WO2023246847A1

WO2023246847A1 - B2m-glun1 blocking peptide, and pharmaceutical composition and use thereof

Info

Publication number: WO2023246847A1
Application number: PCT/CN2023/101626
Authority: WO
Inventors: 王鑫; 高月; 黄莉红
Original assignee: 厦门大学
Priority date: 2022-06-24
Filing date: 2023-06-21
Publication date: 2023-12-28
Also published as: CN117327168A

Abstract

Provided are a drug for reducing a B2M level in a human brain, a drug for reducing a level of an amyloid precursor protein in the human brain, a drug for inhibiting the binding of GluN1 and B2M in the human brain, or a drug for repairing synaptic damage caused by the increase of B2M in the human brain.

Description

B2M-GluN1 blocking peptide, pharmaceutical compositions and uses thereof

Technical field

The invention belongs to the field of biomedicine and relates to B2M-GluN1 blocking peptide, its pharmaceutical composition and uses.

Background technique

Down syndrome (DS), also known as Trisomy 21, is one of the most common intellectual disabilities. About 1 in 800 newborns worldwide suffer from this disease. Patients with Down syndrome have one or part of chromosome 21. The increase in gene copy number on chromosome 21 leads to abnormal gene expression and ultimately causes symptoms of a variety of diseases, including developmental delay, intellectual disability, language delay, immune and endocrine systems. Abnormalities and defects in the bones, heart, and digestive system, among others. Intellectual retardation is the most prominent and serious symptom of Down syndrome. The vast majority of children have varying degrees of intellectual development disabilities. Most of the children have IQs of about 25 to 50 (normal people are above 90). It becomes more and more obvious with age, and language problems , memory, abstract thinking, etc. will be damaged. In addition, almost all patients with Down syndrome will develop neuropathological features similar to Alzheimer's disease (AD) after the age of 40, and 60% of patients with Down syndrome will show symptoms by the age of 65. Significant symptoms of Alzheimer's disease dementia. However, currently, there is still a lack of effective drug treatment and intervention methods for the intellectual disability of Down syndrome patients.

Alzheimer's disease is one of the most common neurodegenerative diseases in humans. According to the World Alzheimer's Disease 2018 Report, 50 million people worldwide were suffering from Alzheimer's disease in 2018. By 2050, this number will will increase to 152 million. The main pathological characteristics of the disease include amyloid deposition formed by the oligomerization of β-amyloid (Aβ) produced by the cleavage of amyloid precursor protein (APP) in the brain, and abnormal phosphorylation of the intracellular microtubule-binding protein tau. Neurofibrillary tangles (NFTs) formed after aggregation, neuronal loss and excessive neuroinflammation. The main clinical manifestations of Alzheimer's disease patients are memory decline and cognitive impairment, and this decline worsens as the disease progresses, eventually leading to the loss of all memory and ability to take care of themselves until death. In view of the global aging population, the continued increase in the incidence of Alzheimer's disease, and the fact that no effective treatment has yet been found, Alzheimer's disease has become one of the diseases that seriously threatens human health.

GluN1 is a component subunit of the NMDA receptor. It is encoded by the human chromosome 9 gene. It contains 938 amino acids and is a three-transmembrane protein, including the N-terminal extracellular segment, C-terminal intracellular segment, transmembrane structure and cell structure. Extracellular loop. NMDA receptors are considered to be a potential pathogenic feature of a variety of neurological diseases, such as ischemic stroke, traumatic brain injury, Alzheimer's disease, epilepsy, mood disorders, and schizophrenia. NMDA receptors are diverse in terms of subunit composition, biophysical and pharmacological properties, interactions and subcellular localization. developing Subunit composition varies in different CNS regions during processes and disease states. Therefore, NMDA receptors have always been a research hotspot and drug target in the field of neuropharmacology. In recent years, interest in NMDA receptor modulators as therapeutic agents has also increased significantly. Therefore, these compounds will provide new tools to study the physiology of NMDA receptor signaling, thereby revealing new therapeutic opportunities. Memantine, a drug currently approved by the FDA for the treatment of Alzheimer's disease, is a reversible blocker of NMDA glutamate receptors, but its mechanism and mode of action are completely different from those of the present invention.

β2-microglobulin (B2M) is a pro-aging factor that has gradually received widespread attention in recent years. B2M is a component subunit of the major histocompatibility complex I (Major histocompatibility complex I, MHC-I) and consists of The gene encoded on human chromosome 15 contains 119 amino acids. Since B2M is not anchored to the cell membrane through a transmembrane domain, B2M can dissociate from the MHC-I complex and enter the intercellular space. Under normal circumstances, B2M protein exists in the form of soluble monomers, but under the influence of some pathological factors, B2M will aggregate and deposit. These pathological factors include aging, long-term renal dysfunction, and inflammation. B2M amyloid deposition is mainly found in bone and joint areas and eventually leads to severe arthritis, fractures and carpal tunnel syndrome. In addition, the levels of B2M in serum and plasma are increased in many disease states. However, there is no research report on whether B2M has a direct or indirect impact on the development of Down syndrome and Alzheimer's disease.

