WO2011072484A1

WO2011072484A1 - Target and medicaments for treatment of brain injuries

Info

Publication number: WO2011072484A1
Application number: PCT/CN2010/002044
Authority: WO
Inventors: 王以政; 杜婉璐; 黄隽波
Original assignee: 中国科学院上海生命科学研究院
Priority date: 2009-12-16
Filing date: 2010-12-15
Publication date: 2011-06-23
Also published as: CN102100913A; CN102100913B

Abstract

The invention discloses the substance used for treating or preventing the ischemic brain injuries or neurodegenerative diseases by sustaining the TRPC6 level of nerve cells. The substance may be calpain inhibitors, reinforcing agents of TRPC6 expression, or NMDA receptor antagonists. The invention also discloses the uses of said substance for manufacturing medicaments for preventing or treating ischemic brain injuries or neurodegenerative diseases.

Description

Description: Targets and drugs for brain injury treatment

The present application relates to targets and drugs for the treatment of brain injury. In particular, the present application relates to a TRPC6 target for treating brain damage, and methods and medicaments for treating brain damage by the target. Background technique

Neurodegenerative diseases are disease states in which cellular neurons of the brain and spinal cord are lost. The brain and spinal cord are made up of neurons, which have different functions, such as controlling movement, processing sensory information, and making decisions. Cells in the brain and spinal cord are generally not regenerated, so excessive damage can be devastating and irreversible. Neurodegenerative diseases are caused by the loss of neurons or their myelin, which worsens over time, leading to dysfunction. Neurodegenerative diseases are divided into two groups according to their phenotype: one class affects exercise, such as cerebellar ataxia; one class affects memory and related dementia. Neurodegenerative diseases usually include: Alzheimer's disease, amyotrophic lateral sclerosis, ataxia telangiectasia, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, Huntington's disease, cerebellar atrophy, multiple Sclerosis, Parkinson's disease, primary lateral sclerosis, and spinal muscular atrophy. Therefore, how to protect neurons from various injuries is very important for the prevention and treatment of neurodegenerative diseases.

Cerebral ischemia, commonly known as stroke, is a pathological process that causes brain cell death or injury due to an insufficient blood supply to the brain. Cerebral ischemia can be divided into focal cerebral ischemia and global cerebral ischemia. The former is due to partial blood vessel embolism or rupture in the brain. The blood supply area of this part of the blood vessels is ischemic, more common in cerebral infarction or cerebral hemorrhage. The latter is more common in the blood supply of the entire brain in the case of drowning or myocardial infarction.

In focal cerebral ischemia, the blood flow can be reduced to less than 15% or even lower in areas where blood is directly supplied by embolized blood vessels. This part of the tissue will undergo irreversible lesions and cell death, called the central infarction area; In the area of indirect blood supply, because there are other collateral circulation blood supply, the blood flow decline is less than the central area, generally lower than 60-80%. This part of the tissue often has non-functional damage or delayed cell death, if timely Restoring blood supply or timely treatment can save cell death and tissue damage in the area; such areas are commonly referred to as penumbras. Due to the delay and reversibility of cell death in the penumbra, scientific research is concentrated in this field, with the aim of reducing cell death in the penumbra and ultimately cerebral infarction. In the late 1980s, the establishment of an animal model of focal cerebral ischemia provided a good platform for the study of ischemic neuronal death mechanisms.

During cerebral ischemia, neurons are the most sensitive and least tolerant type of cells. How to protect neurons from

1

Confirmation Damage and death are an important issue in the field of cerebral ischemia research. In recent decades, there have been numerous studies on the mechanism of ischemic neuronal death, mainly summarized as follows: glutamate excitotoxicity, apoptosis, inflammatory response, and cell damage caused by imbalance of ion concentration. In response to these neuronal death mechanisms, researchers have developed a number of drugs to interfere with the initiation or progression of these disruptive pathways. However, in the clinical treatment and application of cerebral ischemia, the effects of these strategies are not satisfactory.

The classic Transient Receptor Potential Channel (TRPC channel) is a family of channel proteins discovered in recent years. Since its first discovery in 1995, TRPC channels have been found in the nervous system, immune system, and blood circulation system. It is expressed in many systems and tissues such as kidney, lung, spleen, ovary and smooth muscle ("TRPC6 and kidney disease", International Journal of Pathology and Clinical Medicine, October 2008, Vol. 28, No. 5). TRPC family members (subunits) include: TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6, TRPC7. According to their sequence similarity, TRPCl, TRPC2, TRPC4, TRPC5 and TRPC3, TRPC6, TRPC7 can be divided into another. As a group of functional TRPC channels, these subunits are composed of homotetramers or heterotetramers, which are non-selective cation channels that can permeate calcium ions. Many cells have TRPC channels on the cell membrane. Distribution, including on neurons. Recent studies have shown that TRPC channels are involved in many important physiology or Process, such as: axon growth orientation, development of neuronal synapses, muscle cell proliferation, kidney disease, and survival of cerebellar granule cells. There are two pathways for activation of TRPC channels, one of which is G-protein coupling on the membrane. The activation of the downstream receptor is mediated by the activation of the downstream PLC (phospholididase C); the second is the activation of the PLC caused by the tyrosine kinase receptor on the membrane. It can be seen that the activation of the PLC is the TRPC channel. Required for opening. PIP2 (phosphatidylinositol 4,5-diphosphate) on the hydrolyzed membrane after PLC activation produces IP3 (inositol triphosphate) and DAG (diacylglycerol), the former causes internal calcium release and activates the membrane Some of the TRPC channels; the latter can act directly on certain TRPC channels on the membrane to open the channel.

Only one drug currently available for clinical treatment of ischemic stroke (focal cerebral ischemia): tPA (tissue plasminogen activator), which can only be used in a small number of cases, and With the risk of cerebral hemorrhage. Therefore, new drugs are urgently needed and new pathogenesis is understood, new targets are discovered and new drugs are being developed. Summary of the invention

The Applicant has found that the protein level of TRPC6 is specifically down-regulated in the cortical penumbra neurons after ischemia, a process mediated by the proteolysis of calpain initiated by NMDA receptor activation. Blocking the downregulation of TRPC6, or upregulating the protein level of TRPC6, has protective neuronal effects on ischemic injury. These results suggest that TRPC6 plays a key role in neuronal survival during focal cerebral ischemia; calpain-mediated down-regulation of TRPC6 promotes neuronal death and aggravates brain damage caused by ischemia. Accordingly, the present application relates to an isolated polypeptide selected from the group consisting of:

(i) SEQ ID NO: 1 or a fragment thereof comprising SLKAAP; or

(Π) A polypeptide derived from (i) by substituting, deleting or adding one or several amino acids in (i) and having the activity of inhibiting calpain degradation of TRPC6.

In one embodiment, the polypeptide is selected from the group consisting of:

(a) GGSLKAAPGA; or

(b) A polypeptide derived from (a) by substituting, deleting or adding one or more amino acids in (a) and having the activity of inhibiting calpain degradation of TRPC6.

In one embodiment, the polypeptide of (b) is selected from the group consisting of: amino acid residues at corresponding positions of 0, 1, or 2 (a) independently extending to the left and right sides of SLKAAP of the polypeptide of (a) a polypeptide derived from the base; and a polypeptide obtained by independently extending 0, 1, 2, 3, 4, 5 or 6 amino acid residues on the left and right sides of GGSLKAAPGA based on SEQ ID NO: 1.

In one embodiment, the polypeptide is selected from the group consisting of:

(1) RRGGSLKAAPGAGTRR; or

(2) The polypeptide derived from (1) in which the amino acid sequence in (1) is substituted, deleted or added with one or several amino acids and has an activity of inhibiting calpain degradation of TRPC6. .

The polypeptide of the present application includes RRGGSLKAAPGAGTRR or a fragment thereof comprising SLKAAP.

In one embodiment, the fragment comprising SLKAAP is GGSLKAAPGA.

The application provides an isolated polypeptide comprising a transmembrane factor and a polypeptide described herein. In this application, the membrane factor can be selected from RKKRRQRRR,

GWTLNS AGYLLGKINLKALAALAKKIL > RQIKIWFQNRRMKWKK, RR RR, RRRRRRRRR R RRRRR RRR, GRKKRRQRRRC. In one embodiment, the transmembrane factor is GRKKRRQR RC.

In one embodiment, the polypeptide of the present application is as set forth in SEQ ID NO: 4, SEQ ID NO: 9, or SEQ ID NO: 10.

The application provides a pharmaceutical composition comprising a polypeptide of the present application and a pharmaceutically acceptable carrier or excipient.

In the pharmaceutical composition of the present application, the polypeptide of the present application may be RRGGSLKAAPGAGTRR or a fragment thereof comprising SLKAAP, and the polypeptide may be linked to a transmembrane factor.

In one embodiment, the pharmaceutical composition of the present application contains SEQ ID NO: 4, SEQ ID NO: 9 and/or SEQ ID NO: 10.

The present application also provides enhanced expression of an NMDA receptor antagonist, a TRPC6 expression vector, and/or TRPC6. Pharmaceutical composition of the agent.

The application provides the use of a polypeptide of the present application in the manufacture of a medicament for increasing the expression level of a subject TRPC6. The polypeptide includes a polypeptide that is or is not linked to a transmembrane factor. Transmembrane factor (penetrating peptide) is a short peptide that can carry macromolecular substances into cells.

The application includes the use of a TRPC6 expression vector, an inhibitor of calpain, a TRPC6 enhancer or an NMDA receptor antagonist for the preparation of a medicament for the protection of neurons.

In one embodiment, the enhancer is selected from the group consisting of OAG, or an analog thereof.

The application includes the use of a TRPC6 expression vector, an inhibitor of calpain, a TRPC6 enhancer or an NMDA receptor antagonist for the preparation of a medicament for the treatment or prevention of a neurodegenerative disease.

In the present application, neurodegenerative diseases usually include: Alzheimer's disease, amyotrophic lateral sclerosis, ataxia telangiectasia, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, Huntington's disease, Cerebellar atrophy, multiple sclerosis, Parkinson's disease, primary lateral sclerosis, and spinal muscular atrophy.

The present application includes the use of a TRPC6 expression vector, an inhibitor of calpain, a TRPC6 enhancer or an NMDA receptor antagonist for the preparation of a medicament for the treatment or prevention of ischemia-induced damage.

In the present application, the injury includes brain damage caused by ischemia.

In the present application, inhibitors of calpain include a polypeptide of the present invention linked to or not to a transmembrane factor. In a specific embodiment, the polypeptide is RRGGSLKAAPGAGTRR or a fragment thereof comprising SLKAAP, or as set forth in SEQ ID NOs: 4 and 9.

The present application provides a method of screening for a medicament for treating or preventing damage caused by ischemia, the method comprising:

(1) adding the substance to be tested to a system containing TRPC6;

(2) adding calpain to the system described in step (1), and

(3) determining whether the substance to be tested inhibits calpain degradation TRPC6,

Among them, a substance capable of inhibiting calpain degradation of TRPC6 is used as a drug candidate for treating or preventing damage caused by ischemia.

The application also provides a method of screening for a TRPC6 expression enhancer, the method comprising:

(1) adding the substance to be tested to a system expressing TRPC6;

(2) The expression level of TRPC6 in the system was determined, and the substance to be tested which increased the expression level of TRPC6 was determined to be a TRPC6 expression enhancer as compared with the experiment in which no substance to be tested was added.

