Targeted NPTX1-Kv7.2 signal axis for inhibiting neuronal hyperexcitation and improving senescence-induced memory formation disorder

Abstract

The invention belongs to the technical field of neurobiology, and particularly relates to a targeted NPTX1-Kv7.2 signal axis for inhibiting neuronal hyperexcitation and improving memory impairment formed by aging. According to the invention, a scene fear memory mouse behavior model is adopted, experimental research is carried out by combining in-vitro brain slice patch clamp recording, single-molecule fluorescence in situ hybridization, immunohistochemistry and other methods, and the result shows that NPTX1 can be used as a cognitive disorder molecular marker, and neuronal hyperactivity caused by aging can be rescued by specifically supplementing NPTX1 in a DG brain region; the activation of DG neurons during scene fear experience is improved, and neuronal activation damage and dysmnesia caused by aging are improved; the retigabine serving as an agonist of the NPTX1 interaction protein Kv7.2 is injected in the intraperitoneal cavity, so that neuron activation damage and dysmnesia caused by senescence can also be effectively improved. The invention discloses the effect of the NPTX1-Kv7.2 signal axis in improving the memory impairment of an aging individual, and provides a new thought and target spot for treating the memory impairment caused by aging.

Description

Technical Field

[0001] This invention belongs to the field of neurobiology, specifically relating to gene overexpression and pharmacological methods to improve age-related memory impairment. Background Technology

[0002] Physiological aging is a spontaneous and irreversible process. Brain aging is often accompanied by a gradual decline in cognitive function, with its core characteristic being an impairment in the formation of episodic memory, commonly known as "memory loss." Various studies have shown that during memory activity, impaired activation of neurons in relevant brain regions and abnormal expression of functional synaptic proteins, leading to an imbalance in neural network excitation / inhibitory (E / I), may be one of the core mechanisms driving age-related memory impairment. Therefore, identifying age-related specific synaptic proteins and targeting their signaling pathways is of significant physiological and clinical importance for improving age-induced memory disorders.

[0003] Neuronal pentraxins (NPTXs) belong to the cell adhesion molecule (CAM) family and consist of NPTX1, NPTX2, and their receptor NPTXR, specifically expressed in excitatory neurons. Members of this family play important roles in mediating glutamate synaptic transmission and the energy balance (E / I) in neural networks. Furthermore, NPTXs are downregulated in the cerebrospinal fluid of patients with Alzheimer's disease (AD), schizophrenia, and frontotemporal dementia, and have been shown to serve as biomarkers for diagnosing cognitive impairment. NPTXs exhibit functional heterogeneity; compared to NPTX2, NPTX1 is highly expressed in the dentate gyrus (DG) of the hippocampus, a core brain region for memory regulation. NPTX1 can improve the signal-to-noise ratio in neural networks by promoting Kv7.2 potassium channel membrane expression, preventing neuronal overexcitation. These results suggest that the NPTX1-Kv7.2 signaling axis plays a crucial role in maintaining normal memory function. Therefore, clarifying the changes in NPTX1 expression in the DG brain region of aging mice and how these changes cause neural network dysfunction could provide a potential strategy for targeting NPTX1-related signaling pathways and specifically intervening in age-induced memory impairment. Summary of the Invention

[0004] The purpose of this invention is to provide the use of NPTX1 in the treatment of age-related memory impairment.

[0005] This invention first investigates the role of the NPTX1-Kv7.2 signaling axis in improving age-induced scene-based fear memory formation disorder through AAV gene delivery system and pharmacological intervention, providing a potential target for the treatment of age-related memory disorders.

[0006] This invention first constructs a mouse model of contextual fear memory (CFC) behavior and conducts experimental studies using methods such as patch-clamp recording of ex vivo brain slices, single-molecule fluorescence in situ hybridization (smFISH), and immunohistochemistry (IHC). Mice are placed in a fearful environment (Context A) and given a foot shock to associate the fear experience with the specific environment. The next day, a memory retrieval test is performed, in which the mice are placed back in the fearful environment without receiving the shock, and their memory ability is measured by the percentage of time spent in freeze.