Contents of the invention

After in-depth research and creative work, the inventor discovered the role of B2M in the occurrence and development of Down syndrome. The inventor surprisingly found that the blocking peptide that hinders the binding of B2M and GluN1 has the potential to prevent and treat Down syndrome or The potential of drugs to treat cognitive impairment caused by Alzheimer's disease. The following invention is thereby provided:

One aspect of the present invention relates to an isolated polypeptide, which is the polypeptide shown in SEQ ID NO: 8 or a truncated fragment of the polypeptide shown in SEQ ID NO: 8; preferably, the truncated fragment comprises SEQ ID NO: The polypeptide shown in 11.

In some embodiments of the present invention, the isolated polypeptide is a polypeptide represented by any one of SEQ ID NO: 11 or SEQ ID NOs: 14-32.

AVRDNKLHAFIWDSAVLEFEASQ(SEQ ID NO:14)

VRDNKLHAFIWDSAVLEFEASQ(SEQ ID NO:15)

RDNKLHAFIWDSAVLEFEASQ(SEQ ID NO:16)

DNKLHAFIWDSAVLEFEASQ(SEQ ID NO:17)

NKLHAFIWDSAVLEFEASQ(SEQ ID NO:18)

KLHAFIWDSAVLEFEASQKCDLV(SEQ ID NO:19)

KLHAFIWDSAVLEFEASQKCDL(SEQ ID NO:20)

KLHAFIWDSAVLEFEASQKCD(SEQ ID NO:21)

KLHAFIWDSAVLEFEASQKC(SEQ ID NO:22)

KLHAFIWDSAVLEFEASQK(SEQ ID NO:23)

RDNKLHAFIWDSAVLEFEASQKCD(SEQ ID NO:24)

RDNKLHAFIWDSAVLEFEASQKC(SEQ ID NO:25)

RDNKLHAFIWDSAVLEFEASQK(SEQ ID NO:26)

DNKLHAFIWDSAVLEFEASQKCD(SEQ ID NO:27)

DNKLHAFIWDSAVLEFEASQKC(SEQ ID NO:28)

DNKLHAFIWDSAVLEFEASQK(SEQ ID NO:29)

NKLHAFIWDSAVLEFEASQKCD(SEQ ID NO:30)

NKLHAFIWDSAVLEFEASQKC(SEQ ID NO:31)

NKLHAFIWDSAVLEFEASQK(SEQ ID NO:32)

Another aspect of the invention relates to an isolated polynucleotide encoding an isolated polypeptide according to any one of the invention.

Yet another aspect of the invention relates to a recombinant expression vector comprising an isolated polynucleotide of the invention.

Yet another aspect of the invention relates to a transformed cell comprising the recombinant expression vector of the invention.

Yet another aspect of the invention relates to a pharmaceutical composition comprising one or more (eg 2, 3, 4 or 5) isolated polypeptides according to any one of the invention.

In some embodiments of the present invention, the pharmaceutical composition further contains one or more pharmaceutically acceptable excipients.

Another aspect of the present invention relates to the use of the isolated polypeptide according to any one of the present invention for the treatment or prevention of Down syndrome, Alzheimer's disease, or the diagnosis caused by Down syndrome or Alzheimer's disease. Use in medicines that prevent damage.

Another aspect of the present invention relates to the use of the isolated polypeptide according to any one of the present invention in the preparation of the following medicines: use:

Drugs that reduce B2M levels in the human brain, drugs that reduce amyloid precursor protein levels in the human brain, drugs that inhibit the binding of GluN1 to B2M in the human brain, or drugs that repair synaptic damage caused by increased B2M in the human brain.

The isolated polypeptide according to any one of the present invention is used to treat or prevent Down syndrome, Alzheimer's disease, or cognitive impairment caused by Down syndrome or Alzheimer's disease.

The isolated polypeptide according to any one of the present invention is used to reduce the level of B2M in the human brain, reduce the level of amyloid precursor protein in the human brain, inhibit the binding of GluN1 to B2M in the human brain, or repair B2M in the human brain. Increased synaptic damage caused.

Yet another aspect of the present invention relates to a method for treating or preventing Down syndrome, Alzheimer's disease, or cognitive impairment caused by Down syndrome or Alzheimer's disease, comprising administering to a subject in need or with an effective amount of an isolated polypeptide of any one of the invention.

Yet another aspect of the present invention relates to a method for reducing B2M levels in the human brain, reducing amyloid precursor protein levels in the human brain, inhibiting the binding of GluN1 to B2M in the human brain, or repairing synaptic damage caused by increased B2M in the human brain. , comprising the step of administering an effective amount of an isolated polypeptide of any one of the invention to a subject in need thereof.

In some embodiments of the present invention, the method of treating or preventing Down syndrome, Alzheimer's disease, or cognitive impairment caused by Down syndrome or Alzheimer's disease, or the method Methods for inhibiting the binding of GluN1 to B2M in the human brain or repairing synaptic damage caused by increased B2M in the human brain, wherein,

The single dosage of the isolated polypeptide according to any one of the present invention is 0.1-100 mg per kilogram of body weight, preferably 5-50 mg or 5-15 mg per kilogram of body weight;

Preferably, it is administered every 3 days, every 4 days, every 5 days, every 6 days, every 10 days, every 1 week, every 2 weeks or every 3 weeks;

Preferably, the administration method is intravenous drip or intravenous injection.