In another aspect, the application includes a substance for maintaining a level of TRPC6 in a neuron to prevent or treat ischemic brain damage or a neurodegenerative disorder.

The substance may be selected from a calpain inhibitor, or a TRPC6 expression enhancer, or an NMDA receptor antagonist Agent.

The inhibitor of calpain may be selected from calpain-specific small interfering RNA, or calpeptin, or a polypeptide comprising a SLKAAP sequence.

The sequence of the polypeptide may be selected from SEQ ID NO: 1 or a fragment thereof.

In the present application, the polypeptide can be linked to a transmembrane factor.

In some embodiments, the sequence of the polypeptide is set forth in SEQ ID NO: 4, SEQ ID NO: 9, or SEQ ID NO: 10.

In the present application, the calpain-specific small interfering RNA may be selected from SEQ ID NO: 5 or SEQ ID NO: 6.

In the present application, the TRPC6 expression enhancer may be selected from the group consisting of TRPC6 expression vectors, OAG or OAG analogs. .

In the present application, the NMDA receptor antagonist may be selected from the group consisting of amantadine, dextrocycline, SEQ ID NO: 7 or SEQ ID NO: 8.

The application also includes the use of a substance as described above, which comprises preventing or treating ischemic brain damage or a neurodegenerative disorder.

The present application also encompasses the use of the aforementioned substances, including the preparation of a medicament or combination of drugs for preventing or treating ischemic brain damage or neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the specificity of the TRPC6 antibody. (a) shows transfection with antibodies against TRPC6

Western blotting of TRPC6 in HEK293 cells of GFP or WT-TRPC6 construct. (b) Western blotting of TRPC6 in HEK293 cells transfected with GFP or TRPC6- _yc constructs using antibodies against the /w e epitope. (c) Western blotting of TRPC6 in rat brain lysates using antibodies against TRPC6, with or without co-cultivation with antigenic peptides. (d) Representative images showing rat brain sections stained with anti-TRPC6 antibodies and anti-NeN antibodies in the presence or absence of antigenic peptides. The scale is 50 μm. (e) Immunoblot analysis of extracts of HEK293 cells transfected with TRPC1-HA, TRPC3-M c, TRPC4, TRPC5-Flag or TRPC6 constructs. The antibody of TRPC6 specifically recognizes the overexpressed TRPC6 protein but does not recognize the overexpressed TRPC1, 3, 4 and 5 proteins.

Figure 2 shows TRPC6 specific down-regulation in neurons after cerebral ischemia. (a) Immunoblots showing extracts using the indicated antibodies, control side (C) or ischemic side (I) cortex (Sham sham operation group: left cortex (L) or right cortex (R)). Tubulin was used as a control for protein loading. The half panel of (a) shows the quantitative fractionation of normalized TRPC6 protein levels (at each time point, n = 5-8 rats, compared to Sham, *p<0.05, **p<0.01. (b) Quantitative analysis of TRPC6, 3 and 4 protein levels at time points indicated after cerebral ischemia. At each time point, n = 3-5 rats, *p<0.05, **p<0.01 compared to Sham. (c) Immunoblotting of TRPC and GluRl after 24 hours of ischemia reperfusion (R24). The right panel shows quantitative analysis of protein levels, n = '5 rats, **p < 0.01 compared to the control side. (d) shows TRPC6 mRNA levels measured by qRT-PCR, n = 3-5 rats. (e). Representative representation of the indicated cortex obtained by double staining with the indicated antibodies after 24 hours of ischemia (R24). The scale is 50 μm. The right panel shows quantitative analysis (η = 5 rats); ** ρ < 0.01 compared to the fluorescence intensity in the control side cortex.

Figure 3 shows that TRPC6 is not downregulated in glial cells after cerebral ischemia. The figure shows a representative map of the control side and the ischemic side cortex obtained by double staining with antibodies against TRPC6 and GFAP after 24 hours of ischemia and reperfusion (R24). The image to the right is an enlarged view taken from the area shown. Two arrows pointing obliquely show GFAP-positive glial cells; two horizontal arrows show adjacent neurons. The figure shows that TRPC6 protein is specifically down-regulated in neurons after ischemia, whereas there is no change in TRPC6 protein levels in glial cells.

Figure 4 shows that the down-regulation of TRPC6 precedes ischemic neuronal death. (a) shows a representation of the cortex shown by double staining with TRPC6 antibody and TU EL, and 24 hours (R24) or 48 hours (R48) after ischemia reperfusion. Hoechst marks the nucleus. The scale is 50μηι. (b) shows the quantitative level of TRPC6. protein and the number of TUNEL-labeled positive cells in the ischemic cortex or sham (Sham) at the indicated time; in each case, n = 3 rats. *p<0.05, **p<0.01 compared to Sham.

Figure 5 shows the down-regulation of TRPC6 in neurons in a simulated ischemic experiment. (a) Immunoblotting of cultured cortical neuron extracts with the indicated antibodies after OGD. The values under the bands are normalized protein levels (n=3). (b) Real-time RT-PCR quantitative mRNA levels of TRPC1, 3 and 6 in neurons at the indicated time points after OGD.

Figure 6 shows the current (I _0AG ) produced by cultured cortical neurons stimulated by OAG. (a) Whole-cell recording of cultured cortical neurons (first trajectory) and recording of cortical neurons transfected with TRPC6 RNAi (second trajectory). The graph to the right shows the quantitative analysis of current density (10 cells per group). The right panel shows the protein levels of TRPC6 in cortical neurons transfected with the control plasmid, random RNAi sequence TRPC6 RNAi, respectively. OAG (1-oleoyl-2-oleoyl-sn-glycerol) ΙΟΟμΜ; SKF96365, 1 (^M _; NMDG: N-methyl-D-glucosamine-free Ca ²⁺ solution. Single asterisk indicates relative control For the group, p < 0.05 o (b) The representative current trajectory recorded by the ramp-up voltage clamp. The diagram below the current trajectory shows the way of recording the voltage. As shown in the figure, the slope from -100V to +80V is applied four times. The voltage of ΙΟΟμΜ was administered after the first ramp-up voltage. (c) Untransfected cortical neurons (control group) and neurons transfected with TRPC6 RNAi, IV curve of OAG-stimulated current.

Figure 7 shows the effect of OAG stimulation on intracellular calcium signaling in cultured cortical neurons. (a) Intracellular calcium elevation as defined by AR/R as defined by F340/F380 and its normalized baseline. The straight line in the figure represents the administration time. SS HPSS buffer was used as a control, and the composition of the zero calcium extracellular fluid was: SS + 2 mM EGTA. OAG ΙΟΟμΜ, SKF1 (^M. Right panel is the quantitative analysis of the area under the curve, data of 15-25 cells per group. Double asterisk indicates p < 0.01 relative to SS group or OAG group. (b) Dominant suppressor TRPC6 pair Effect of intracellular calcium elevation induced by OAG. Cortical neurons transfected with control plasmid or dominant suppressor TRPC6 plasmid were transfected with ΙμΜ OAG ,, and the increase in calcium was detected. The straight line in the figure represents the time of OAG administration. The figure shows the area of each group of curves under the fixed-halo analysis, data of 20 cells per group. Double asterisks indicate relative control < 0.01.

Figure 8 shows that oxygen sugar deprivation specifically inhibits OAG-stimulated currents in neurons. The left panel shows the results of quantitative analysis of the current density of the corresponding stimulus, and the right panel shows the statistical results of the inversion potential of OAG stimulation under control or oxygen deprivation. A single asterisk indicates p < 0.05 relative to the control group.

Figure 9 shows that down-regulation of TRPC6 aggravates neuronal death due to oxygen deprivation. The cultured cortical neurons transfected with the corresponding plasmids were reperfused for 24 hours after oxygen deprivation treatment, and the cell death was quantitatively counted (3 trials). A single asterisk indicates p < 0.05 for the GFP group or the control RNAi group, and a double asterisk indicates p < 0.01.

Figure 10 shows the effect of pharmacological methods on the effects of TRPC6 on ischemic injury. (a) The control group shown by PI staining, the OAG (100 M) treatment group and the SKF 96365 (10 M) treatment group, had an effect on cell death caused by oxygen deprivation treatment. The statistical data represents three independent experiments, with a single asterisk indicating p < 0.05 versus the control group and a double asterisk indicating p < 0.01. (b, c) TTC (2,3,5-triphenyltetrazolium) staining area (photograph) of brain slices after 24 hours of cerebral ischemia reperfusion, and quantified cerebral infarction volume (columnar) Figure): (b) R2, 4 or sham surgery: control (veh, DMSO 5μΜ/5μ1) or OAG 100ηιΜ/5μ1, data for 8-1 1 rat per group. Double asterisks indicate p < 0.01. (c) R24 or sham surgery: Control (veh, DMSO 5μΜ/5μ1) or SKF96365 20πιΜ/5μ1, data for 8-1 1 rat per group. Double asterisks indicate ρ < 0.01.

Figure 11 shows neurons overexpressing TRPC6 specifically protecting oxygen glucose deprivation. (a) Representative maps of cultured cortical neurons transfected with wild-type TRPC6 and quantitative analysis of transfection efficiency. The results of immunoblot imprinting of neuronal lysates transfected with GFP or wild type T PC6 were performed using antibodies to TRPC6. (b) TRPC6, but not TRPC3, protects neurons from oxygen deprivation. Neuronal death was observed by PI (propidium iodide) staining after transfection of the corresponding plasmid with an oxygen glucose deprivation treatment. The illustration is an immunohybrid imprinting map that overexpresses TRPC3. The statistical analysis data was obtained from three different experiments. Double asterisks indicate p < 0.01 relative to GFP.

Figure 12 shows that the cyclic adenosine monophosphate response element binding protein (CREB) is a downstream signaling molecule of TRPC6. (a) Immunoblot imprinting analysis of neurons transfected with GFP or wild-type TRPC6 using the corresponding antibodies. (b) Results of cell death by PI staining after oxygen-glucose deprivation treatment of neurons transfected with the corresponding plasmid. KCREB is a dominant inhibitory CREB, and each group is statistically independent of three experimental data. The double asterisk indicates a relative GFP p < 0.01.

Figure 13 shows that TRPC6 is degraded by calcium ion-dependent proteases. (a) Rat brain lysate and calcium ions and After incubation with the corresponding inhibitors, the corresponding antibodies were used for immunoblotting. Calpeptin: 20 μ M; leupeptin: 100 μM; PMSF: 100 l M; EGTA: 5 mM. The right panel shows the statistical results of TRPC6 protein levels in three independent experiments. Double asterisks indicate p < 0.01 relative to control. (b) The left panel shows the time course of TRPC6 degradation of rat brain lysate co-incubated with I mM calcium at 37 ° C. The right panel shows that rat brain lysate was co-incubated with the corresponding concentration of calcium at 37 ° C for 30 minutes. Dose curve for degradation of TRPC6 protein. (c) The rat brain lysate was incubated with calcium ions and the corresponding inhibitor or control group, and the results of immunoblot imprinting were performed using TRPC6 or α-spectrin antibody. Calpeptin: 20 μ M; leupeptin: ΙΟΟ μ Μ; MDL28170 (calpain inhibitor 3): 60 μ M; cpm-VAD-CHO: 20 μM; Lactacystin (proteasome inhibitor): ΙΟ μ Μ; EGTA: 5 mM.

