial testing phase from day 4 to day 5. During the identification phase from day 6 to day 17 tasks, control mice quickly learned to differentiate scenes; however, AD mice exhibit a short but very significant deficiency during the acquisition of the identification task. This deficiency in AD mice was manifested by an increase in the rigidity response in the non-shocked chamber B. In contrast, AD mice showed no rigidity defect for the shock-present scenario (chamber a). By day 17 (day 12 of the identification task), AD mice were also unable to distinguish between the two scenes. Demonstrating that it shows significant impairment in the identification task. We also found that siP-DC treatment significantly improved the discrimination in AD mice, but not in wild type mice.
(2) TAT-siP-DC improved the inhibition of LTP in APP/PS1 mice. See fig. 4.
Hippocampal Moss Fiber (MF) synapses play a key role in gated information transfer in the DG-CA3 network and are central to pattern segregation and spatial memory establishment. We first measured long-term potentiation (LTP) in the MF-CA3 pathway using the MED64 recording system, functionally evaluating the changes in MF-CA3 projection.
Mice were sacrificed by decapitation and brain tissue was immediately immersed in ice-cold artificial cerebrospinal fluid (125mM NaCl,2.0mM KCl, 2.5mM CaCl)2,1.2mM MgSO4,1.2mM KH2PO4,26mM NaHCO3And 11mM glucose), with a continuous 95% O feed2And 5% CO2A lateral sagittal section of 300 μm was cut with a vibrating microtome (Leica). Sections were preincubated in oxygenated artificial cerebrospinal fluid at 32 ℃ for 1.5 hours and then transferred to a recording tank constantly perfused with artificial cerebrospinal fluid. A planar multi-electrode recording setup (MED 64; Alpha Med Sciences, Tokyo, Japan) was used to record the field excitatory post-synaptic potential (fEPSP). Stimulation and recording electrodes were placed in the hippocampal DG and CA3 regions, respectively. Stimulation of the DG region induces field potentials. The appropriate stimulation and recording electrode locations are selected. An input-output relationship (I/O curve) was generated by providing 10-to 100- μ a electrical stimulation and measuring the amplitude of the peak value fEPSP. The stimulus intensity was selected to be 40% of the maximum amplitude of evoked fEPSPs. After 30 minutes to stabilize the baseline, LTP was induced using a series of high frequency stimuli (100Hz,1s) and recorded for 120 minutes. Statistical analysis was performed using the last 5 minutes of data recorded.
AD mice showed inhibition of LTP in MF-CA3 projections, which was reported in 6 month AD mice. Here we used 6-month old APP/PS1 mice and we found that long-term potentiation of the MF-CA3 pathway was inhibited and that this inhibition was improved after administration of the polypeptide TAT-siP-DC.
(3) TAT-siP-DC ameliorated MFB damage in APP/PS1 mice. See fig. 5.
Morphologically, granulosa cells in the Dentate Gyrus (DG) are known to project towards CA3 pyramidal neurons with multiple terminal types: large moss fiber knots (MFB) and small filamentous pseudopodia extensions emanating from these knots. We analyzed the formation of MFBs and filopodia under different treatment regimes to assess the morphological changes projected by MF-CA 3.
The mice were anesthetized and perfused to collect brains, after postfixation and dehydration, sectioned in a cryomicrotome (Leica 1950, Wetzlar, Germany), and brain slices were stored in PBS for future use. Brain slices were blocked with BSA at room temperature for 30min to block non-specific sites, followed by overnight incubation with primary antibody at 4 deg.C, next day PBS washing for 30min, secondary antibody incubation at room temperature for 1h, and finally scanning imaging of the hippocampal region with a laser confocal microscope (LSM 780; Carl Zeiss) for statistical analysis of the imaging results.
As expected, the characteristic shape of MFBs is formed in wild-type mice, i.e. consisting of a large major knob and several presynaptic filamentous pseudopodas. MFB density was significantly reduced in AD mice, the major bouton region was reduced, and the number of presynaptic filopodia was reduced. These results indicate that AD mice present lesions of MFB density, size and complexity. siP-DC treatment restored the size of the main bouton area and the number of presynaptic filopodia.
(4) TAT-siP-DC improves the aggregation and phosphorylation of beta-catenin in APP/PS1 mice. See fig. 6.
Previous studies have shown that the accumulation of β -catenin, regulated by its phosphorylation, is critical for synaptic formation, and that phosphorylation of β -catenin at the Ser552 site increases its stability and may induce its accumulation.