The present invention discovered for the first time that the expression of B2M in the brain tissue of patients with Down syndrome is significantly increased. Increased B2M will damage synaptic plasticity and cognitive function. Furthermore, the inventors found that there is a direct interaction between B2M and the extracellular segment of GluN1. Using a truncated GluN1 amino acid sequence as a blocking peptide can hinder the binding of B2M to GluN1 and inhibit the function of B2M from damaging NMDA receptors. In vivo experiments show that blocking The peptide can significantly inhibit the binding of B2M and GluN1 in the brain and enhance synaptic plasticity. This discovery provides a potential clinical treatment for Down syndrome Drug targets and new treatments based on these targets.

In some embodiments of the invention, the amino acid sequence of GluN1 is shown in SEQ ID NO: 1.

The amino acid sequence (N-terminus to C-terminus) of rat GluN1 protein is as follows:

In some embodiments of the invention, the amino acid sequence of GluN1 is shown in SEQ ID NO: 2.

The amino acid sequence (N-terminus to C-terminus) of human GluN1 protein is as follows:

After comparison, the homology (similarity) between human GluN1 protein and rat GluN1 protein is 99.25%.

In the present invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used in this article are routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

In the present invention, when referring to the amino acid sequence of GluN1, it includes the full length of GluN1 and also includes its fusion protein. However, those skilled in the art understand that in the amino acid sequence of GluN1, mutations or variations (including but not limited to substitutions, deletions and/or additions) can occur naturally or be introduced artificially without affecting its biological function. In one embodiment of the invention, the amino acid sequence of GluN1 is shown in SEQ ID NO: 1. In one embodiment of the invention, the amino acid sequence of GluN1 is shown in SEQ ID NO: 2.

In the present invention, the terms "isolated" or "isolated" refer to those obtained by artificial means from the natural state. If an "isolated" substance or ingredient occurs in nature, it may be that the natural environment in which it is located has changed, or that the substance has been separated from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the high purity of the same polynucleotide or polypeptide isolated from this natural state is called isolation. of. The term "isolated" or "isolated" does not exclude the admixture of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

In the present invention, the term "host cell" refers to the cell into which the vector is introduced, including many cell types, such as prokaryotic cells such as Escherichia coli or Bacillus subtilis, yeast cells or fungal cells such as Aspergillus, such as S2 Drosophila cells or Insect cells such as Sf9, or fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or animal cells such as human cells.

In the present invention, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide that inhibits a certain protein can be inserted. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); phages such as lambda phage or M13 phage and animal viruses, etc. The types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses. Viruses (such as SV40). A vector may contain multiple elements that control expression.

The term "disease and/or disorder" refers to a physical state of the subject that is associated with the disease and/or disorder of the present invention.

The term "subject" may refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, that receives the pharmaceutical composition of the present invention to treat, prevent, alleviate and/or alleviate the disease or condition described in the present invention. , horses, etc.

As used herein, the term "pharmaceutically acceptable excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, as is well known in the art (See, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers. For example, pH adjusters include but are not limited to phosphate buffer; surfactants include but are not limited to cationic, anionic or nonionic surfactants such as Tween-80; ionic strength enhancers include but are not limited to sodium chloride.

As used herein, the term "effective amount" refers to an amount sufficient to obtain, at least in part, the desired effect. For example, an effective amount to prevent a disease (such as Down syndrome or Alzheimer's disease) is an amount sufficient to prevent, prevent, or delay the occurrence of a disease (such as Down syndrome or Alzheimer's disease); treat a disease An effective amount is an amount sufficient to cure or at least partially prevent disease and its complications in a patient already suffering from the disease. Determining such effective amounts is well within the capabilities of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall status of the patient's own immune system, the patient's general condition such as age, weight and gender, the manner in which the drug is administered, and other treatments administered concurrently etc.

The term "blocking peptide" refers to a protein that competitively binds to B2M with the full-length GluN1 protein, thereby inhibiting B2M binding. The biological effects of GluN1 on synaptic plasticity impairment.

In the present invention, unless otherwise specified, the concentration unit μM represents μmol/L, mM represents mmol/L, and nM represents nmol/L.

In the present invention, when referring to the amount of drug added to cells, unless otherwise specified, it generally refers to the final concentration of the drug after addition.

Beneficial effects of the invention

The present invention provides new drug targets for preventing, treating or improving cognitive function impairment caused by Down syndrome or Alzheimer's disease. Inhibiting or blocking beta2-microglobulin activity can effectively prevent and treat Down syndrome, Alzheimer's disease, or cognitive impairment caused by Down syndrome or Alzheimer's disease.

Description of the drawings

Figure 1A to Figure 1D: B2M expression levels are increased in Down syndrome fetal brain and Down syndrome mouse tissue. in:

Figure 1A: Western blot detection results of B2M expression in fetal brain tissue of Down syndrome patients.

Figure 1B: Image J analysis of B2M expression levels in Figure 1A, n=6 human brain tissues in the control group, n=7 human brain tissues in the Down syndrome group. Data were statistically analyzed using student’s t test. ns, no significant difference, P>0.05; *P<0.05; **P<0.01; ***P<0.001.

Figure 1C: Western blot detection results of B2M expression in brain tissue of Dp16 mice.