Figure 14 shows that the K16 and A17 amino acids of the TRPC6 protein are cleaved by calpain. (a) Overexpression of the N-flag-second loop-HA-C-myc protein (as shown in the schematic) in HEK293 cells, co-incubating the cell lysate with the corresponding concentration of μ-calpain, using the corresponding tag antibody Immunoblot blot analysis. (b) Purified NUS-labeled product of 203 amino acids before TRPC6 was digested with calpain and subjected to Coomassie blue staining after SDS-PAGE gel separation. The arrow shows a small fragment of 22 kD cleaved by calpain, and the right panel shows the cleavage site of calpain on TRPC6 by sequencing with Edman N-end sequencing. The TAT-C6 sequence with the TAT transmembrane sequence synthesized according to this site and containing this site is followed by the rat brain lysate with ImM calcium ion and the corresponding concentration of TAT within 30 minutes. After incubation with -C6, the corresponding antibodies were used for immunoblot blotting analysis.

Figure 15 shows the degradation of TRPC6 by calpain in a NMDA receptor-mediated cell ischemia model. (a) Left panel: Cortical neuron lysates treated with oxygen glucose deprivation treated with control (Veh.), calpeptin (20 μM) or MDL28170 (MDL, 60 μM), and treated with TRPC6 antibody Immunoblot blot analysis. Middle panel: TRPC6 protein level statistics from three independent experiments. A single asterisk indicates p < 0.05 for the control group and a double asterisk indicates p < 0.01. Right panel: The three treatments of oxygen glucose deprivation lead to cell death statistics, three independent experiments, double asterisks indicate p < 0.01. (b) Immunoblot analysis of calpain proteins by RNAi (CAPN i-1, CAPN i-2) transfected with control nonsense siRNA and transfected with two calpains. The graph below shows the calpain protein level statistics for three experiments. Double asterisks indicate p < 0.01. Right: Both RNAs of calpain can block the degradation of TRPC6 protein caused by oxygen deprivation. The figure below shows the TRPC6 protein level statistics, three independent experiments, and the double asterisk indicates p < 0.01. (c) Oxygen glucose deprivation treatment of cultured cortical neurons after co-incubation of control or lOuM dextrozine. Cell lysates were subjected to immunoblot blot analysis using TRPC6 antibody. (d) Immunoblot analysis of NTU protein was performed on RNAi (NRl i_l, NRl i_2) transfected with control nonsense siRNA and transfected with two NMDA receptor NR1 subunits. The graph below shows the NR1 protein level statistics for three experiments. Double asterisks indicate p < 0.01. Right: Both RNAs of NR1 block the degradation of TRPC6 protein caused by oxygen deprivation. The figure below shows TRPC6. Protein level statistics, three independent experiments, double asterisks indicate p < 0.01. (e) Immunoblot imprinting experiments showed that the calpaeptin inhibitor calpeptin and the NMDA receptor inhibitor amantadine can block the ischemia-induced degradation of TRPC6 protein. The following figure shows the TRPC6 protein level statistics, three independent experiments, two stars. The number indicates p < 0.01.

Figure 16 shows that TRPC6 protein levels are specifically up-regulated in the forebrain in TRPC6 transgenic mice. (a) Immunoblot imprinting of cortical extracts of transgenic mice (tg) or wild type mice (wt) was performed using the corresponding antibodies. The figure below shows the genotype identification of the corresponding mice, and the right panel shows the quantitative analysis of TRPC6 protein levels. For each of the three genotypes, the double asterisk indicates p < 0.01 relative to the wild type. (b) Transgenic mice (tg) from three independent transgenic lines or wild-type mice (wt) from littermates using antibodies against TRPC6, 3, 4, 5, NR2A, GluR2/3 and PSD95, respectively Cortical extracts were subjected to immunoblot blotting analysis. The graph on the right shows the corresponding protein level quantitative analysis. Three mice per genotype, single asterisk indicates p < 0.05 relative to wild type. (c) Immunohistochemical analysis of brain frozen sections of transgenic mice (tg) or wild type mice (wt) with TRPC6 antibody. The picture below shows different brain regions. CTX cortex, HIP hippocampus, CBM cerebellum. The scale bar is 200 microns.

Figure 17 shows that increasing TRPC6 protein levels reduces cerebral ischemic injury in mice. Left: TTC-stained brain slices and cerebral infarction volume statistics (bar graph) of the corresponding genotype mice after focal cerebral ischemia, 14 mice per genotype, double asterisk relative to wild type p < 0.01. Right panel: Survival rates of transgenic and wild-type mice after cerebral ischemia. 14 mice per genotype.

Figure 18 shows a histogram of cerebrovascular conditions in transgenic and wild-type mice. Coronal sections of transgenic and wild-type mice were cultured with antibodies against vascular endothelial cell-specific protein CD31, and Hoechst stained the nucleus. The scale is 50 microns. The right panel shows cerebral blood flow analysis of transgenic and wild-type mice after ischemia. Double asterisks indicate p < 0.01 relative to ischemia at 0 minutes.

Figure 19 shows that the amount of TRPC6 protein is inversely related to cell death caused by ischemia. Left panel: Immunohistochemical double-labeled representation of the ischemic lateral cortex penumbra using TRPC6 antibody and TUNEL staining. Hoechst stains the nucleus. Scale bar 50 microns. Right panel: Statistical analysis of the immunofluorescence intensity of TRPC6 and the number of TUNEL-labeled positive cells. Data for three mice per group, double asterisk indicates p < 0.01 relative to wild type.

Figure 20 shows that (a) immunological hybridization imprinting of ischemic or sham-operated mouse cortex extracts with antibodies to TRPC6, the right graph is statistical results, with 6 mice per group, double asterisks indicating relative to sham surgery ( Transgenic) p < 0.01. (b, c) Cortical extracts of wild-type or transgenic mice after ischemia or sham surgery were immunoblotted with p-CREB, CREB, p-CaMKII α, CaMKII α and NOS1 antibodies, respectively. The right panel shows the protein level ratio of p-CREB/CREB, p-CaMKII a /CaMKII α, and the relative protein level of NOS l. A single asterisk indicates p < 0.05, and a double asterisk indicates p < 0.01 relative to the corresponding group.

Figure 21 shows that inhibition of calpain degradation of TRPC6 can reduce cerebral ischemic injury in rats. Focal cerebral ischemia or sham surgery was performed in rats injected with control peptide or TAT-C6 in the lateral ventricle, left: TRPC6 and spectrin The antibody was subjected to immunoblotting imprinting experiments on brain tissue extracts. The lower panel shows the statistical analysis of T PC6 protein levels. For each group of three rats, the single asterisk indicates < 0.05 relative to the control. Right: TTC stained brain slices and cerebral infarction volume (histogram). A control (Veh) control TAT-peptide (TAT-ctd) or TAT-C6 peptide (TAT-C6) was injected into the lateral ventricle of rats before ischemia. A single asterisk indicates p < 0.05 relative to the control.

: Figure 22 shows TTC stained brain slices and lesion volume (bar graph). The ventricle was injected with a control TAT-peptide (TAT-empty) or TAT-C6-2 peptide (TAT-C6-2). A single asterisk indicates p < 0.05 relative to the control.

Figure 23 shows a photograph of TTC stained brain slices and lesion volume (bar graph). The lateral ventricle was injected with a control TAT-peptide (TAT-empty) or TAT-C6-3 peptide (TAT-C6-3). A single asterisk indicates p < 0.05 relative to the control.

Figure 24 shows that intraperitoneal injection of TAT-C6 improves learning and memory in a model of Alzheimer's disease.

In the drawings, "control", and "ctd" refer to the control, "Tubulin" refers to tubulin, "No inh. (No Ca ²⁺ )" refers to "no inhibitor (no Ca ²⁺ ) " , "No inh ""Noinhibitor"," Veh. " means "carrier" (eg distilled water). "TAT-empty" means TAT, "naive" means "untested", "scramble RNAi" is "control" Nonsense RNAi". Detailed Description

The singular forms "a", "the", and "the" Thus, the use of "a polypeptide" includes mixtures of two or more polypeptides and the like.

The following amino acid abbreviations are used herein:

Alanine: Ala(A) Arginine: Arg(R)

Asparagine: Asn(N) Aspartic acid: Asp(D) Cysteine: Cys(C) Glutamine: Gln(Q) Glutamate: Glu(E) Glycine: Gln(Q)

Histidine: His(H) Isoleucine: Ue(I) Leucine: Leu(L) Lysine: Lys(K)

Methionine: Met(M) Phenylalanine: Phe(F) Proline: Pro(P) Serine: Ser(S)

Threonine: Thr(T) Tryptophan: Trp(W) Tyrosine: Tyr(Y) Proline: Val(V)

The terms "polypeptide" and "protein" refer to a polymer of amino acid residues and are not limited to the minimum length of the product. Thus, peptides, oligopeptides, dimers, polymers, and the like are included in this definition. Full length proteins and fragments thereof are included in this definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like. Another For the purposes of this application, "polypeptide" refers to modifications including natural sequences, such as deletions, additions and substitutions (usually nature-conservative), so long as the protein maintains the desired activity. These modifications can be designed by site-directed mutagenesis, or can be accidental, such as by host mutations that produce proteins, or errors due to PCR amplification.

The term "analog" refers to a compound having a native polypeptide sequence and structure, and one or more amino acid additions, substitutions (usually nature conserved) and/or deletions relative to the natural molecule, as long as the modification does not destroy the original polypeptide from which the analog was derived. Activity. Methods of preparing polypeptide analogs and muteins are known in the art and are further described below.

Particularly preferred analogs include those which are conservative in nature, i.e., these substitutions occur in a class of amino acids associated with their side chains. Specifically, amino acids are generally classified into four categories: (1) acid-aspartic acid and glutamic acid; (2) basic one-lysine, arginine, histidine; (3) non-polar Alanine, valine, leucine, isoleucine, valine, phenylalanine, methionine, tryptophan; (4) uncharged polar mono-glycine, Asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonable to predict: leucine alone with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similarly conserved with structurally related amino acids Amino acids, such substitutions will not have a significant impact on biological activity. For example, a polypeptide of interest may comprise up to about 2-6 conservative or non-conservative amino acid substitutions, even up to about 5-10 conservative or non-conservative amino acid substitutions, or any integer between 2-10, As long as the desired function of the molecule remains intact. One skilled in the art can readily determine regions of the molecule of interest that are tolerant to alteration, in conjunction with Hopp/Woods and Kyte-Doolittle plots well known in the art.

The polypeptide or nucleotide sequence of the present application can be defined by "identity" or "homology." "Identity" or "homology" refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid sequence of two polynucleotide or polypeptide sequences. By arranging the sequences of two molecules to directly compare their sequence information, the exact number of matches between the two aligned sequences is calculated, divided by the length of the shortest sequence, and then multiplied by 100 to obtain the percent identity.