[0007] This invention demonstrates in experiments that, compared to 3-year-old young mice, the expression of Nptx1 transcripts in the DG brain region of 24-year-old aged mice was significantly downregulated, accompanied by increased neuronal excitability, including increased action potential firing frequency and input impedance, as well as decreased base strength and half-width. Neuronal activation in aged mice with impaired neural network excitability during scene-based fear experiences leads to memory impairment.

[0008] The invention further demonstrates in experiments that using the AAV gene delivery system to specifically replenish NPTX1 in the DG brain region can salvage age-induced neuronal hyperexcitability, thereby increasing the activation of DG neurons during scene-based fear experiences and improving the memory impairment phenotype in aged mice.

[0009] The invention also showed in experiments that intraperitoneal (ip) injection of NPTX1 interacting protein Kv7.2 agonist half an hour before CFC can also successfully improve age-related DG neuron activation impairment and memory impairment.

[0010] The Kv7.2 agonist is, in particular, retigabine, but is not limited to, retigabine.

[0011] In this invention, the Kv7.2 agonist (retegabin) also includes any pharmaceutically acceptable salt, solvate, or prodrug formulation thereof.

[0012] In summary, the main contributions of this invention include:

[0013] As a biomarker for predicting neurodegenerative diseases, NPTX1 is downregulated in patients with mild cognitive impairment (MCI), with changes occurring long before the abnormal accumulation of β-amyloid (Aβ) and the hyperphosphorylation of Tau protein. This invention proposes for the first time that targeting NPTX1 can effectively prevent and improve cognitive impairment caused by natural aging, preventing further progression to Alzheimer's Disease (AD).

[0014] Retigabine, an agonist of the potassium channel Kv7.2, was initially used clinically as an antiepileptic drug. In recent years, with further research, it has been found that this type of agonist also shows promising potential in the treatment of depression. This invention reveals for the first time that, in aging mice with downregulated NPTX1 in the brain, retigabine can effectively prevent and improve memory impairment caused by natural aging by inhibiting abnormal neuronal excitability. Attached Figure Description

[0015] Figure 1 Illustration of downregulated Nptx1 expression in the DG brain region of aged mice, accompanied by increased neuronal excitability.

[0016] Figure 2 Illustration of scene-based fear memory impairment caused by impaired neuronal activation in the DG brain region of aged mice.

[0017] Figure 3 Illustration of neuronal hyperexcitability caused by aging to be reversed by NPTX1 replete in the DG brain region via AAV delivery system.

[0018] Figure 4 Illustration of how NPTX1 replenishment in the DG brain region via AAV delivery system can salvage age-related neuronal activation impairment and scene-based fear memory disorder.

[0019] Figure 5 Illustration of how intraperitoneal injection of retigabine improves age-related neuronal activation impairment and scene-related fear memory disorder.

[0020] Figure 6 This is a schematic diagram illustrating how the present invention targets the NPTX1-Kv7.2 signal axis to inhibit neuronal overexcitation and improve age-related memory formation disorders. Detailed Implementation

[0021] Experimental Materials and Methods

[0022] 1. Laboratory animals

[0023] Young male C57BL / 6J mice were purchased from a laboratory animal company in Shanghai, and older male C57 / BL6 mice were purchased from a biotechnology company in Jiangsu Province. They were 3 and 24 months old, respectively, at the time of the experiment. All mice were housed in a clean-grade IVC system. The temperature in the animal room was controlled at 22-26℃, and the humidity was maintained at 30%-60%. A 12-hour day / night cycle (8:00-20:00 night, 20:00-8:00 day) was used to simulate the circadian rhythm of mice. 3-5 same-sex mice were housed per cage, and the mice had free access to food and water. All animal husbandry and procedures during the experiment strictly adhered to laboratory animal management regulations.

[0024] 2. Single-molecule fluorescence in situ hybridization (smFISH)

