Application of urolithin A in Alzheimer's disease

Abstract

The invention provides application of urolithin A in Alzheimer's disease, belongs to the technical field of medicinal chemistry and treatment of mental diseases, and aims to solve the problem of social anxiety caused by the Alzheimer's disease. According to the application disclosed by the invention, the mitochondrial autophagy effect of urolithin A is utilized, A beta is eliminated, a key brain region loop aBLA-vCA1 is repaired, and the anxiety symptom of a patient suffering from the Alzheimer's disease is relieved. According to the present invention, with the application of the urolithin A in the treatment of Alzheimer's disease, through the mitochondrial autophagy function of the urolithin A, the targeted removal of A beta and the repair of the damaged basal outer side amygdala nucleus and hippocampus loop are achieved so as to effectively relieve the anxiety symptom of the patient and provide the new strategy for the AD treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical chemistry and treatment of mental illnesses, and specifically relates to the application of urolithin A in Alzheimer's disease. Background Art

[0002] Alzheimer's disease is the main cause of dementia, which leads to cognitive decline and gradual memory loss. In the early stages of Alzheimer's disease, due to cognitive decline caused by neuronal damage, patients will have compensatory emotional reactions such as anxiety, and continue to damage social relationship functions as a psychological response to cognitive decline. Anxiety and depression often overlap in patients with mild Alzheimer's disease, and anxiety disorders are a psychological response to the diagnosis of Alzheimer's disease. Anxiety and depression are associated with cognitive decline, and the progression of anxiety and depression will also accelerate cognitive decline and promote cognitive defects in Alzheimer's disease. Symptoms of anxiety and depression can be directly observed in the middle stage of Alzheimer's disease. Due to the sudden change in lifestyle and the rapid progression of the disease, changes occur at the psychological level. One-third of patients with mild cognitive impairment will show obvious anxiety and depression. Therefore, controlling the patient's anxiety state can help alleviate the progression of Alzheimer's disease and has great clinical significance.

[0003] Anxiety disorders and depressive symptoms increase with the progression of Alzheimer's disease, but the cause of the relationship between anxiety disorders and AD has not been clearly explained. Possible reasons are related to the neurobiological mechanisms shared by the two diseases. Neuropathological changes in Alzheimer's disease, including changes in transmitter and protein secretion or damage and cell death of cortical cells, affect the neural circuits related to the control of emotions in the brain. So far, only a few studies have studied the reasons why anxiety disorders and depression co-progress in Alzheimer's disease. In addition, the appearance of neuroinflammatory plaques composed of amyloid β protein and hyperphosphorylated forms of tau protein in neurons, two different brain changes, are hallmark pathological changes in the progression of AD. Amyloid β (Aβ) causes a strong correlation between the brain and anxiety-like behaviors, and individuals with high Aβ concentrations are more likely to have anxiety symptoms. There are few means of diagnosing and treating anxiety disorders in AD patients in clinical practice, and it is difficult to evaluate the role of the relationship in disease progression. Treatment of anxiety disorders caused by AD generally uses a combination of medications, such as using drugs donepezil or memantine to treat AD, while using antidepressants such as trazodone and sertraline to treat the psychotic and behavioral symptoms of anxiety disorders and depression.

[0004] The basolateral amygdala (BLA) and hippocampus (HP) play an important role in the processing of anxiety and depression. The anterior basolateral amygdala (aBLA) has an anti-anxiety effect through nonspecific activation of glutamatergic activity, while the ventral hippocampus (vHP) is also closely related to anxiety. There are anxiety-related cells in the CA1 region of the ventral hippocampus, and optogenetic inhibition of them can produce an anti-anxiety effect.