In the analysis of homology and identity, it is possible to assist in the use of readily available computer programs such as ALIGH,

Dayhoff, MO (Atlas of Protein Sequence and Structure, MODayhoff, ed., 5 Suppl., 3: 353-358, National Biomedical Research Foundation, Washington, DC), which applies to the local homology algorithm for peptide analysis by Smith and Waterman ( Advances in Appl. Math., 2: 482-489, 1981). Procedures for determining nucleotide sequence homology, such as BESTFIT, FASTA, and GAP programs, are also available from the Wisconsin Sequence Analysis Package (version 8, available from Genetics Computer Group, Madison, WI), which also rely on Smith and Waterman. algorithm. These programs can be easily used using the default parameters suggested by the manufacturer and described in the Wisconsin Sequence Analysis Package above. E.g, N2010/002044 The percent homology of the nucleotide sequence to the reference sequence as determined by the default score table of the homology algorithm of Smith and Warerman and the gap penalty of 6 nucleotide positions.

Another method for establishing percent homology in the present application is to use the MPSRCH package, which is copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). The Smith-Waterman algorithm can be used in this package, where default parameters are used in the score table (for example, interval penalty = 12, interval extension penalty = 1, interval = 6). The "match" value generated from this batch of data reflects "sequence homology". Other suitable procedures for calculating percent identity or percent similarity between sequences are generally known in the art. For example, another alignment procedure is BLAST, using default parameters. For example, BLASTN and BLASTP can be used with the following default parameters: Gene coding = standard; Filter = None; Chain = two; Interception = 60; Expected value = 10; Matrix = BLOSUM62 _; Description = 50 sequences; Sort = HIGH SCORE; = No redundancy, GenBank+ EMBL+ DDBJ+ PDB+ GenBank CDS Translation + Swiss Protein + Spupdate+ PIR. A detailed description of these procedures can be found at http:AVww.ncbi.nim.gov/cgi-bin/BLAST.

Alternatively, polynucleotide hybridization is carried out under conditions in which a stable double strand is formed between homologous regions, followed by digestion with a single-strand specific nuclease, and then the size of the digested fragment is measured to thereby measure homology. In a Southern hybridization assay performed under stringent conditions (as defined for a particular system), a substantially homologous DNA sequence can be identified. Determining appropriate hybridization conditions is within the knowledge of those skilled in the art. See, for example, Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.

The present application preferably has 90% or more, 95% or more, 96% or more, 98% or more, or 99% or more sequence identity with the polypeptide of the present application, and retains the therapeutic or prophylactic activity described herein or retains the activity of inhibiting calpain degradation of TRPC6. Peptide.

The polypeptides of the present application can be prepared in a variety of ways. For example, it can be prepared by conventional chemical synthesis or recombinant expression.

The polypeptide sequence isolated herein is based on MSQSPRFVTRRGGSLKAAPGAGTRRNESQD (SEQ ID NO. 1, see Figure 14b) and includes this sequence and its fragment containing SLKAAP. The term "fragment containing SLKAAP" refers to a sequence obtained by independently truncating any number of amino acids on the left and right sides of the amino acid sequence of SEQ ID NO: 1, but still retaining the SLKAAP sequence. The fragment still retains the activity of inhibiting calpain degradation of TRPC6. The arbitrary number refers to an integer between 1 and 13 for the left side and an integer between 1 and 1 1 for the right side, wherein the shortest segment may be SLKAAP. The present application also encompasses conservative substitution products of the above polypeptides and fragments thereof, in particular, one or more amino acid residues in the above polypeptides or fragments are substituted by amino acid residues in the nature of the phase (see above), while being conservative Substitution of the resulting amino acid sequence retains the activity of inhibiting calpain degradation of TRPC6. The polypeptide isolated in the present application may be selected from: (a) GGSLKAAPGA; or (b) the amino acid sequence in (a) is substituted, deleted or added with one or several amino acids and has an activity of inhibiting calpain degradation of TRPC6 (a) ) a derived polypeptide.

The polypeptide of the above item (b) includes, but is not limited to, a polypeptide which independently extends 0, 1 or 2 amino acid residues at the corresponding positions shown in (a) on the left and right sides of SLKAAP of the above (a), and Based on ID NO: 1, a polypeptide obtained by independently extending 0, 1, 2, 3, 4, 5 or 6 amino acid residues on the left and right sides of GGSLKAAPGA. For example, when one amino acid residue is extended on each of the left and right sides of SLKAAP, the sequence is GSLKAAPG; when two amino acid residues are extended on the left and right sides of SLKAAPGAGTR, the sequence is GGSLKAAPGAGTRRN; when the left side of SLKAAPGAGTR When 6 amino acid residues are extended and 4 amino acids are extended to the right, the sequence is VTRRGGSLKAAPGAGTRR ES; and so on. The present application also includes conservative substitution products of these amino acid sequences, in particular, one or several amino acid residues in the above polypeptides are substituted by amino acid residues of the same nature (see above), while the amino acid sequence obtained by conservative substitution is still The activity of inhibiting calpain degradation of TRPC6 is retained.

The application also provides an isolated polypeptide selected from the group consisting of: (c) RRGGSLK AAPG AGTRR; or (d) the amino acid sequence in (C) is substituted, deleted or added with one or several amino acids and has inhibition of calpain degradation TRPC6 The active polypeptide derived from (c).

The polypeptide of the above item (d) includes, but is not limited to, a polypeptide obtained by independently reducing 0, 1, 2, 3 or 4 amino acid residues on both sides of the above (c) sequence; and based on SEQ ID NO: 1, A polypeptide obtained by independently extending 0, 1, 2, 3, 4, 5 or 6 amino acid residues on the left and right sides of RRGGSLKAAPGAGTRR. For example, when only one amino acid residue extends to the left of the sequence (c), the sequence is TRRGGSLKAAPGAGTRR; when two amino acid residues are extended on both sides of the sequence of (c), the sequence is VTRRGGSLKAAPGAGTRRNE; analogy. The present application also includes conservative substitution products of these amino acid sequences, in particular, one or several amino acid residues in the above polypeptides are substituted by amino acid residues of the same nature (see above), and the amino acid sequence obtained by conservative substitution is still The activity of inhibiting calpain degradation of TRPC6 is retained. In one embodiment, the application includes a separate CRRGGSLKAAPGAGTRR. As mentioned above, amino acids with similar properties of C and T are qualitatively similar, and their substitution does not affect the activity of the resulting polypeptide to inhibit calpain degradation of TRPC6.

In a specific embodiment, the application provides an isolated polypeptide selected from the group consisting of RRGGSLKAAPGAGTRR and fragments thereof comprising SLKAAP. In a specific embodiment, the SLKAAP-containing fragment is GGSLKAAPGA.

The application also provides a peptide sequence comprising SLKAAP and having an activity of inhibiting calpain degradation of TRPC6. In one embodiment, the peptide sequence has 6 to 30 amino acid residues, for example, It can be 6-25, 6-20, 6-16, 6-10, etc. In one embodiment, the sequence comprising SLKAAP is SEQ ID NO: 1 or a fragment thereof. In another embodiment, the sequence comprising SLKAAP is R GGSLKAAPGAGTRR or a fragment thereof. In another embodiment, the sequence comprising SLKAAP is GGSLKAAPGA or a fragment thereof.

The aforementioned TRPC6 shown in Fig. 14b is MSOSPRFVTRRGGSJ ^ ^G ^l ^G7 ^ ^RNE SOD, and the aforementioned amino acid residues which can be independently extended or reduced on both sides of the SLKAAP, (a), (c) and (e) sequences. And its location corresponds to this TRPC6 sequence one by one. The one-to-one correspondence means, for example, that when an amino acid residue is extended, the extended amino acid residue is from the base sequence (ie, SLKAAP, (a) term sequence, (c) term sequence, and (e) term sequence) The last amino acid residue on the side is extrapolated to the specified number of amino acid residues, respectively. For example, when two amino acid residues are extended, two amino acid residues are extrapolated in order from the last amino acid residue on both sides of the base sequence. The extended amino acid residue is identical to the amino acid residue at the corresponding amino acid position in TRPC6 of Figure 14b. When amino acid residues are reduced, it is meant that a specified number of amino acid residues are reduced starting from the first amino acid on either side of the base sequence.

In a specific embodiment, the present application is preferably the following amino acid sequence:. SLKAAP, SLKAAPGAGTR, GSLKAAPGAGTR, GGSLKAAPGAGTR, RGGSLK A APGAGTR R GGSLKAAPGAGTR, RRGGSLKAAPGAGTRR, TRRGGSLK A APGAGTR, VTR GGSLKAAPGAGTR, SLKAAPGAGTRR, SLKAAPGAGTRRN, SLKAAPGAGTRRNE, GSLKAAPGAGTRR, GGSLKAAPGAGTRR, RGGSLKAAPGAGTRR TRRGGSLK A APGAGTRR, CRRGGSLKAAPGAGTR, VCRRGGSLKAAPGAGTRR F VCRRGGSLK A APG AGTRRN, GGS AAPGA, etc.

The isolated polypeptide of the present application can be linked to a transmembrane factor to facilitate passage through the cell membrane. A transmembrane factor refers to a polypeptide that is less than about 30 amino acids in length and that is capable of passing through the cell membrane and capable of bringing the various substances it carries into the cell. Various transmembrane factors known in the art can be used in this application, including but not limited to RKKRRQRRR,

GWTLNSAGYLLGKINLKALAALAKKIL^ RQIKI WFQNRRMKWKK:, RRRRRRR, RRRRRR RR, RRRR RRR RR, GRKKRRQRRRC, etc. The polypeptide isolated in the present application can be directly linked to a transmembrane factor. The polypeptide of the present application linked to a transmembrane factor can be prepared by various methods known in the art, for example, by conventional chemical synthesis.

The application provides a pharmaceutical composition comprising an isolated polypeptide of the present application and a pharmaceutically acceptable carrier or excipient. In one embodiment, the polypeptide is linked to a transmembrane factor.

The application also provides a pharmaceutical composition comprising a TRPC6 expression enhancer and a pharmaceutically acceptable carrier or excipient. The TRPC6 expression enhancer refers to a substance capable of increasing the expression level of TRPC6. Expression enhancers include the TRPC6 expression vector, OAG, or an analog thereof. The application also provides a pharmaceutical composition comprising an inhibitor of an NMDA receptor and a pharmaceutically acceptable carrier or excipient. In the present application, the MDA receptor antagonist may be selected from the group consisting of dexamethapine (MK801, Sigma), amantadine, and the like.

The application also provides a pharmaceutical composition comprising a TRPC6 expression vector. In one embodiment, the expression vector is an expression vector driven by a CaMKIIa promoter. The expression vector can be administered to a subject using various conventional techniques, for example, transfection techniques and the like.

The application also provides a pharmaceutical composition comprising an inhibitor of calpain and a pharmaceutically acceptable carrier or excipient. The inhibitor is a polypeptide of the present application, calpepthu leupeptin, or MDL28170, or any combination thereof. The polypeptide can be linked to a transmembrane factor.

The pharmaceutical composition of the present application also contains one or more other preparations dissolved or dispersed in a pharmaceutically acceptable carrier or excipient. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that, when used in animals, such as humans, do not produce side effects, allergies or other adverse effects. Those skilled in the art will be aware of the preparation of pharmaceutical compositions containing at least one polypeptide, and in some embodiments one or more other active ingredients, as disclosed herein, for example, see Remington Drugs. Science, 18th ed., Mack Printing Company, 1990 (incorporated in this article). In addition, administration to animals such as humans is understood to be consistent with sterile, pyrogen-free, overall safety and purity standards.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (eg, antibacterial, antifungal), isotonic agents, absorption. Delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegrating agents, lubricants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof, which are common in the art The skilled artisan is known (see, for example, Remington's Pharmaceutical Sciences, 18th ed., Mack Printing Company, 1990, pages 1289-1329, incorporated herein by reference). It is considered to be useful in therapeutic or pharmaceutical compositions, except in conventional carriers which are incompatible with the active ingredient.