Figure 1D: Image J analysis of B2M expression level in Figure 1C, n=6 mouse brain tissues in WT group, n=6 mouse brain tissues in Dp16 group. Data were statistically analyzed using student’s t test. ns, no significant difference, P>0.05; *P<0.05; **P<0.01; ***P<0.001.

Figure 2: NMDAR EPSC amplitude statistical chart. Under in vitro conditions, WT mouse brain slices were incubated with B2M protein (concentration 10 μg/ml) and ACSF for two hours respectively. After incubation, electrophysiological detection was performed to record the NMDAR EPSC amplitude of the Schaeffer collateral loop in the hippocampus. The stimulation electrode was placed close to In the CA3 area, the current of pyramidal cells in the CA1 area was recorded. 50 μM PTX and 20 μM CNQX were added to the perfusate to block GABA _A receptor and AMPA receptor ion channels respectively, and the clamping voltage was +40 mV. The number of recorded cells was n=16 in the ACSF group and n=14 in the B2M group. The data represent the mean ± standard error (SEM). The statistical method was one-way ANOVA, ns. There was no significant difference; P>0.05; *P <0.05;**P<0.01;***P<0.001;****P<0.0001.

Figure 3A to Figure 3D: Co-immunoprecipitation (co-IP) experimental results of B2M and GluN1 extracellular cyclic peptide binding to each other. The B2M plasmid with HA tag was combined with GluN1, N-terminal deleted GluN1, C-terminal deleted GluN1, GluN1 extracellular cyclic peptide plasmid was co-transfected into HEK293T cells. An equal amount of protein lysate was taken and incubated with IgG (control group), HA antibody and Protein G beads overnight for co-immunoprecipitation. Western blot detection and analysis were performed the next day. in:

Figure 3A: Immunoprecipitation of B2M and GluN1 with anti-HA antibodies.

Figure 3B: Immunoprecipitation of B2M and N-terminally deleted GluN1 using anti-HA antibodies.

Figure 3C: Immunoprecipitation of B2M and C-terminally deleted GluN1 using anti-HA antibodies.

Figure 3D: Immunoprecipitation of B2M and GluN1 extracellular cyclic peptides using anti-HA antibodies.

Figure 4A: In order to further confirm the minimum region of the GluN1 extracellular cyclic peptide that binds to B2M, the GluN1 extracellular cyclic peptide sequence was truncated into three short peptides without overlapping sequences.

Figure 4B: The GST-pull down experiment detects that the GST fusion protein of GluN1 extracellular cyclic peptide segment L2 has a stronger binding ability to B2M.

Figure 4C: In order to further narrow the scope of the amino acid sequence that exerts the inhibitory effect, the inventor further divided the GluN1 extracellular cyclic peptide segment L2 into three short peptides without overlapping sequences.

Figure 4D: The above three short peptides without overlapping sequences were pre-incubated with Ni-NTA Agarose in PBS solution for 8 hours at 4°C, and then hB2M protein was added and incubated at 4°C overnight. Western blot detection and analysis were performed the next day.

Figure 4E: GluN1-P2 truncated peptide and GST-B2M fusion protein were pre-incubated in PBS with rotation at 4°C for 8 hours, then 800 μg of 293T cell lysate transfected with GluN1 plasmid was added and incubated at 4°C overnight, and immunized the next day. Blots were detected and analyzed.

Figure 5A: NMDAR EPSC amplitude statistical graph. The hippocampus of 6-month-old Dp16 mice and control WT mice were stereotaxically injected with 1 μl (1 μg/μl) GluN1-P2 truncated peptide or Non-sense peptide (the left and right brains of each mouse were controlled), and electroporation was performed one day after injection. Physiological recording. Record the NMDAR EPSC amplitude of the Schaeffer collateral loop in the hippocampus. The stimulating electrode is placed near the CA3 area to record the pyramidal cell current in the CA1 area. 50 μM PTX and 20 μM CNQX are added to the perfusate to block GABA _A receptor and AMPA receptor ions respectively. channel, the clamping voltage is +40mV. The number of recorded cells is WT Scrambled n=14, WT GluN1-P2n=14, Dp16Scrambled n=15, Dp16GluN1-P2n=16. The data represent the mean ± standard error (SEM). The statistical method is one-way ANOVA, ns , no significant difference, P>0.05;*P<0.05;**P<0.01;***P<0.001;****P<0.0001.

Figure 5B: NMDAR EPSC amplitude statistical graph. The hippocampus of 3-month-old C57BL/6 mice was stereotaxically injected with 1 μl (1 μg/μl) GluN1-P2 truncated peptide or Non-sense peptide (the left and right brains of each mouse were controlled). One day after the injection, the mice were brain-dissected. Under in vitro conditions, the brain slices were incubated with B2M protein (concentration 10 μg/ml) and ACSF for two hours respectively, and electrophysiological recording was performed after incubation. Record the NMDAR EPSC amplitude of the Schaeffer collateral circuit in the hippocampus and place the stimulating electrode In the area close to CA3, the current of pyramidal cells in the CA1 area was recorded. 50 μM PTX and 20 μM CNQX were added to the perfusate to block GABA _A receptor and AMPA receptor ion channels respectively, and the clamping voltage was +40 mV. The number of recorded cells was WT-ACSF n=15, WT-B2M n=15, WT-B2M Scrambled n=16, WT-B2M GluN1-P2n=16. Data represent the mean ± standard error (SEM). Statistical methods It is one-way ANOVA, ns, no significant difference, P>0.05;*P<0.05;**P<0.01;***P<0.001;****P<0.0001.