[0005] Urolithin A belongs to the urolithin family and is a natural compound. It is a small molecule polyphenol converted from ellagitannins and ellagic acid by intestinal bacteria in the colon. Ellagitannins and ellagic acid are natural polyphenols widely present in the diet. They have low bioavailability and can only be converted into other derivatives or metabolized out of the body. The microorganisms that convert ellagitannins and ellagic acid into UA are not clear, but UA quickly enters the blood circulation after intestinal absorption. Direct supplementation of UA can bypass the microbial-mediated precursor conversion process and play a positive role in cells and their mitochondria in age-related diseases in the blood circulation. UA has been reported to be able to observe lasting improvement in mitochondrial health in a variety of model organisms and humans, and can regulate the clearance and circulation of dysfunctional mitochondria, that is, promote the occurrence of mitochondrial autophagy. Mitochondrial autophagy is blocked in age-related diseases. UA can be used as an effective mitochondrial autophagy agonist to restore normal mitochondrial autophagy levels and offset age-related cell damage. Summary of the invention

[0006] In response to the above problems, the present invention proposes the application of urolithin A in Alzheimer's disease, using the mitochondrial autophagy of urolithin A to remove Aβ, repair key brain circuits, and relieve anxiety symptoms in patients with Alzheimer's disease. It aims to effectively relieve the anxiety symptoms of patients and provide a new strategy for AD treatment.

[0007] The technical solution adopted by the present invention is:

[0008] First, the present invention protects the use of urolithin A in the preparation of drugs for treating / alleviating Alzheimer's disease. Urolithin A activates mitochondrial autophagy, targets and clears Aβ, and repairs the damaged aBLA-vCA1 circuit.

[0009] Secondly, the present invention also protects a drug for treating / alleviating Alzheimer's disease comprising urolithin A, wherein the drug comprises a therapeutically effective amount of urolithin A and a pharmaceutically acceptable carrier.

[0010] In some embodiments, the Alzheimer's disease mentioned above is social anxiety symptoms induced by Aβ.

[0011] Finally, the present invention also protects a mitochondrial autophagy activator, which comprises urolithin A and a pharmaceutically acceptable carrier.

[0012] In some embodiments, the pharmaceutically acceptable carrier includes any one or more of alumina, aluminum stearate, lecithin, serum protein, phosphate, glycerol, sorbic acid, potassium sorbate, a partial glyceride mixture of saturated vegetable fatty acids, water, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, beeswax and lanolin.

[0013] In some embodiments, the drug or activator further comprises at least one vitamin derivative or a salt thereof selected from vitamin C derivatives, vitamin E derivatives, and vitamin P derivatives.

[0014] In some embodiments, the vitamin derivative or salt thereof is at least one vitamin derivative or salt thereof selected from ascorbic acid phosphate or a salt thereof, fatty acid ester of ascorbic acid phosphate or a salt thereof, tocopherol phosphate or a salt thereof, and methyl hesperidin or a salt thereof.

[0015] In some embodiments, the drug or activator is in the form of tablets, capsules, powders, syrups, solutions, suspensions or aerosols.

[0016] Beneficial effects of the present invention:

[0017] 1. The present invention found that urolithin A directly acts on the aBLA-vCA1 circuit which is closely related to anxiety behavior, reduces the toxic effects of Aβ, and thus effectively alleviates anxiety, depression and other emotional disorders in patients with Alzheimer's disease.

[0018] 2. The present invention also found that urolithin A can have a positive effect on the repair of cognitive function by reducing Aβ deposition and improving mitochondrial function.

[0019] 3. The present invention also found that the antioxidant, anti-inflammatory and neuroprotective properties of urolithin A can protect neurons from further damage and slow down the progression of Alzheimer's disease.