Administration of a diseased animal The actual dosage of the composition of the present application is determined by physical and physiological factors such as body weight, severity of the disease, type of disease to be treated, original and common therapeutic measures, subject's specific disease and route of administration. . The physician responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dosage of the individual subject.

In certain embodiments, the pharmaceutical compositions may contain, for example, at least about 0.001% by weight of active ingredient. In other embodiments, the pharmaceutical compositions may contain, for example, 0.01 to 99.9% by weight, 0.01 to 50% by weight, 0.01 to 10% by weight, and the like, of the polypeptide of the present application. In a specific embodiment, the concentration of the polypeptide in the administered pharmaceutical composition may range from 0.01 to 5 mM, such as from 0.01 to 3 mM, from 0.05 to 1 mM. The mode of administration is conventional and can be determined by the clinician based on the specific circumstances of the patient. For example, it can be injected directly into the lateral ventricle or subarachnoid space. Alternatively, it can also be administered by intraperitoneal injection.

The pharmaceutical compositions of the present application may contain various antioxidants to prevent oxidation of one or more components. In addition Preservatives are used to prevent the action of microorganisms, such as various antibacterial and antifungal agents, including but not limited to p-hydroxyphenylpropionates (eg, methyl p-hydroxyphenylpropionate, propyl p-hydroxyphenylpropionate), chlorine Butanol, phenol, sorbic acid, thimerosal or a combination thereof.

The therapeutic polypeptide can be formulated as a free base, neutral or salt form. The pharmaceutically acceptable salts include acid addition salts such as those formed with the free amino group of the protein component, or salts with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium, ammonium, calcium or iron; or organic bases such as isopropylamine, trimethylamine, histamine or procaine.

In embodiments where the composition is in liquid form, the carrier can be a solvent or dispersion medium including, but not limited to, water, polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (eg, Triglycerides, vegetable oils, liposomes) and combinations thereof. For example, by using a coating such as lecithin; by maintaining a desired particle size with a carrier such as a liquid polyol or a lipid dispersion; using a surfactant such as hydroxypropylcellulose; or a combination of these methods to maintain proper flow Sex. In many cases, it will be preferable to include isotonic agents such as sugars, sodium chloride or combinations thereof.

The pharmaceutical compositions of the present application can be formulated using methods routine in the art.

The composition must be stable under the conditions of manufacture and storage to prevent contamination by microorganisms such as bacteria and fungi. It is important to keep endotoxin contamination to a minimum and at a safe level, for example less than 0.5 ng/mg protein.

The present application includes the use of the pharmaceutical composition of the present application for the preparation of a medicament for increasing the expression level of TRPC6 in a subject. .

The application also includes the use of an inhibitor of calpain in the manufacture of a medicament for increasing the expression level of TRPC6 in a subject.

In the present application, inhibitors of calpain include the polypeptide of the present application, calpain-specific small interference

RNA, calpeptin, leupeptin, or MDL28170, or any combination thereof. The polypeptide can be linked to a transmembrane factor. The calpain-specific small interfering RNA may be selected from SEQ ID NO: 5 or SEQ ID NO: 6, or a combination thereof.

The present application also encompasses the use of a TRPC6 enhancer in the manufacture of a medicament for increasing the expression level of TRPC6 in a subject. Enhancers include OAG, analogs thereof, or any combination thereof. The present application includes the use of an NMDA receptor antagonist for the preparation of a medicament for increasing the amount of TRPC6 expression in a subject. The NMDA receptor antagonist includes memantine, dextrozine, SEQ ID NO: 7, or SEQ ID NO: 8. The application includes the use of a TRPC6 expression vector for the preparation of a medicament for increasing the expression level of TRPC6 in a subject.

This application of course also includes the TRPC6 expression vector itself. In a specific embodiment, the expression vector is an expression vector driven by a CaMKIIa promoter.

The present application relates to the use of a polypeptide of the present invention for the manufacture of a medicament for the treatment or prevention of damage caused by ischemia. The polypeptide can be linked to a transmembrane factor. The damage includes brain damage caused by cerebral ischemia. The polypeptide of the present application can also be used for the preparation of a medicament for treating or preventing calpain and TRPC6-mediated various diseases, and the therapeutic or prophylactic purpose is achieved by inhibiting calpain degradation of TRPC6. The diseases include damage caused by ischemia and various neurodegenerative diseases.

The polypeptides of the present application are useful in the preparation of a medicament for protecting neurons from injury. The injury can be caused by a variety of causes, including injuries caused by ischemia.

The polypeptide of the present application can be used for the preparation of a medicament for treating or preventing various neurodegenerative diseases.

Here, neurodegenerative diseases include Alzheimer's disease, amyotrophic lateral sclerosis, ataxia telangiectasia, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, Huntington's disease, cerebellar atrophy, Multiple sclerosis, Parkinson's disease, primary lateral sclerosis, and spinal muscular atrophy.

Accordingly, the present application also encompasses a method of treating or preventing ischemia-induced damage, the method comprising increasing the expression of TRPC6 in a subject in need thereof.

The present application also encompasses a method of treating or preventing a neurodegenerative disease, the method comprising increasing the expression of TRPC6 in a subject in need thereof.

The present application also encompasses a method of protecting a neuron from injury, the method comprising increasing the expression of TRPC6 in a subject in need thereof.

Methods for increasing the expression of TRPC6 in a subject include: (1) administering an inhibitor of calpain to a subject to inhibit degradation of TRPC6 expression by the enzyme; (2) providing an NMDA receptor antagonist; (3) providing a TRPC6 expression vector; and / Or (4) providing a TRPC6 expression enhancer.

Inhibition of administration of a subject calpain comprises administering to the subject a polypeptide of the present application, calpain-specific small interfering RNA, calpeptin, leupeptin, or MDL28170, or any combination thereof. The polypeptide can be linked to a transmembrane factor. Enhancers include OAG, analogs thereof, or any combination thereof. NMDA receptor antagonists include acesulfamide and dextrocycline.

The application also includes a method of increasing the expression of TRPC6 in a subject.

The present application also encompasses a method of treating or preventing calpain and TRPC6 mediated various diseases, the method comprising administering a polypeptide of the present application to a subject in need thereof, and achieving the therapeutic or prophylactic purpose by inhibiting calpain degradation of TRPC6. .

In this context, subjects include a variety of mammals, especially humans.

(1) adding the substance to be tested to a system containing TRPC6 or expressing TRPC6;

(2) adding calpain to the system described in step (1);

(3) determining whether the substance to be tested inhibits calpain degradation of TRPC6, Among them, a substance capable of inhibiting calpain degradation of T PC6 is used as a drug candidate for treating or preventing damage caused by ischemia.

The method further comprises testing whether the candidate drug affects other activities of calpain other than degradation of TRPC6, wherein a drug candidate that does not affect other activities of calpain is a preferred drug.

The method further comprises subjecting the measured preferred drug to an in vivo experiment.

The application also relates to a method of screening for a TRPC6 expression enhancer, the method comprising:

(1) adding the substance to be tested to a system expressing TRPC6;

The system for expressing TRPC6 may be, for example, a cell (or cell culture) system, and the cell may be a cell that endogenously expresses TRPC6; or may be a cell that recombinantly expresses TRPC6. The system for expressing TRPC6 may also be, but is not limited to, a subcellular system, a solution system, a tissue system, an organ system or an animal system (such as an animal model). The TRPC6-containing system may be, for example, a solution system containing TRPC6. The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent that treats, alleviates or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect. One skilled in the art will be able to assess whether a treatment achieves the desired therapeutic purpose using conventional methods. The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. Implementation of the Invention Unless otherwise stated, conventional methods of chemistry, biochemistry, recombinant DNA techniques and immunology known to those skilled in the art will be used. These techniques are fully explained in the literature. See, for example, Fundamental Virology, Second Edition, Volumes I and II (edited by BNFields and DMKnipe); Handbook of Experimental Immunology, Volumes I-IV (DMWeir Wo) Π CC Blackwell, Blackwell Scientific Publications); TE Creighton, Proteins: Structures and Molecular properties (WH Freeman and Company, 1993); AL Lehninger, Biochemistry (Worth Publishers) , Inc., latest edition); Sambrook et al., Molecular Cloning: a Laboratory Manual, Second Edition, 1989; Methods in Engymology (S.Colowick) Edited by Kaplan, Academic Press, Inc.). Percentages and parts are by weight unless otherwise stated. For reagents that do not indicate a source, conventional reagents that are commonly commercially available are used. N2010/002044

Specific embodiment

1. Experimental materials and methods

1.1 Animal model of cerebral ischemia

Rats (male SD rats, body weight 250-280 g) or mice (male C57BL6 strain mice, 25-30 g) were anesthetized with 10% chloral hydrate, then the neck skin was cut and the neck was separated. Arterial (CCA) and external carotid artery (ECA), ligature at both ends of the ECA and then cut the small opening, insert the tying line, tie the tying line and blood vessel, and then release the ligature line near the CCA end, push the tying line into CCA And push the ICA up until there is a slight resistance. In the experiment, the rats were ischemia for 2 hours, and after 3 hours of ischemia in the mice, the suture was withdrawn and the perfusion (re-irrigation) was resumed. After refilling for different times, the rat brain was removed, sectioned at intervals of 2 mm (rat) or 1 mm (mouse), and then the brain was sectioned in 2% 2,3,5-triphenyltetrazolium chloride (TTC, Stain in 0.9% saline to determine the size of the infarct area.

When the drug was injected into the lateral ventricle, the rats were fixed with a stereotactic locator from Stoelting. According to the brain mapping, the drug (5 μΐ) was injected into the side with a microsyringe pump (Stoelting Co.) at a rate of 0.5 μΐ/min. In the ventricle, leave the needle for lOmin after injection: then suture.

1.2 Western blot and immunohistochemistry

Cells or tissues in lysate (in mM, 10 Tris-Cl, pH 7.4, 150 NaCl, 5 EDTA, 1% Triton-X100, 1 sodium orthovanadate, 50 NaF, 1 PMSF, 1 aprotinin, 1 bright The homogenate was lysed in aprotinin and 5 DTT), and the homogenate was centrifuged at 13,000 rpm, 4 degrees for 15 minutes, and the supernatant was separated after separation. The sample protein concentration was measured with a spectrophotometer and adjusted to the same concentration. Add the loading buffer, mix and heat at 95 °C for 5~8 minutes, denature the protein, pass the protein SDS polyacrylamide gel electrophoresis, transfer to the nitrocellulose membrane, and seal with 5% skim milk at room temperature. Hours, dilute the primary antibody to the appropriate concentration (1:200~1:2000) with antibody dilution (5% BSA + 0.05% NaN ₃ + PBS), hybridize overnight with diluted primary antibody, and wash 3 times with PBS ( 3*10 min), dilute the HRP-conjugated goat anti-rabbit (mouse) secondary antibody with antibody dilution and incubate for 2 hours at room temperature. After washing 3 times with PBS (3*10 minutes), ECL was exposed to development. Western-blot strip grayscale was scanned by film and analyzed by software ImageQuant (Amersham).