Figure 5C: Analysis results of LTP recording in CA1 area of brain slices. The hippocampus of 6-month-old Dp16 mice and control WT mice were stereotaxically injected with 1 μl (1 μg/μl) GluN1-P2 truncated peptide or Non-sense peptide (the left and right brains of each mouse were controlled), and electroporation was performed one day after injection. Physiological recording. The number of recorded brain slices are WT Scrambled n=9, WT GluN1-P2n=8, Dp16Scrambled n=8, Dp16GluN1-P2n=8. The data represents the mean ± standard error (SEM). The statistical method is one-way ANOVA. ns, no significant difference, P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Figure 5D: Statistical analysis results of fEPSP slope in the last 10 minutes of the LTP recording results in Figure 5C. The n value of each group is the same as in Figure 5C.

Figure 5E: Analysis results of LTP recording in CA1 area of brain slices. Inject 1 μl (1 μg/μl) GluN1-P2 truncated peptide or Non-sense peptide into the hippocampus of 8-month-old 5×FAD mice through brain stereotaxic injection (the left and right brain controls of each mouse), one day after injection Perform LTP recording. A total of 5 mice were injected, and the number of recorded brain slices were 5×FAD Scrambled n=8 and 5×FAD GluN1-P2n=10. Data represent mean ± standard error (SEM), statistical method is unpaired t test, ns, no significant difference, P>0.05; *P<0.05; **P<0.01; ***P<0.001.

Figure 5F: Statistical analysis results of fEPSP slope in the last 10 minutes of the LTP recording results in Figure 5E. The n value of each group is the same as in Figure 5E.

Detailed ways

The embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If the specific conditions are not specified in the examples, the conditions should be carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.

5×FAD mice are transgenic model mice of Alzheimer’s disease. The brains of these mice will show the pathological characteristics of β-amyloid plaques unique to Alzheimer’s disease; 5×FAD mice were purchased from Jackson Laboratory (Ellsworth, ME, USA), No. 034840-JAX.

Dp16 mice are Down syndrome model mice, purchased from The Jackson Laboratory in the United States, No. 013530-JAX.

WT control mice were C57BL/6 mice, purchased from the Experimental Animal Center of Xiamen University.

Example 1: Increased expression levels of B2M in human Down syndrome fetal brain and Down syndrome mouse tissues

Brain tissue from patients with Down syndrome and non-Down syndrome-like control human brain tissue were collected (obtained from the Women and Children's Hospital of Xiamen University, informed consent was obtained before the brain tissue samples were stored), Dp16 mice and WT controls. Mouse hippocampus tissue was ground and lysed with RIPA protein lysis solution to extract total protein, BCA concentration was measured and samples were prepared, and then immunoblotting was performed (the detection antibody was from Abcam, Cat. No. ab75853).

The results are shown in Figures 1A to 1D.

The results showed that compared with the control group, total B2M protein in the cerebral cortex of patients with Down syndrome was significantly increased. B2M protein was significantly elevated in hippocampal tissue of Dp16 mice compared with controls.

In addition, it was detected that the expression level of amyloid precursor protein (APP) encoded by chromosome 21 in the brain tissue of Down syndrome patients and Dp16 mice was significantly higher than that of the respective control groups.

Example 2: Incubation with B2M protein impairs NMDA receptor function

Under in vitro conditions, WT mouse brain slices were incubated with B2M protein (concentration 10 μg/ml) and artificial cerebrospinal fluid (ACSF) for two hours respectively. After incubation, electrophysiological detection was performed to record the NMDAR of the Schaeffer collateral circuit in the hippocampus. EPSC, the stimulating electrode is placed in the area close to CA3, and the current of pyramidal cells in the CA1 area is recorded. 5mM QX-314 is added to the inner solution of the electrode, and 50μM PTX and 20μM CNQX are added to the perfusate to block GABA _A receptor and AMPA receptor ions respectively. channel, the clamping voltage is +40mV.

The experimental results are shown in Figure 2.

The results showed that compared with the control group, B2M protein treatment significantly reduced the NMDAR EPSC amplitude of pyramidal neurons in the CA1 area of the hippocampus, indicating that B2M protein treatment could damage the NMDA receptor function of hippocampal neurons.

Example 3: Interaction between GluN1 extracellular loop segment and B2M

Under in vitro conditions, HA antibody (Sigma, Cat#H6908) and IgG (control) antibody (Invitrogen, Cat#02-6102) were incubated with protein lysate expressing GluN1 and its truncated protein and B2M-HA, respectively. Co-immunoprecipitation experiments were performed, and then Western blotting was used to analyze whether there was a direct interaction between the two in vitro. The specific operations are as follows:

The B2M plasmid with HA tag was co-transfected into HEK293T cells with GluN1 (SEQ ID NO: 1), GluN1 C-terminal intracellular segment deletion, GluN1 N-terminal extracellular segment deletion or GluN1 extracellular cyclic peptide plasmid for expression. 24 hours. Take an equal amount of protein lysate and incubate it with IgG (control group), HA antibody and Protein G magnetic beads (used to bind to the Fc region of antibody IgG, Thermo Fisher Scientific, Cat#88848) overnight for co-immunoprecipitation. Western blotting was performed and analyzed the next day. Take 3% of the mass of the immunoprecipitated protein lysate as Input (positive control).