[0025] Ultrathin sections of 10 μm were prepared using a Leica cryostat. The sections were carefully adhered to positively charged, detachable slides. After drying briefly at room temperature, the sections were dried at 60 °C for 30 minutes to ensure close adhesion between the brain slices and the slides. The brain slices were outlined with an oil-based hydrophobic pen. After the pen mark dried completely, 5-8 drops of H2O2 were added to the brain slices, completely covering them. The slices were treated at room temperature for 10 minutes to remove endogenous catalase. The slices were washed twice in distilled water to remove excess H2O2. The target retrieval solution from the Advanced Cell Diagnostics RNAscope® Multiplex Fluorescent Reagent Kit v2 was boiled, and the brain slices were immediately placed in the solution, maintaining a temperature of 99 °C for 3 minutes. The slices were then placed in anhydrous ethanol for 3 minutes and air-dried at room temperature. 5-8 drops of proteinase III were added to the brain slices, completely covering them, and incubated at 40 °C for 30 minutes. The slices were washed twice with 1× Washing buffer. Add the preheated Nptx1 probe (505421) and incubate at 40 °C for 2 hours to allow the probe to hybridize with the target RNA. Wash twice with 1× Washing buffer to remove excess probe. Amplify the signal stepwise using AMP1, AMP2, and AMP3 from the kit, labeling each channel with a different fluorescent dye. Finally, mount the slides with anti-quenching mounting medium. Acquire images using a Nikon A1 confocal microscope and perform subsequent analysis using Image-Pro Plus 6.0 software.

[0026] 3. Immunohistochemistry (IHC)

[0027] Mice were anesthetized with isoflurane gas and perfused with physiological saline and 4% paraformaldehyde (PFA) to remove their brains. The brains were fixed in 4% PFA at 4 °C for 4 hours, then transferred to 30% sucrose / PBS solution for dehydration for 3 days. Frozen sections were prepared into 30 μm thick brain slices, washed three times with PBS, and incubated overnight at 4 °C with primary antibody (anti-c-Fos, 1:1000). The next day, after rinsing with PBS, the slices were incubated with the fluorescently conjugated secondary antibody Alexa-555 at room temperature for 1.5 hours. Anti-quenching mounting medium was added to the brain slices, and coverslips were placed on top. Images were acquired using a Nikon A1 confocal microscope, and subsequent analysis was performed using Image-Pro Plus 6.0 software.

[0028] 4. Stereotactic AAV injection into the brain

[0029] After anesthetizing, the mice had their scalp hair removed and their heads fixed to the stereotaxic adapter. The scalp was cut open and disinfected with iodine. The anterior and posterior fontanelle heights were recorded, and the adapter height was adjusted to ensure they were the same. Then, using the anterior fontanelle as the origin, the corresponding brain region was injected with a virus at a rate of 0.1 μL / min and a volume of 0.3 μL. After injection, the injection was paused for approximately 5 minutes, and then the microinjector was slowly withdrawn upwards. The scalp was then sutured. Finally, the mice were placed in a clean cage and heated with an electric blanket to ensure they did not feel too cold post-surgery. They were allowed free access to food and water. Two days later, they were returned to their original independent ventilation system. Mice underwent at least 3 weeks of recovery before starting experiments. The DG brain region coordinates are as follows: AP -1.85 mm, ML ± 1.1 mm, DV – 2.1 mm.

[0030] 5. Electrophysiological recordings of brain slices

[0031] Mice were perfused with frozen sectioning solution, and 300 μm brain slices of the hippocampus were excised using a vibrating microtome in oxygen-saturated frozen sectioning solution. The slices were first incubated in sectioning solution at 32 °C for 12 minutes, then transferred to a brain slice incubation solution. Patch-clamp recording was performed in artificial cerebrospinal fluid using an EPC-10 amplifier and Pulse v8.78 software. Neuronal location, morphology, size, and fluorescence expression were recorded, with electrode resistance ranging from 5 to 7 MΩ. Action potentials were recorded using current-clamp mode, and membrane potentials were measured when intracellular currents were injected (duration 1000 ms, amplitude from -150 pA to 250 pA, step size 10 pA). 20 μM CNQX, 50 μM D-AP5, and 100 μM PTX were added to the artificial cerebrospinal fluid, and recording was performed using KCl electrode internal solution. All electrophysiological experimental signals were acquired at a sampling frequency of 10 kHz, followed by filtering at a cutoff frequency of 2 kHz. The measured series resistance was less than 30 megohms. Data analysis was performed by an uninformed participant using pCLAMP 10.7 software (Molecular Devices, San Jose, CA, USA).

[0032] 6. CFC Training

[0033] Before subjecting mice to conditioned fear stimuli, they were placed in an adaptation room for three days, for two hours each day, and the mice were allowed to become familiar with the experimenter's scent by being touched appropriately.