In the immunohistochemistry experiment, after anesthetizing the animals, the rats were perfused with PBS at 37 degrees Celsius, then fixed with 4% PFA (paraformaldehyde) at 4 degrees Celsius, and finally the brain tissue was removed; the tissue blocks were embedded with OCT (ice frozen slices) The agent, which is a water-soluble mixture of polyethylene glycol and polyvinyl alcohol, is embedded and then frozen and sliced. The thickness of the slice is generally 12-16 μηΐ; the frozen section is blocked with 5% normal sheep serum for 1 hour at room temperature, and then added. 5% normal sheep serum diluted primary antibody, 4 degrees Celsius overnight, the next day PBS washed 3 times, add the corresponding fluorescing The photosecondary antibody was incubated in the dark for 1 hour, washed 3 times with PBS, fixed with a sealing tablet, and observed under a fluorescence microscope. 1.3 Neuron culture, plasmid transfection and oxygen glucose deprivation (OGD) experiments

Plasmid pregnant SD rats (E17), taking embryonic mouse cortical, trypsinized, 4χ 10 ⁶ cells by electroporation Plasmid 4 g, electricity gyroscope the rat neuron Nucleofector Kit (Amaxa, Koeln, Germany) the DNA Or an RNA plasmid is introduced into the cell. In the oxygen glucose deprivation experiment, the extracellular fluid was first replaced with a glucose-free Earle's balanced salt solution (see 1.7 Antibodies and Drugs for the experiment) and placed in a closed OGD chamber (Forma Scientific, Marietta, OH, US A). Medium, flush 10 molecules of nitrogen (5% C0 ₂ and 95% N ₂ ), then incubate the chamber in a 37-inch incubator for 2 h; end the OGD treatment, switch back to the original extracellular fluid and place it in a normal incubator. Can be incubated in the middle. Cell death was calculated by propidium iodide (PI) staining.

1.4 Electrophysiology and calcium imaging experiments

Electrical signals were recorded on cultured cortical neurons using whole cell patch clamp using a computer controlled 700A instrument (Axopatch 700A, Molecular Devices, Foster City, CA). The liquid content in the electrode was as follows (in mM): CsCl 140, CaCl ₂ 0.3, EGTA 10, MgCl ₂ 1 , HEPES 10, pH 7.2. The normal extracellular fluid contained (in mM): NaCl 140, KCl 5, MgCl ₂ 1, CaCl ₂ 1 , D-glucose 10, and HEPES 10, pH 7.4o During the recording, the clamping voltage was -70 mV.

The method of calcium imaging experiments is mainly based on the article by Zhu et al. 1996 [Zhu, X. et al., trp, a novel mammalian gene family essential for agonist-activated capacitative Ca2+ entry. Cell, 1996. 85(5): p. 661-71 .]. Briefly, cortical neurons and calcium ion dye Fura-2 AM (dissolved in 0.0125% polyether acid and diluted in DMSO) were incubated for 30 min, and the entire reaction was in HEPES buffered saline (HPSS: (in mM) 120 NaCl) , 5.3 KCl, 0.8 MgS0 ₄ , 1.8 CaCl ₂ , 1 1. Glucose and 20 HEPES, pH 7.4), after incubation, wash with HPSS 2-3 times, then incubate with HPSS for half an hour. The Nikon eclipse Te2000-e microscope was then used to detect changes in intracellular calcium concentration [Ca ²⁺ ]i (F340/F380 ratio).

1.5 in vitro calpain assay

Adult rat brain tissue was homogenized with HEPES buffer (in mM: 20 HEPES, pH 7.4, 5 KCl, 1.5 MgCl ₂ , 1 dithiothreitol, 1 EGTA), extract with 1 mM Ca ^{2 +} 37 ° C in the reaction; or with purified μ- calpain (Biovision, Palo Alto, CA, USAS) incubation in order to determine _D calpain cleavage site on TRPC6 in I mM Ca ^2+, 37 ° C conditions, the HEK293 cells were transfected with 3⁄4g-loop-H^-TRPC6-"yc plasmid (pcDNA3.1TRPC6-myc plasmid, inserting flag before, inserting HA in the second loop region of intermediate TRPC6, inserting the label with QuikChange of Stratagene, USA) ® Site-Directed Mutagenesis Kit), then extract the cell lysate with HEPES buffer and add μ-calpain digestion. The purified protein NUS-C6W03 was extracted with a column (Qiagen, Hilden, Germany), then subjected to μ-calpain digestion, and the digested fragments were used for mass spectrometry and Edman sequencing. 1.6 Construction and culture of TRPC6 transgenic mice

The promoter of CaMKIIa was used to drive expression of the mouse TRPC6 gene on neurons in the brain of mice (especially the forebrain). On plasmid p279 (Joe Z Tsian et al, Neuron, March 4, 2004) contains approximately 8.5 kb of promoter region of CaMKIIa derived from the mouse genome, followed by a cDNA fragment of mouse TRPC6, and polyA tail. The plasmid was linearized and then microinjected into the fertilized eggs of the C57BL6J and FBN strain mice, and finally the injected fertilized eggs were implanted into the surrogate mothers. The genotype of transgenic mice was identified by polymerase chain reaction (PCR). The primers used for PCR detection are:

P279 F, GTTCTCCGTTTGCACTCAGG ( SEQ ID NO: 2);

Trpc6-flag R, CGGGATCCCTTGTCGTCATCGTCTTTGTAGTCTCTGCGG ( SEQ ID NO: 3 )

Finally, 3 parental mice (F0) were obtained. F0 mice were mated with C57BL6J strain mice to produce progeny, and the mice used in the experiment were all 3 generations (F3).

1.7 Antibodies and drugs used in the experiment

The commercial antibodies used in the experiments were from the following companies:

Alomone Labs rabbit anti-TRPC 1, 3, 4, 5, 6 antibodies;

Millipore's rabbit anti-TRPC6 antibody, mouse anti-neuN, anti-GFAP, anti-ghost protein, anti-NR2A and anti-CD31 antibody;

Upstate rabbit anti-GluRl, GluR2/3, GREB and phospho-GREB Serl33 antibodies, mouse anti-myc and anti-PSD95 antibodies;

Sigma mouse anti-CaMKIIo anti-alpha-tubulin antibody, and anti-TRPC6 antibody;

Santa Cruz goat anti-capain 1 antibody, mouse anti-phospho-CaMKIIa Thr286 antibody and anti-NOS 1 antibody. .

Unless otherwise stated, other drugs and reagents are from Sigma.

The TAT-C6 peptide of the present application was directly synthesized by Jill Biochemical (Shanghai) Co., Ltd. (see Figure 14b).

1.8 Statistics The software used for data analysis mainly includes: Clampfit 9.0 (Axon, USA), Origin 7.0 ( Originlab corporation, USA), and Excel 2003 (Microsoft). The experimental data is expressed as mean ± sem. Differences between the two sets of data were compared using the t test (student's t-test, including paired and unpaired tests); variance analysis (ANOVA) was used to compare differences between sets of data. p represents the significance value, and n represents the number of experimental cases. A significant difference was considered when p < 0.05. In addition, the survival curve was analyzed using the nonparametric Kaplan-Meier method.

2. Experimental results

2.1 TRPC6 protein-specific down-regulation in neurons after cerebral ischemia

The rat model of focal cerebral ischemia uses middle cerebral artery occlusion (middle cerebral artery occlusion,

MCAO) method [Longa, EZ et al, Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke, 1989. 20(1): p. 84-91 ], ischemia for 2 hours and then re-irrigation for different time; ischemic injury The condition is detected by TTC staining. First, changes in protein expression of molecules associated with survival were screened in the brain of ischemic rats. Proteins were extracted from the penumbra of the ischemic side of the ischemic rat brain and the corresponding normal lateral brain regions, and the protein changes were detected by immunoblotting. Using a specific TRPC antibody (Fig. 1) (see 1.7 Antibodies and Drugs for the Experiment), it was found that the expression level of TRPC6 protein decreased to 74% of the control side protein at 0, 6, 12, and 24 hours after ischemia. 58 %, 40 %, and 32 % (at each time point, n = 5-8 mice, * p < 0.05, * * p < 0.01, Figure 2a), while TRPC3 and TRPC4 proteins were not significant at these time points Change (Figure 2b). Although the amount of TRPC6 protein was significantly reduced 24 hours after reperfusion, the expression levels of TRPC 1, C3, C4, C5 and GluRl were not significantly reduced (Fig. 2c;). In addition, real-time enzyme-chain polymerization also showed no significant change in TRPC6 mRNA levels after ischemia-reperfusion (Fig. 2d), suggesting that a significant down-regulation of protein levels occurred at the post-translational level. In summary, it can be inferred that TRPC6 protein has a specific down-regulation in the penumbra after ischemia.

Next, immunohistochemistry was used to investigate whether down-regulation of TRPC6 protein occurred in neurons. The experiment showed that the immunopositive of TRPC6 was significantly decreased in NeuN-positive cells (neurons) on the ischemic side 24 hours after reperfusion (Fig. 2e). However, in GFAP-positive cells (glial cells), TRPC6 was immunopositive but not Significant changes (Figure 3). This suggests a specific down-regulation of TRPC6 protein in neurons after ischemia.

2.2 Down-regulation of TRPC6 protein precedes neuronal death

To investigate whether down-regulation of TRPC6 protein is a passive result of neuronal death, brain sections of ischemic rats were simultaneously treated with antibodies to TRPC6 protein (see 1.7 Antibodies and Drugs for Experiments) and TUNEL Kit for Detection of Cell Death. To do the staining mark. The experimental results show that after 24 hours of ischemia and reperfusion, the penumbra The TRPC6 protein has been significantly down-regulated, but the positive cells detected by TU EL did not increase significantly after 48 hours of reperfusion (Fig. 4a). Further statistical analysis showed a good negative correlation between the amount of TRPC6 protein and the number of TU EL positive cells during reperfusion after ischemia. As shown in Figure 4b, TRPC6 protein was significantly reduced at 0 hours after ischemia, and the protein expression was significantly reduced at 12, 24, and 48 hours. TUNEL-labeled positive cells began to appear 24 hours after reperfusion and increased significantly at 48 hours. This suggests that downregulation of TRPC6 protein precedes neuronal death, and downregulation of TRPC6 protein may play an important role in subsequent ischemic neuronal death. ,

2.3 Functional down-regulation of TRPC6 protein in cell mimic ischemia assay

In order to better study the down-regulation of TRPC6 protein, the widely used cells were used to simulate ischemic experiments, ie, Oxygen-Glucose Deprivation (OGD) on cultured neuronal cells [Goldberg, MP and DW Choi, Combined oxygen and glucose deprivation in cortical cell culture: calcium-dependent and calcium-independent mechanisms of neuronal injury. J Neurosci, 1993. 13(8): p. 3510-24]. As a result, in the OGD-treated cultured neurons, the amount of TRPC6 protein was specifically down-regulated, while the amount of TRPC3 protein was not significantly changed (Fig. 5a). Analysis by quantitative RT-PCR showed that TRPC6 did not change at the mRNA level (Fig. 5b), which is consistent with the previous experimental results in animals, indicating that in the conditions of simulated ischemic stimulation, in neurons The TRPC6 protein also undergoes a specific down-regulation.