[0020] 4. The use of the mitochondrial autophagy activator urolithin A proposed in the present invention in the preparation of drugs for treating / alleviating Alzheimer's disease provides clinicians and patients with new and potential treatment options. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 UA improves impaired social ability caused by Aβ. (a) Schematic diagram of virus injection and fiber implantation, rAAV-CaMKII-Cre was injected into vCA1, AAV-CaMKII-DIO-GCaMP6f was injected into aBLA, and the fiber was implanted into aBLA after viral expression; (b) Representative images of GCaMP6f in aBLA, scale bar = 200 μm; Peri-event time histogram (PETH) of normalized ΔF / F of aBLA CaMKII neurons projecting to vCA1 in 5×FAD group (c) and 5×FAD+UA group (d). (e) Quantitative analysis of the average ΔF / F of 5×FAD group and 5×FAD+UA group (independent t test, t = 13.33, df = 6, P < 0.0001. 4 animals in each group). (f) Quantitative analysis of AUC of 5×FAD, 5×FAD+UA and 5×FAD+UA groups (independent t test, t = 9.93, df = 6, P < 0.0001. 4 mice in each group). (g) Schematic diagram of the three-chamber social experiment test. Mice in the 5×FAD and 5×FAD+UA groups were tested in three chambers. (h, i) UA treatment increased the investigation time of 5×FAD mice in the social area (independent t test, t = 7.656, df = 14, P < 0.0001) and the social: non-social ratio (independent t test, t = 6.725, df = 14, P < 0.0001). 8 mice in each group. (j) There was no significant difference in distance between groups.

[0023] Figure 2 To show that UA regulates the aBLA-vCA1 circuit to restore the social ability impaired by Aβ; (a) Schematic diagram of the three-chamber social test test, in which 5×FAD and 5×FAD+UA mice expressing rhodopsin 2 (ChR2) were tested; (b, c) After light stimulation, the social zone time (one-way ANOVA, F(2,21)=58.06, P<0.0001) and social: non-social ratio (one-way ANOVA, F(2,21)=35.85, P<0.0001) of 5×FAD+UA mice were reduced, with 8 mice in each group. (d) No difference was detected in the distance between the two groups of mice. DETAILED DESCRIPTION

[0024] To make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0025] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores.

[0026] Although UA has been widely studied and used, the impact of UA on aging and disease has only recently been noticed, especially in the treatment of anxiety disorders in AD. In addition, anxiety has been detected in the early stages of Alzheimer's disease and can accelerate disease progression, but the mechanism behind anxiety is unclear and there are no effective interventions.

[0027] Urolithin A, as one of the end products of natural polyphenol ellagitannin metabolism by intestinal microorganisms, exhibits significant mitochondrial autophagy promotion and has multiple biological activities such as anti-aging, anti-cancer, anti-diabetes and neuroprotection. This embodiment aims to target the removal of Aβ, repair the damaged aBLA-vCA1 circuit function, restore the disrupted protein homeostasis, and enhance the adaptability of the circuit through the mitochondrial autophagy stimulating effect of urolithin A, thereby effectively alleviating the anxiety symptoms of Alzheimer's patients.

[0028] This example focuses on the use of urolithin A as a mitochondrial autophagy agonist in the treatment of Alzheimer's disease (AD)-related anxiety disorders caused by the pathological process of β-amyloid protein (Aβ). Alzheimer's disease, a common progressive dementia in the elderly, has core pathological features including memory and cognitive decline, as well as neuroinflammatory plaques formed by β-amyloid protein deposition in the brain and abnormal phosphorylation of tau protein. Aβ is not only a hallmark molecule of early AD pathology, but also mediates complex pathological processes such as neurotoxicity, neuronal inhibition, impaired synaptic function, cellular autophagy disorders, mitochondrial dysfunction and neuroinflammation.

[0029] AD patients are often accompanied by psychiatric symptoms such as anxiety and depression, and these emotional disorders significantly affect social interactions. Studies have shown that there is a close connection between Aβ accumulation and anxiety-like behavior, and patients with high Aβ levels are more likely to show anxiety symptoms. In particular, the basolateral amygdala (BLA) and hippocampus (HP), especially the ventral hippocampus (vHP), play a key role in regulating anxious behavior, and abnormal function of both is closely related to social anxiety. Experimental evidence indicates that abnormal neural network activity between the BLA and vHP, especially overexcitation of the aBLA-vCA1 circuit, can induce anxious behavior.