Rat GluN1 C-terminal intracellular segment deletion (99.16% homology with human GluN1 C-terminal intracellular segment deletion)

Rat GluN1 N-terminal extracellular segment deletion (100% homology to human GluN1 N-terminal extracellular segment deletion)

Rat GluN1 extracellular cyclic peptide (100% homology with human GluN1 extracellular cyclic peptide)

HA-tagged B2M proteins

For plasmid construction, cell transfection and protein sample preparation, please refer to the following methods.

Plasmid construction:

(1) PCR target gene fragment (such as GluN1 N-terminal extracellular segment deletion, GluN1 extracellular cyclic peptide segment, GluN1 extracellular cyclic peptide segment). (2) Double-enzyme digestion of the PCR product and vector (pcDNA3.1(+)/myc-His A) (endonucleases are EcoR1 and Xba1 respectively) at 37°C for 1 hour. (3) Separate the digested vector by agarose gel electrophoresis. Then stain with Gel Green, perform gel cutting and recovery under UV light, and use the SanPrep column DNA gel recovery kit to recover and purify the digested plasmid DNA fragments. (4) Connect the annealed oligonucleotide double strands to the pcDNA3.1(+)/myc-His A vector after digestion and recovery, use T4DNA Ligase for ligation, and ligate at 22°C for 1 hour. (5) Transform the ligation product into E.coli DH5α competent cells, place on ice for 30 minutes, heat shock at 42°C for 90 seconds, incubate on ice for 3 minutes, add antibiotic-free LB medium, and incubate for 45 minutes at 37°C with shaking (250 rpm). Then spread the recovered E.coli DH5α bacteria on a plate containing 50 μg/ml Amp/LB, incubate it upright in a 37°C incubator for 30 minutes, and then invert it for 12-16 hours. (6) Pick a single clone colony and culture it in 4-6ml medium containing 50μg/ml Amp/LB medium on a shaking table at 37°C (250rpm) for 12-16 hours, and then use the SanPrep column plasmid DNA mini-extraction kit ( Shanghai Sangon Biotechnology Co., Ltd. (Cat#HC17KA2946) performed plasmid DNA extraction. (7) The plasmid was sent to Xiamen Primus Biotechnology Co., Ltd. for sequencing and identification.

Cell transfection:

Use PEI for cell transfection. (1) One day before transfection, seed the cells into a 60mm culture dish. The cell density should be 70-90% during transfection; (2) Add the plasmid to be transfected: Opti-MEM:PEI (1:50: 5.5, w/v/v), transfect 4 μg of plasmid into a 60 mm petri dish), mix well, and let stand at room temperature for 10 minutes; (3) Add the mixture drop by drop to the culture medium evenly, shake gently, and incubate at 37°C , continue culturing in a 5% CO ₂ incubator; (4) Change to fresh medium after 8 hours of transfection, and continue culturing for 24 hours before collecting the cells.

Protein sample preparation:

(1) Aspirate the culture medium in the culture dish, add pre-cooled PBS, shake well, aspirate the PBS, repeat three times; (2) Add 500 μl RIPA cell lysis buffer, scrape the cells with a cell scraper, and transfer to the pre-cooled In a centrifuge tube, centrifuge at 1000g for 5 minutes at 4°C to collect the cells; vortex for lysis at 4°C for 30 minutes; (3) After lysis, centrifuge at 12000g for 10 minutes at 4°C and the supernatant obtained is the required protein lysis solution. Transfer the supernatant to a new 1.5ml centrifuge tube; (4) Determine the protein concentration using the BCA method, and prepare the sample based on the obtained protein concentration. For protein concentration determination, use BCA Protein Assay Kit for protein concentration determination.

The experimental results are shown in Figure 3A to Figure 3D.

The results showed that GluN1 interacted with B2M (Fig. 3A).

GluN1 is a three-transmembrane protein, including an N-terminal extracellular segment, a C-terminal intracellular segment, a transmembrane structure and an extracellular loop. In order to detect the binding region between B2M and GluN1, the inventors deleted the C-terminal intracellular segment and the N-terminal extracellular segment of GluN1 respectively, and conducted co-immunoprecipitation experiments with B2M respectively. The results showed that: compared with the control group, B2M Interacts with both N-terminal deletion (GluN1-N terminal deletion-myc) (Figure 3B) and C-terminal deletion GluN1 (GluN1-C terminal deletion-myc) (Figure 3C).

GluN1 with N-terminal deletion and C-terminus deletion contain a common extracellular cyclic peptide region. In order to detect whether the GluN1 extracellular cyclic peptide fragment binds to B2M, a co-immunoprecipitation experiment was performed on the GluN1 extracellular cyclic peptide fragment and B2M. The results showed that the GluN1 extracellular cyclic peptide fragment can interact with B2M (Figure 3D).

In summary, there is an interaction between the extracellular loop segment of GluN1 and B2M.