Electrophysiological experiments were performed in order to investigate whether down-regulation of TRPC6 protein affects the function of the TRPC6 channel on the membrane. Using a whole-cell patch clamp recording method, OAG (an analog of DAG (diacylglycerol), an enhancer of TRPC6, TRPC3 channel) in cultured neurons can cause a slow and small inward current (I _OAG ). This current can be completely inhibited by SKF96365 (a broad inhibitor of TRPCs channels, available from Sigma) or with 0 Ca ²⁺ external solution plus NMDG (instead of Na ⁺ ). Further, transfection of a specific RNAi plasmid (RNAi_C6, see the literature by Zhou j* et al., aimed at knockdown of the expression level of TRPC6 protein) against the TRPC6 protein in neurons can suppress this current caused by OAG (Fig. 6a, b) ). Similarly, in calcium imaging experiments, it was found that stimulation with OAG caused a slow and small increase in intracellular calcium concentration ([Ca ²⁺ ]i) in cultured cortical neurons. This increase in [Ca ²⁺ ]i can be inhibited by SKF96365; if in 0 Ca ²⁺ external solution, 0AG does not cause intracellular calcium elevation; in addition, if the mutant overexpresses TRPC6 ( DN-TRPC6 has three mutations in the channel pore region, which can inhibit the opening of the TRPC6 channel [Hofmann, T., et al., Subunit composition of mammalian transient receptor potential channels in living cells. Proc Natl Acad Sci USA, 2002. 99 (1 1): p. 7461 -6]) , then the increase in [Ca2+]i caused by OAG is also significantly inhibited (Fig. 7). And, according to the relationship between current and voltage, the bidirectional rectification of the channel is shown. The quality (Fig. 6c) suggests that the I _OAG current is mainly mediated by the TRPC6 channel protein. In the OGD-treated neurons, it was found that I _0AG was significantly inhibited (Fig. 8). As a control, it was consistent with the report that the current mediated by the acid ion channel ΙρΗβ.Ο apparently increased the force [Xiong, ZG , et al., Neuroprotection in ischemia: blocking calcium-permeable acid-sensing ion channels. Cell, 2004. 1 18(6): p. 687-98]. This suggests that the TRPC6 channel protein in neurons is specifically down-regulated under experimental conditions that mimic ischemia, resulting in a specific down-regulation of TRPC6 channel function on the membrane.

The following plasmids were overexpressed in neurons: GFP (control protein), TRPC6 (functional channel protein), DN-TRPC6 (mutant non-functional channel), RNAi_C6 (knockdown of TRPC6 protein expression) (Zhou J*, Du WL*, Zhou KC, Tai YL, Yao HL, Jia YC, Ding YQ, Wang YZ. Critical role of TRPC6 channels in the formation of excitatory synapses. Nat Neurosci. 2008 Jul;ll(7):741-3), and OGD treatment of neurons. It was found that overexpression of TRPC6 protein reduced OGD-induced cell death, while overexpression of DN-TRPC6 or overexpression of RNAi_C6 plasmid knockdown endogenous TRPC6 protein significantly increased OGD-induced cell death. This suggests that increasing the expression of TRPC6 protein has a protective effect on neurons under simulated ischemic conditions, while decreasing the amount of TRPC6 protein aggravates cell damage (Fig. 9). Correspondingly, we pretreated the cells with OAG and then OGD stimulation, and the cell death rate was significantly reduced. However, if the cells were pretreated with SKF96365 and then stimulated with OGD, the cell death rate increased significantly (Fig. 10a). Similarly, in MCAO rats (Sprague-Dawley, 250-280 g, Shanghai Slack Laboratory Animal Co., Ltd.), if OAG is injected into the lateral ventricle, the area of infarction caused by ischemia is significantly reduced; on the contrary, if SKF96365 is injected in advance , aggravating ischemic injury (Fig. 10b, c). The above experimental results show that up-regulation of TRPC6 has a good neuroprotective effect in the case of simulated ischemic stimuli, while down-regulation of TRPC6 aggravates neuronal damage.

Furthermore, overexpression of TRPC3 protein in cultured neurons was found to have no protective effect on OGD-induced cell death (Fig. 11). Further experiments suggest that the specific protection of TRPC6 protein depends on the activation of CREB protein, because, first, overexpression of TRPC6 protein can increase the expression of p-CREB, that is, the activated form of CREB increases (Fig. 12a); Second, if KCREB (dominantly inhibited cyclic adenosine monophosphate response element binding protein, unable to bind to DNA, no function) is co-expressed with TRPC6, the protective effect of TRPC6 protein is completely inhibited (Fig. 12b).

2.4 TRPC6 protein is degraded by calpain

Rapid ischemic downregulation of TRPC6 in ischemic reperfusion suggests that this may be a protease-mediated rapid degradation process. The next experimental screening is a protease that is involved in the downregulation of the TRPC6 protein.

In rat brain tissue extract, the addition of calcium ions can induce specific down-regulation of TRPC6 protein, while TRPC3 The protein does not have this calcium-induced down-regulation, and pre-addition of EGTA blocks this protein degradation (Fig. 13a). Time-history analysis showed that the degradation of this protein was very evident after 5 minutes of calcium addition, and it was found that this degradation occurred when the calcium ion concentration started from ΙμμΜ (Fig. 13b). It is known that such a calcium ion concentration can activate μ-type calpain, which is also a type of calpain mainly distributed in neurons [Goll, DE, et al., The calpain system. Physiol Rev, 2003. 83(3) : p. 73 1 -801 ]. These results suggest that the TRPC6 protein is hydrolyzed by a calcium-activated protease. To further verify which protease is involved in this process, the following inhibitors were used: PMSF, serine protease inhibitor, cpm-VAD-CHO, caspase protease inhibitor, and lactacystin, a protein degradation inhibitor (see 1.7 for antibody and Drugs) However, none of these inhibitors blocked the degradation of TRPC6 protein by calcium ions in this in vitro system. In contrast, calpain inhibitors, including: calpeptin, leupeptin, and MDL28170 (see 1.7 Antibodies and Drugs for Experiments), all block calcium-induced degradation of TRPC6 protein (Figure 13a, c; ). These results indicate that the degradation of TRPC6 protein is due to the proteolysis of calpain. In addition, the degradation of spectrin protein was detected by immunoblotting (can be used as a marker of calpain activation [Siman, R., M. Baudry, and G. Lynch, Brain fodrin: substrate for calpain I, an Proc Natl Acad Sci USA, 1984. 81(1 1): p. 3572-6] ) , confirmed that calpain is indeed activated in this in vitro system.

Next, it was verified whether the TRPC6 protein was directly degraded by calpain. First, N- 7ag-second \oo >-HA-C-myc ( pcDN A3. 1 TRPC6-myc plasmid was expressed in HEK293 cell line, inserting flag before, inserting HA in the second loop region of intermediate TRPC6, inserting label The TRPC6 plasmid (Figure 14, schematic panel) was labeled by Stratagene's QuikChange® Site-Directed Mutagenesis Kit. After 24 hours, the cellular protein was collected and calpain in vitro digestion experiments were performed. In the immunoglobulin imprinting experiment, it was found that the band representing the full-length TRPC6 protein decreased with the increase of calpain concentration (Fig. 3.4.2a), and two bands appeared under the original main band. . Using the ?ag antibody assay, the full-length TRPC6 protein band disappeared rapidly with increasing calpain concentration, while the full-length band detected with the m_yc antibody disappeared significantly later than the band recognized by the 3⁄4 _g antibody. And as the concentration of calpain increases, a strip appears immediately below the full-length strip, and all of the strips disappear. This suggests that the N-terminus of the TRPC6 protein is more susceptible to degradation by calpain than the C-terminus. Therefore, it is speculated that the N-terminus is the place where degradation begins. With the degradation of the N-terminus, the TRPC6 protein undergoes sequential degradation until the C-terminal fragment is completely degraded.

Next, the N-terminal amino acid sequence of TRPC6 protein was expressed in prokaryotic cells. Co/ (the N-terminal sequence of TRPC6 was subcloned from M ¹ to D ^{2Q3 and} then ligated into the vector NUS- tag (pET-43. 1 a vector, Novagen, Germany), ie NUS_C6 _WC) 3), after isolation and purification of this protein, add calpain digestion. The results showed that calpain cleaves this protein in a concentration-dependent manner and finds a fragment that has been cleaved. Mass spectrometry and N-terminal sequencing were performed and it was shown that the fragment was indeed derived from TRPC6. The sequencing results also confirmed that the cleavage site of calpain on TRPC6 was at the N-terminus of the AAPGA sequence (Fig. 14b). Based on this site, a polypeptide was constructed, the sequence of which is the amino acid sequence of a segment of TRPC6 containing this cleavage site, with a TAT sequence (in order to make it well across the cell membrane [Vives, E., P. Brodin, and B. Lebleu, A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J Biol Chem, 1997. 272(25): p. 16010-7]). The polypeptide was named TAT-C6 and its sequence was GRKKRRQRRRCRRGGSLKAAPGAGTRR (SEQ ID NO: 4). The next step is to specifically block the proteolytic action of calpain on TRPC6 without affecting other functions of calpain. It was found that this TAT-C6 peptide can effectively inhibit the degradation of TRPC6 protein in rat brain lysates caused by Ca ions, but does not affect the degradation of the target substrate spectrin of calpain (Fig. 14b, right picture). This suggests that this peptide is very specific and effective in inhibiting the process by which calpain degrades TRPC6. 2.5 NMDA receptors are involved in the degradation of TRPC6 protein under ischemic conditions

According to reports in the literature, the activation of calpain during ischemia is most likely due to the excessive opening of NMDA receptors.

To investigate whether NMDA receptors are involved in calpain-mediated TRPC6 proteolysis under ischemic conditions, two inhibitors of calpain were first added to cultured neurons: calpeptin and MDL28170 (see 1.7 Antibodies and Drugs for Experiments) ), the cells were treated with OGD, and the changes in protein and cell death were detected 24 hours later. The experimental results show that the degradation of TRPC6 protein caused by OGD can be blocked by the inhibitor of precalcinase. Moreover, both inhibitors of calpain are effective in reducing OGD-induced cell death (Fig. 15a).

A specific two-stage siRNA against calpain (CAPN i_l : 5 ' -GCUUCUUGUUGGCCCUC AUTT-3 ' ( SEQ ID NO: 5 ); CAPN i_2 , 5 '-GAAUCAUUAGCAAAC ACAATT-3 ' was previously transfected into cultured cortical neurons. (SEQ ID NO: 6), synthesized by Shanghai Gemma Co., Ltd.) Knockdown of calpain expression, OGD stimulation and statistics of TRPC6 protein degradation. It was found that both siRNAs blocked OGD-induced degradation of TRPC6 protein (Fig. 15b).

These results indicate that the downregulation of TRPC6 protein under cellular ischemic conditions is indeed due to the hydrolysis of calpain.

In addition, dextrocycline (MK801, a blocker of NMDA receptors purchased by Sigma) was found to effectively block the degradation of TRPC6 protein (Fig. 15c), suggesting that NMDA receptors may be involved in this process. further, Pre-transfection of siRNA (RANi synthesized by Shanghai Gemma, NR li-1 : 5'-GGCAGUUCACGAACUCCUATT-3' (SEQ ID NO: 7); NR li-2: 5 ' -GACU AAAGAUAGUGAC AAUTT-3 ' ( SEQ The method of ID NO: 8)) reduces the protein expression level of an essential subunit NR1 of the NMDA receptor, and as a result, OGD-induced degradation of TRPC6 protein is effectively inhibited (Fig. 15d). This suggests that NMDA receptors are also involved in the degradation of TRPC6 protein. In addition, in the rat model of ischemia, if the NMDA receptor blocker (calpeptin) is injected into the cerebral ventricle, the degradation of TRPC6 protein caused by ischemia can be effectively inhibited ( Figure 15e). NMDA receptors and calpain were involved in the degradation of TRPC6 protein, both in ischemic animal models and in cell models.