[0030] Animal experiments

[0031] 1. Six-month-old mice were anesthetized with sodium pentobarbital (1%, 30 mg / kg) and fixed in a stereotaxic syringe (RWD 68046 & 68055, China). After the scalp was disinfected and balanced, a small hole was gently made. Under a stereotaxic instrument (World Precision Instruments, USA), the virus was injected into the aBLA (with bregma as the origin, bregma ML value (X axis), bregma direction AP value (Y axis), depth DV value (Z axis)) (AP: -1.34mm, ML: ±3.4mm, DV: -4.8mm) and vCA1 (AP: -3.28mm, ML: ±3.3mm, DV: -4.6mm) using a 10μL gastight mixing syringe (KF019, China). The injection rate was 50–100 nL / min (for virus injection) or 30 nL / min (for CTB injection). After each injection, the needle was left in place for 5 minutes to avoid virus spread and aspiration. Lincomycin lidocaine gel was evenly applied to the skull, and the skin was then sutured with absorbable sutures. The mice were placed on a heated blanket and waited for awakening. Finally, the injection site was confirmed by fluorescent expression. Mice with incorrect virus expression were excluded.

[0032] 2. Choleratoxin subunit B (CTB) was used to retrogradely track the social control ability of the aBLA-vCA1 circuit. Cholera toxin subunit B (recombinant) was obtained from BrainVTA (CTB-555). CTB (100 nl) was stereotactically delivered to vCA1 (AP: -3.28 mm, ML: ±3.3 mm, DV: -4.6 mm).

[0033] After 3 and 7 days, the mice were anesthetized and perfused, the brains were removed, and the expression of CTB in the aBLA was detected under a confocal microscope (LSM780, Zeiss, Germany).

[0034] 4. AAV5-Ca MKIIa-eYFP was purchased from Brain Case Co., Ltd., Shenzhen, China. The virus (150–200 nL, >1012 vg / mL) was diluted in 4% paraformaldehyde at 0.1 μL min -1 Optogenetic manipulations were performed in freely moving mice by injecting virus into the aBLA at a speed of -1.34 mm (AP: -1.34 mm, mL: ±3.4 mm, DV: -4.8 mm). Virus injections were balanced between the left and right aBLA.

[0035] 5. Approximately 4 weeks later, an optical fiber (core = 200 μm; numerical aperture = 0.37) was implanted into vCA1 (AP: -3.28 mm, ML: ±3.3 mm, DV: -4.1 mm). The mice were given a week to recover before behavioral testing.

[0036] 6. After the behavioral test, mice were gavaged with 50 mg / kg UA per mouse per day.

[0037] 7. Tandem mass tags (TMT) were then used to label proteomic experiments in UA-treated 5×FAD mice and 5×FAD mice. Each sample was digested into peptides using mass spectrometry trypsin (Promega, V5072), then labeled with different TMTs (ThermoFisher 90406), mixed together, and separated into 15 fractions by high-performance liquid chromatography (HPLC) for subsequent experiments.

[0038] 8. The dried fractions were dissolved in 0.1% formic acid (FA) and captured with a silica gel capillary column filled with C18 resin (Varian, Lexington, MA, USA) for subsequent analysis by a Q Exactive (Thermo Scientific, NJ, USA) mass spectrometer with the following parameters: 400-1800 m / z, 70,000 resolution; MS / MS scan (100-1800 m / z). Proteome Discoverer 2.1 software (Thermo Scientific) was used to retrieve MS / MS data against the Uniport human database.

[0039] 9. The three-chamber social test was used to identify anxiety disorders in UA-treated 5×FAD mice and 5×FAD mice. The three-chamber social test is derived from the inherent tendency of mice to engage in social interactions and investigate unfamiliar stimuli. The test apparatus was constructed using a custom rectangular plexiglass resin (60 cm long × 40 cm wide × 30 cm high). The dimensions of each chamber were 20 cm by 40 cm. The custom-designed resin cup (8 cm in diameter) was inverted and symmetrically placed in the two conical parts of the chamber, allowing adolescent mice (3-4 weeks) to move while restricting their activities to the designated area.