The mouse is dislocated and killed, brain tissues are taken out rapidly, bilateral hippocampus is separated rapidly in 0.05M Tris buffer salt solution (TB, pH7.0) on ice, 10% of protein homogenate is prepared, centrifugation is carried out for 5 minutes at 1000rmp, and supernatant is taken out and is reserved after protein concentration is determined. Detecting the protein level of the beta-catenin and the phosphorylation level of the beta-catenin, namely PS 552-beta-catenin, wherein the beta-actin is an internal reference. We found that AD mice show accumulation/phosphorylation of beta-catenin, and siP-DC treatment reduces accumulation/phosphorylation of beta-catenin
Example 4
Application of TAT-siP-DC in senile dementia cell model
(1) The polypeptide TAT-siP-DC improves the aggregation and phosphorylation of beta-catenin induced by A beta. See fig. 7.
First, a β oligomers were prepared: the a β polypeptide (Qiangyao, Shanghai, China) was dissolved in 100% hexafluoroisopropanol to 1mM and the hexafluoroisopropanol was removed by vacuum. The peptide was resuspended in dimethyl sulfoxide to 5mM, further diluted with F12 (phenol red free) medium to 100. mu.M, and incubated at 4 ℃ for 24 hours. The solution was centrifuged at 13,000rpm for 20 minutes and the supernatant was collected for use or stored at-20 ℃. After 24h of A.beta.treatment, cells were harvested, centrifuged at 1000rmp for 5 min, and the supernatant was removed and assayed for protein concentration. We found that administration of a β treatment resulted in aggregation of β -catenin and increased phosphorylation of the PS552 site; siP-DC can reduce the accumulation and over-phosphorylation of beta-catenin caused by A beta.
(2) TAT-siP-DC ameliorated the decrease in mEPSC frequency induced by A β. See fig. 8.
Accumulation of β -catenin decreases the rate of synaptic vesicle release, which is critical for presynaptic maturation.
Whole cell recording pipettes (3-5 M.OMEGA.) containing 120mM CH4SO3,20mM CsCl，4mM NaCl 10mM HEPES，0.05mM EGTA，4mM Mg2ATP，0.2mM Na3GTP, 5mM QX-314 and w290mOsm solution. The bath solution contained 124mM NaCl, 3mM KCl, 26mM NaHCO3,1.2mM MgCl2·6H2O，1.25mM NaH2PO4·2H2O，10mM C6H12O6,2mM CaCl2pH 7.4 and 305 mOsm. mEPSC was recorded at 270mV in the presence of 1. mu.M tetrodotoxin and 10. mu.M bicuculline. The pceps were distinguished from baseline noise using pClamp 10(Molecular Devices corp., Sunnyvale, CA). The mini post-synaptic current was automatically measured by setting an appropriate threshold of-6 pA (2.5 times the noise SD). Cumulative probability maps were used to compare the spacing and amplitude of mepscs in neurons from the sham surgical sections. The Kolmogorov-Smirnov double sample test was used to compare the distributions of interval and amplitude. Data was analyzed using the entire 3 minute standard acquisition cycle.
Through a whole-cell patch clamp experiment, we find that the mEPSC frequency is reduced when A beta treatment is given, and the presynaptic function is proved to be damaged; siP-DC reduced the decreased mEPSC frequency caused by A.beta.thereby ameliorating the damage.
Example 5
TAT-siP-DC did not affect the activity of DAPK 1.
Sample preparation was the same as in FIG. 6.
The results show that the treatment of AD mice with siP-DC did not change DAPK1 itself and its substrate P-MLC, i.e. siP-DC did not change DAPK1 activity, demonstrating that the way in which it acts was not by changing DAPK1, but only interfered with the binding of DAPK1 and beta-catenin.
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.
Sequence listing
<120> brain-penetrating polypeptide and application thereof in preparing medicine for preventing and treating senile dementia
<141> 2018-11-30
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 32
<212> PRT
<213> Artificial Sequence
<400> 1
Met Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala His Gln Asp
1 5 10 15
Thr Gln Arg Arg Thr Ser Met Gly Gly Thr Gln Gln Gln Phe Val Glu
20 25 30
<210> 2
<211> 32
<212> PRT
<213> Artificial Sequence
<400> 2
Met Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Ala Thr His Arg Gln
1 5 10 15
Arg Gln Ser Phe Gln Gly Glu Thr Gln Asp Gln Met Val Gly Thr Arg
20 25 30
Claims (5)
1. An artificially synthesized polypeptide, the sequence of which is shown in SEQ ID NO. 1.
2. The use of the polypeptide of claim 1 for the preparation of a medicament for the treatment of senile dementia.
3. The use of the polypeptide of claim 1 in the manufacture of a medicament for improving cognitive deficits characteristic of early-stage Alzheimer's disease.
4. The use of the polypeptide of claim 1 for the preparation of a medicament for ameliorating the loss of function of the isolated senile dementia pattern.
5. The use of the polypeptide of claim 1 in the preparation of a medicament for improving synaptic function in Alzheimer's disease.
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