2.6 Increase the expression of TRPC6 protein can effectively reduce brain damage in ischemic mice

To further demonstrate the relationship between the amount of TRPC6 protein and cerebral ischemic protection, a TRPC6 transgenic mouse was constructed. Using the CaMKIIa promoter-driven expression vector, exogenous TRPC6 protein can be specifically expressed in forebrain neurons, including cortical and hippocampal neurons, but not cerebellar neurons.

The expression level of TRPC6 protein in the cerebral cortex proteins of transgenic mice was firstly identified as compared with that of wild-type mice, while other protein levels were unchanged (Fig. 16a, b). Secondly, immunohistochemistry experiments showed that TRPC6 protein was highly expressed in cortex and hippocampus in transgenic mice, but not in cerebellum (Fig. 16c). This all indicates that the TRPC6 protein specific in transgenic mice is highly expressed in forebrain neurons. Next, an ischemic experiment was performed with transgenic mice (Tg) and their littermates of non-transgenic mice (WT) (here, a double-blind trial was used, ie the person who did the surgery did not know the mice. Grouped, and the person who is statistically ischemic is not aware of the grouping of the mice). The results showed that the area of cerebral infarction in TRPC6 transgenic mice was significantly smaller than that in non-transgenic wild-type mice, and the survival rate of transgenic mice was also higher than that in wild-type mice (Fig. 17). This further confirms that increasing the expression of T PC6 protein has a significant reduction in brain loss in ischemic mice and can improve survival after cerebral ischemia. At the same time, the vascular structure and density in the brains of transgenic mice and wild-type mice were also examined by immunohistochemistry by labeling CD31 protein, a marker for vascular endothelial cells, which is often used to display the structure and density of blood vessels. The results showed no significant differences (Figure 18). On the other hand, laser Doppler flowmetry was used to detect changes in blood flow in the brains of transgenic mice and wild-type mice before and after ischemia, and the results showed no significant difference (Fig. 18, right panel). Therefore, we believe that the tolerance of transgenic mice to ischemic injury may be due to the maintenance of a certain amount of TRPC6 protein, but not for other reasons.

To further confirm that the tolerance of TRPC6 transgenic mice in cerebral ischemia is due to their neurons

The amount of TRPC6 protein is high. We used the TUNEL kit to detect cell death by immunohistochemical staining of TRPC6 protein in ischemic mouse brain slices. The results showed that TRPC6 protein in transgenic mice Cells with high expression levels had fewer positive TU EL numbers, whereas cells with low TRPC6 protein expression in wild-type mice had significantly more TUNEL positive numbers than the former (Fig. 19). This suggests that maintaining a certain amount of TRPC6 protein in the cells can effectively increase its tolerance to ischemic injury.

As shown in Figure 20a, in the sham operation group, the amount of TRPC6 protein in the brain of transgenic mice was significantly higher than that in wild-type mice; after ischemia, although the amount of TRPC6 protein in the brain of transgenic mice and wild-type mice was Significantly reduced, but the amount of TRPC6 protein in the brain of transgenic mice was still higher than that of wild-type mice. This suggests that ischemic protection may be dependent on maintaining a certain amount of TRPC6 protein.

2.7 The ischemic protective effect of TRPC6 may be dependent on the activation of CREB protein

In the cortical proteins of TRPC6 transgenic mice, the protein levels of p-CREB and CREB before and after ischemia were detected. The results showed that the level of p-CREB/CREB in the brain of transgenic mice was significantly higher than that of wild type in the sham operation group. Rats, and after ischemia, the level of p-CREB/CREB in the cortex of transgenic mice was also higher than that of control mice (Fig. 20b). As a control, other Ca2 ⁺ -regulated downstream proteins, such as the amount of p-CaMKIIa/CaMKIIa and NOS 1 protein, were also detected, and the results showed that these two proteins were expressed in transgenic mice and wild-type mice. There was no significant difference in the expression levels before and after ischemia (Fig. 20c;). This suggests that the ischemic injury and long survival rate of transgenic mice is probably due to the higher level of TRPC6 protein in the brain, and the downstream activated form of CREB, which is a higher level of p-CREB protein, as a promoting cell survival. Protein, continuous activation of CREB plays an important role in protecting the brain from ischemic injury [Kitagawa, K., CREB and cAMP response element-mediated gene expression in the ischemic brain. Febs J, 2007. 274(13): p. 3210-7].

2.8 inhibition of calpain degradation TRPC6 can effectively reduce cerebral ischemic injury in rats

The foregoing experiments show that, TAT- C6 brain lysate this peptide can be caused by Ca ^{2 +} TRPC6 degradation without affecting protein effective to inhibit calpain degradation of other substrates. Therefore, detecting whether the TAT-C6 peptide can inhibit the decrease of TRPC6 protein in the brain caused by cerebral ischemia, thereby maintaining the amount of endogenous TRPC6 protein and protecting the brain from ischemic injury. The results showed that the injection of TAT_C6 peptide into the anterior ischemic ventricle can effectively reduce the degradation of TRPC6 protein induced by ischemia without affecting the degradation of spectrin (Fig. 21), that is, it does not affect other functions of calpain, but only specifically inhibits calcium. Protease degradation of TRPC6 is a process. In addition, rats were divided into three groups and given Veh. (distilled water), T AT- Ctrl (GRKKRRQRRRC - PPYGYYPSFRG E RL directly synthesized by Gil Biochemical (Shanghai) Co., Ltd.) and TAT_C6 peptide (ischemic anterior ventricle injection) Then, the ischemic injury was evaluated after reperfusion for 2 hours after ischemia for 2 hours. It was found that only the third group, the TAT_C6 peptide treatment group, had the smallest brain damage compared with the previous two groups (Fig. 21, Right)). In summary, use targeted The sexual peptide (TAT_C6) inhibits calpain degradation of TRPC6 protein, a strategy that effectively protects against ischemic brain damage.

3. Other peptides also inhibit calpain degradation TRPC6

In previous experiments we have demonstrated that RRGGSLKAAPGAGTRR prevents calpain from cleavage of TRPC6 and that injection of this peptide in the lateral ventricle protects ischemic brain damage. To further demonstrate that the cleavage site of calpain to TRPC6 is the key to this protection, we synthesized a shorter peptide TAT-C6-2 (grkkrrqrrrcGGSLKAAPGA (SEQ ID NO: 9) containing this site, grkkrrqrrrc is TAT sequence (transmembrane factor), KA is the amino acid on both sides of the tangent point). Analysis of the lesion area after lateral ventricle injection (I mM peptide X 5 U 1 in each rat lateral ventricle) showed that the short peptide fragment before ischemia had significant protective effect on ischemic injury (*ρ<0.05) (See Figure 22).

To further demonstrate that calpain cleavage of TRPC6 is a key factor in this protection, we synthesized a shorter peptide T AT-C6-3 ( TAT-C6-3: grkkrrqrrrcSLKAAP ( SEQ) containing this site ID NO: 10), grkkrrqrrrc is the TAT sequence (transmembrane factor), and KA is the amino acid on both sides of the tangent point). The intracerebral ventricle of each rat was injected with 1 mM peptide Χ 5 μ 1, and the analysis of the lesion area after lateral ventricle injection showed that the short peptide fragment before ischemia still had significant protective effect on ischemic injury (*ρ<0.05). ) (see Figure 23).

'In addition, in a cell-free system, calpain was incubated with purified TRPC6 protein at 37 ° C in the presence of calcium ions while adding different concentrations of polypeptide. Western blotting was then used to detect whether a polypeptide inhibits calpain degradation of TRPC6 protein in a concentration-dependent manner.

The following polypeptides were detected by the above method: SLKAAPGAGTR, RGGSLKAAPGAGTR,

TRRGGSLKAAPGAGTR, VCRRGGSLKAAPGAGTRR,

FVCRRGGSLKAAPGAGTRRN, these polypeptides are expected to inhibit the specific competitive binding of calpain to the site of TRPC6, thereby inhibiting calpain degradation of TRPC6. 4. Intraperitoneal injection of TAT-C6 improves learning and memory in rats with Alzheimer's disease

Wild type (WT) and APP/PS1 senile dementia model mice (APP/PS1) were selected at a dose of about 1 month, and TAT-C6 peptide and corresponding saline were intraperitoneally injected at a dose of 20 mg/kg. , divided into four groups (n=5 in each group): WT injection of TAT-C6 peptide group (WT+ peptide), WT injection of saline group (WT), APP/PS 1 injection of TAT-C6 peptide group (APP/PS1+ peptide) ), APP/PS1 injection of saline group (APP/PS1). The injection was performed twice a week for a total of three months, and then a water maze test was performed to test the learning and memory ability of the mice.

The results show (Fig. 24) that the APP/PS 1 group takes more time during the training phase than the WT group. The hidden platform was found (Fig. 24b), and the platform position was traversed less frequently during the test phase (Fig. 24d), suggesting that APP/PS1 mice showed significant loss of learning and memory at 14 months. Compared with APP/PS1, the APP/PS 1+ peptide group only needs less time to find the hidden platform during the training phase, and more than the platform position in the test phase ( *p<0.05 ; * *p<0.01;***p<0.001). This indicates that the TAT-C6 peptide can significantly improve the learning and memory ability of APP/PS 1 mice. The invention has been described above in terms of specific embodiments. However, these specific examples are merely illustrative and not intended to limit the scope of the invention. Various changes and modifications of the invention may be made by those skilled in the art without departing from the spirit of the invention. The scope of protection of this application is defined by the claims.

Claims

Claim

1. A substance used to maintain the level of TRPC6 in nerve cells to prevent or treat ischemic brain damage or degenerative brain damage.

The substance according to claim 1, wherein the substance is an inhibitor of calpain, or a TRPC6 expression enhancer, or an NMDA receptor antagonist.

3. The substance according to claim 2, wherein the inhibitor of calpain is a calpain-specific small interfering RNA, or calpeptin, or a polypeptide comprising a SLKAAP sequence.

The substance according to claim 3, wherein the polypeptide has the sequence shown as SEQ ID NO: 1, or a fragment thereof.

The substance according to claim 2 or 3, wherein the polypeptide is linked to a transmembrane factor.

The substance according to claim 5, wherein the sequence of the polypeptide is as shown in SEQ ID NO: 4, SEQ ID NO: 9 or SEQ ID NO: 10.

The substance according to claim 3, wherein the sequence of the calpain-specific small interfering RNA is as shown in SEQ ID NO: 5 or SEQ ID NO: 6.

The substance according to claim 2, wherein the TRPC6 expression enhancer is a TPC6 expression vector, an analog of OAG or OAG.

The substance according to claim 2, wherein the NMDA receptor antagonist is rugamine, dextrozine, or as shown in SEQ ID NO: 7 or SEQ ID NO: 8.

10. Use of a substance according to claim 1 to 9, wherein the use comprises the preparation of a medicament or combination of drugs for preventing or treating ischemic brain damage or neurodegenerative diseases.