Example 4: GluN1 truncated peptide can be used as a blocking peptide to prevent GluN1 from binding to B2M and prevent B2M. GluN1 acts to produce inhibition

In order to clarify the amino acid sequence where GluN1 interacts with B2M, as shown in Figure 4A, the inventors truncated the GluN1 extracellular loop sequence into three non-overlapping amino acid sequences, and then synthesized a GST fusion expression protein of these amino acid sequences. .

Rat GluN1-S2loop-L1 (100% homology to human GluN1-S2loop-L1)

Rat GluN1-S2loop-L2 (100% homology to human GluN1-S2loop-L2)

Rat GluN1-S2loop-L3 (100% homology to human GluN1-S2loop-L3)

The above three GluN1 extracellular loop sequence GST fusion proteins were incubated with 0.5 μg hB2M protein at approximately 1:1 (molar ratio) overnight at 4°C, and Western blotting was performed the next day. As shown in Figure 4B, GluN1-S2loop-L2 interacts more strongly with B2M.

As shown in Figure 4C, on the basis of GluN1-S2loop-L2, the inventors further shortened the amino acid sequence to narrow the effective binding range. The inventors truncated the GluN1-S2loop-L2 sequence into three non-overlapping amino acid sequences (see Table 1 below for specific sequences), and then synthesized these small peptides. It is stated that, considering that rat GluN1-S2loop-L2 is 100% homologous to human GluN1-S2loop-L2, the truncated fragments GluN1-P1, GluN1-P2, and GluN1-P3 in Table 1 are identical to human GluN1-S2loop. The homology of the corresponding truncated fragments in -L2 is also 100% respectively.

Table 1

Pre-incubate 2.5 μg of the above three short peptides without overlapping sequences with Ni beads in PBS solution at 4°C for 8 hours, and then add 1 μg of B2M protein and incubate overnight at 4°C. Immunoblot analysis was performed the next day. As shown in the picture As shown in 4D, B2M specifically binds to the GluN1-P2 short peptide.

In order to further verify whether the GluN1-P2 short peptide can competitively bind GluN1 in vivo. First, incubate 0.055 μg, 0.55 μg, 1.1 μg, and 2.2 μg mass gradient GluN1-P2 short peptide and GST-B2M fusion protein (approximately 5 μg) in PBS solution at 4°C for 8 hours. GluN1 plasmid (recombinant plasmid of rat GluN1 and pcDNA3.1) was transferred into 293T cells. An equal amount of protein lysate was added to the above system and continued to incubate at 4°C overnight. Western blot detection was performed the next day. analyze. Take 0.3% of the mass of the IP protein lysate as the input control. As shown in Figure 4E, as the mass of the GluN1-P2 short peptide increases, its ability to bind B2M becomes stronger.

In summary, the GluN1-P2 short peptide can be used as a blocking peptide to prevent GluN1 from binding to B2M, making B2M unable to inhibit NMDA receptor function.

Example 5: GluN1-P2 blocking peptide blocks the binding of B2M to GluN1, thereby reducing synaptic damage

GluN1 is an essential subunit of NMDA receptors. Impairment of its function will severely damage synaptic plasticity. GluN1-P2 blocking peptide can prevent B2M from binding to GluN1. Therefore, it is necessary to study whether GluN1-P2 blocking peptide can prevent B2M from damaging NMDA. Receptor function in turn impairs excitatory synaptic function.

The hippocampus of 6-month-old Dp16 mice and control WT mice were stereotaxically injected with 1 μl (1 μg/μl) GluN1-P2 short peptide or Non-sense peptide (the left CA1 area of each mouse was injected with the GluN1-P2 short peptide, and the right Non-sense peptide) was injected into the lateral CA1 area, and electrophysiological recording was performed one day after injection. Record the NMDAR EPSCs of the Schaeffer collateral circuit in the hippocampus. The stimulating electrode is placed near the CA3 area to record the pyramidal cell current in the CA1 area. 5mM QX-314 is added to the electrode internal solution, and 50μM PTX and 20μM CNQX are added to the perfusion solution to block respectively. GABA _A receptor and AMPA receptor ion channels, clamping voltage is +40mV.

The experimental results are shown in Figure 5A. The results showed that in Dp16 mice, compared with the group injected with the nonsense peptide, the NMDA EPSC amplitude of the GluN1-P2 truncated peptide injection group was significantly increased, while the injection of GluN1-P2 truncated peptide had a significant increase in the NMDA EPSC amplitude of WT mice. There was no obvious effect, suggesting that GluN1-P2 truncated peptide significantly improved NMDA receptor function in Dp16 mice.

The hippocampus of 3-month-old C57BL/6 mice was stereotaxically injected with 1 μl (1 μg/μl) GluN1-P2 truncated peptide or Non-sense peptide (the left and right brains of each mouse were controlled). One day after the injection, the mice were brain-dissected. Under in vitro conditions, the brain slices were incubated with B2M protein (concentration 10 μg/ml) and ACSF for two hours respectively, and electrophysiological recording was performed after incubation. Record the NMDAR EPSC amplitude of the Schaeffer collateral loop in the hippocampus. The stimulating electrode is placed near the CA3 area to record the pyramidal cell current in the CA1 area. 5mM QX-314 is added to the electrode internal solution, and 50μM PTX and 20μM CNQX are added to the perfusate to block the current. Block GABA _A receptor and AMPA receptor ion channels with a clamping voltage of +40mV.