[0040] 10. In the initial phase, the test mouse was placed in the center of the chamber and allowed 5 min for unrestricted exploration (habituation). During the next 5 min, the test mouse was restrained in the center of the chamber for 30 s, while an adolescent mouse (3-4 weeks) was placed in an inverted resin cup in the designated social chamber.

[0041] 11. An empty resin cup was placed on the side designated as the non-social chamber. Subsequently, the test mouse was placed in the exploration chamber for 5 min. Each test mouse participated in two days of optogenetic stimulation of social interaction testing. The position of the adolescent mice and the sequence of light stimulation of the test mice were randomly assigned in a balanced manner. Social behavior was characterized by the exploration of the test mouse within a 2 cm radius, especially by sniffing or orienting its nose or head, around the inverted resin cup containing the adolescent mouse. Checking the empty inverted resin cup on the opposite side was considered non-social behavior. The complete experimental process was recorded by using tracking software and a camera system. After each experiment, the bottom of the three chambers and the resin cups were thoroughly cleaned with 70% ethanol.

[0042] 12. 90 minutes after the sociability test, mice were killed with a lethal dose of sodium pentobarbital and the brains were immunofluorescently stained. The mice were intracardially perfused with saline and 4% paraformaldehyde (PFA), and the brains were removed and postfixed overnight in 4% PFA. Afterwards, they were transferred to a 25-30% sucrose solution in PBS for 3 hours and cut into 30 μm thick coronal sections. The sections were washed with PBS-T (PBS containing 0.1% TritonX-100) and infiltrated with PBS containing 0.5% TritonX-100 for 20-30 minutes.

[0043] 13. Use QuickBlock TM Blocking buffer (P0260, Beyotime) was used for blocking. Polyclonal rabbit c-Fos antibody (1:200; catalog number sc-52, Santa Cruz) or monoclonal mouse 6E10 antibody (1:300; catalog number 803001, BioLegend) was then used for immunostaining in QuickBlock TM The sections were incubated in primary antibody dilution buffer (P0262) for 20 hours. Afterwards, the sections were washed with PBS-T (three times, 5 minutes each time) and then incubated with secondary antibodies (1:500, catalog number A-21206 or A-21202 Invitrogen) for 1 hour at 37°C. Finally, the sections were washed three times and counterstained with DAPI.

[0044] Results Analysis

[0045] There is a clear CTB signal in the aBLA region ( Figure 1 a), indicating a physical connection between the aBLA and vCAl. Notably, 5×FAD mice exhibited a higher number of c-Fos in the aBLA compared with WT mice. + and CTB + Neuron ( Figure 1 b), Demonstrating overactivation of the aBLA-VCA1 circuit in 5×PAD mice.

[0046] Adeno-associated virus AAV5-CaMK1a-eNpHR3.0-eYFP was introduced into the aBLA region (referred to as NpHR-5×FAD mice, as Figure 1 c). As a control group, we administered DAAV5-CaMKla eYFP (eYFP-5×FAD mice). After a 4-week viral expression period, expression in aBLA neurons was confirmed by green fluorescence (eYFP) signals in the aBLA ( Figure 1 d). Optical fibers were then implanted bilaterally above the vCAl region in NpHR-5×FAD and eYFP-5×FAD mice. Subsequently, the mice were allowed a one-week recovery period after the surgical procedure. Behaviorally, in the three-chamber sociability test, there was no difference in the time spent in the social zone between NpHR-5×FAD and eYFP-5×FAD mice during the light trial. NpHR-5×FAD mice spent more time in the social zone during the light period compared to eYFP-5×FAD mice ( Figure 1 e, f). In addition, NpHR-5×FAD mice showed higher social / non-sociality during the light period, indicating that they spent more time exploring the social area during light stimulation ( Figure 1 g). In contrast, the social / non-social ratio of YFP-5×xFAD mice did not change significantly between the lights-on and lights-off periods ( Figure 1 h). In addition, we analyzed the locomotor activity during the two periods and found no difference in the distance traveled between the two groups during the lights-on period and the lights-off period ( Figure 1 i).