The experimental results are shown in Figure 5B. The results showed that compared with brain slices incubated with ACSF, brain slices incubated with B2M significantly reduced the NMDA EPSC amplitude of WT mice, and injection of GluN1-P2 truncated peptide significantly improved the NMDA EPSC amplitude after B2M incubation.

Six-month-old Dp16 mice and control WT mice were stereotaxically injected with 1 μl (1 μg/μl) GluN1-P2 truncated peptide or Non-sense peptide into the hippocampus (each mouse was injected with GluN1-P2 short peptide in the left CA1 area, Non-sense peptide) was injected into the right CA1 area, and 8-month-old 5×FAD mice were subjected to the same operation as above. Electrophysiological recording was performed one day after injection. After the mice were anesthetized, the brain tissue was quickly removed and placed in ice-cold and oxygen-saturated artificial cerebrospinal fluid (ACSF), and then transferred to a vibrating microtome for coronal sectioning. The thickness of the brain slices was 400 μm. The brain slices were incubated in oxygen-saturated ACSF at 32°C for 1 hour, and then transferred to room temperature for 1 hour. The recording electrode was placed in the stratum radiatum of the CA1 area of the Schaffer collateral pathway, and the stimulating electrode was placed in the CA3 area. The stimulation intensity was 30% of the maximum amplitude of the excitatory postsynaptic potential (fEPSP). After the fEPSP baseline was stably recorded for 20 minutes, high-frequency stimulation (HFS) induced LTP (2 series of stimulations, each containing 100 stimulation pulses, with an interval of 30 seconds between each series of stimulations), and continuous recording for 60 minutes.

The experimental results are shown in Figure 5C to Figure 5F. The results showed that in Dp16 mice, compared with the injection of nonsense peptide group, the injection of GluN1-P2 blocking peptide group significantly improved LTP in the CA1 area of the hippocampus of Dp16 mice, while the injection of GluN1-P2 blocking peptide had an effect on WT mice. LTP had no obvious effect, suggesting that GluN1-P2 blocking peptide reversed synaptic damage in Dp16 mice. Likewise, similar results would be seen in 5xFAD mice.

In summary, GluN1-P2 blocking peptide blocks the binding of B2M to GluN1, thereby reducing synaptic damage.

Although specific embodiments of the invention have been described in detail, they will be understood by those skilled in the art. According to all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are within the protection scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

An isolated polypeptide, which is the polypeptide shown in SEQ ID NO:8 or a truncated fragment of the polypeptide shown in SEQ ID NO:8.
The isolated polypeptide of claim 1, wherein the truncated fragment comprises the polypeptide shown in SEQ ID NO: 11.
The isolated polypeptide according to any one of claims 1 to 2, which is a polypeptide represented by any sequence in SEQ ID NO: 11 or SEQ ID NOs: 14-32.
An isolated polynucleotide encoding an isolated polypeptide according to any one of claims 1 to 3.
A recombinant expression vector comprising the isolated polynucleotide of claim 4.
A transformed cell comprising the recombinant expression vector of claim 5.
A pharmaceutical composition comprising one or more isolated polypeptides according to any one of claims 1 to 3.
The pharmaceutical composition according to claim 7, further comprising one or more pharmaceutically acceptable excipients.
The isolated polypeptide according to any one of claims 1 to 3 is used in the preparation of a method for treating or preventing Down syndrome, Alzheimer's disease, or cognitive impairment caused by Down syndrome or Alzheimer's disease. Uses in medicines.
The use of the isolated polypeptide according to any one of claims 1 to 3 in the preparation of drugs that inhibit the binding of GluN1 to B2M in the human brain or repair the synaptic damage caused by the increase in B2M in the human brain.
The isolated polypeptide according to any one of claims 1 to 3, which is used to treat or prevent Down syndrome, Alzheimer's disease, or symptoms caused by Down syndrome or Alzheimer's disease. Know the damage.
The isolated polypeptide according to any one of claims 1 to 3, which is used to reduce the level of B2M in the human brain, reduce the level of amyloid precursor protein in the human brain, inhibit the binding of GluN1 to B2M in the human brain, or repair the human brain. Synaptic damage in the brain due to increased B2M.
A method of treating or preventing Down syndrome, Alzheimer's disease, or cognitive impairment caused by Down syndrome or Alzheimer's disease, comprising administering an effective amount to a subject in need thereof. The step of isolating the polypeptide of any one of claims 1 to 3.
A method for reducing the level of B2M in the human brain, reducing the level of amyloid precursor protein in the human brain, inhibiting the binding of GluN1 to B2M in the human brain, or repairing synaptic damage caused by increased B2M in the human brain, including giving it to a subject in need The step of administering to a subject an effective amount of the isolated polypeptide of any one of claims 1 to 3.
The method according to claim 13 or 14, wherein,

The single dosage of the isolated polypeptide is 0.1-100 mg per kilogram of body weight, preferably 5-50 mg or 5-15 mg per kilogram of body weight;

Preferably, it is administered every 3 days, every 4 days, every 5 days, every 6 days, every 10 days, every 1 week, every 2 weeks or every 3 weeks;

Preferably, the administration method is intravenous drip or intravenous injection.