[0047] To determine the necessity of the aBLA-vCA1 circuit in ameliorating the effects of UA on Aβ-impaired sociability, we injected AAV5.CaMKla-hChR2(H134R)-eYFP into the aBLA region of 5xFAD mice with UA. The control group received an AAV5-CaMKIa-eYFPA injection 4 weeks after viral expression, and an optical fiber was implanted above the vCAl region. Then, mice were given a one-week recovery period after surgery. A three-chamber sociability test was performed to assess social behavior. During a 10-min blue light activation period ( Figure 2 a), Social interaction time of UA group ( Figure 2 b) and social / non-social ratio ( Figure 2 c) was significantly reduced during the light period. In addition, there was no statistically significant difference in the distance traveled between the two groups, suggesting that their motor abilities remained unaffected ( Figure 2 d). These findings suggest that UA improves Aβ-impaired sociability in mice via the aBLA-vCA1 circuit.

[0048] in conclusion

[0049] 1. Urolithin A directly acts on the aBLA-vCA1 circuit which is closely related to anxiety behavior, reduces the toxic effects of Aβ, and thus effectively alleviates anxiety, depression and other emotional disorders in Alzheimer's patients.

[0050] 2. Reducing Aβ deposition and improving mitochondrial function are essential for neuronal function and survival.

[0051] 3. Through the mitochondrial autophagy function of urolithin A, Aβ is targeted for removal, the damaged basolateral amygdala and hippocampus circuits are repaired, the patient's anxiety symptoms are effectively relieved, and a new strategy is provided for the treatment of AD.

[0052] The embodiments described above are only preferred specific implementation modes of the present invention, and the protection scope of the present invention is not limited thereto. Any simple changes or equivalent replacements of the technical solutions that can be obviously obtained by any technician familiar with the field within the technical scope disclosed in the present invention belong to the protection scope of the present invention.

Claims

1. The use of urolithin A in the preparation of a drug for treating / alleviating Alzheimer's disease, characterized in that: Urolithin A activates mitochondrial autophagy, targets and clears Aβ, and repairs the damaged aBLA-vCA1 circuit.

2. A drug for treating / alleviating Alzheimer's disease comprising urolithin A, characterized in that: The medicine comprises a therapeutically effective amount of urolithin A and a pharmaceutically acceptable carrier.

3. The use according to claim 1 or 2, characterized in that: Alzheimer's disease is a symptom of social anxiety induced by Aβ.

4. A mitophagy activator comprising urolithin A, characterized in that: The activator comprises urolithin A and a pharmaceutically acceptable carrier.

5. The use according to claim 2 or 4, characterized in that: The pharmaceutically acceptable carrier includes any one or more of aluminum oxide, aluminum stearate, lecithin, serum protein, phosphate, glycerol, sorbic acid, potassium sorbate, a partial glyceride mixture of saturated vegetable fatty acids, water, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, beeswax and lanolin.

6. The use according to claim 2 or 4, characterized in that: The medicine also includes at least one vitamin derivative or its salt selected from vitamin C derivatives, vitamin E derivatives, and vitamin P derivatives.

7. The use according to claim 6, characterized in that: The vitamin derivative or salt thereof is at least one vitamin derivative or salt thereof selected from ascorbic acid phosphate or salt thereof, fatty acid ester of ascorbic acid phosphate or salt thereof, tocopherol phosphate or salt thereof, and methyl hesperidin or salt thereof.

8. The use according to claim 2 or 4, characterized in that: The drug is in the form of tablets, capsules, powders, syrups, solutions, suspensions or aerosols.

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