[0034] On the day of the conditioned fear stimulus (day 0), the mice were first placed in an adaptation room for 30 minutes. Then, they were placed sequentially into the conditioned fear training chamber (context A, a square plexiglass observation room with stainless steel bars around the edges, and a base through which an electric shock generator could be applied). The shock procedure was as follows: a total of 180 seconds, with a mild electric current stimulus of 0.3 mA for 1 second at the 120th second. The mice were removed from the conditioned fear chamber after 60 seconds. If an immunohistochemical experiment was to be performed subsequently, the brain was perfused and harvested 1 hour after the training ended. If a behavioral test was to be performed, the mice were placed back in environment A (without electric shock) for 3 minutes of free exploration the day after the training ended (day 1).

[0035] The percentage of rigidity in mice was analyzed using Med-Associates software, and rigidity was defined as a mouse remaining motionless for more than 1 second.

[0036] 7. Open Field Test (OFT)

[0037] Before the test, the mice were placed in an acclimatization room for half an hour. After acclimatization, the mice were placed in an open field testing box (40×40×40 cm). 3 The mice were placed in the center of the test chamber for 20 minutes of free exploration under 15 lux light intensity. Behavioral data were recorded by a camera above the test chamber and analyzed using Etho Vision XT 8.5 software. The distance the mice traveled and the time spent in the central area were recorded; the central area was defined as half of the total area.

[0038] Example:

[0039] 1. Detection of Nptx1 expression and neuronal excitability changes in the DG brain region of aged mice.

[0040] smFISH analysis revealed that, compared with young mice, the expression of Nptx1 transcripts in the DG brain region of aged mice was significantly downregulated. Figure 1 A-1B, Two-tailed unpaired t-test, t = 5.681, P < 0.001). Brain slice action potential recording using current-clamp mode revealed that aging caused increased excitability of DG neurons. Figure 1 C-1M) includes an increase in action potential firing frequency (1D, Two-way RM ANOVA, P < 0.001), input impedance (1J, Two-tailed unpaired t-test, t = 2.435, P = 0.020), and a decrease in base strength (1E, Two-tailed unpaired t-test, t = 3.340, P = 0.002) and half-width (1H, Two-tailed unpaired t-test, t = 3.993, P < 0.001).

[0041] 2. Neuronal activation impairment in the DG brain region of aged mice leads to scene-based fear memory impairment.

[0042] To investigate whether NPTX1 downregulation-induced neuronal hyperexcitability affects DG neuron activation and thus impairs memory in aged mice, we used a CFC model (0.3 mA, 1 s, 1 shock) to conduct behavioral training on young and aged mice. Figure 2 A) Brain samples were perfused one hour after training ended for c-Fos staining, or memory retrieval tests were performed the day after training ended. Results showed reduced neuronal activation in aged mice ( Figure 2B-2C (Two-tailed unpaired t-test, t = 4.008, P = 0.005), decreased freezing levels indicate impaired memory. Figure 2 D, two-tailedunpaired t-test, t = 2.603, P = 0.019).

[0043] 3. AAV delivery of NPTX1 reverses senescence-induced neuronal hyperexcitability

[0044] To investigate whether reintroducing NPTX1 in aged mice could salvage age-induced neuronal hyperexcitability, we used the AAV gene delivery system to specifically introduce the Nptx1 gene sequence into the DG brain region of aged mice. Current-clamp recordings were performed 21 days after viral expression. Figure 3 A). The results showed that NPTX1 reinjection in the DG brain region successfully reversed neuronal hyperexcitability in aged mice. Figure 3 B-3L) includes a decrease in action potential firing frequency (3C, Two-way RM ANOVA, P = 0.003), a decrease in input impedance (3I, Two-tailed unpaired t-test, t = 2.435, P = 0.031), and an increase in base strength (3D, Two-tailed unpaired t-test, t = 3.340, P < 0.001).

[0045] 4. AAV delivery of NPTX1 improves age-related neuronal activation impairment and scene-related fear memory formation disorders.

[0046] We then investigated the effects of NPTX1 reinjection in the brains of aged mice on neuronal activation and memory formation. Figure 4 A). c-Fos staining analysis, a neuronal activation marker, showed that overexpression of NPTX1 in the DG nucleus of aged mice increased neuronal activation. Figure 4 B-4C, Two-tailed unpaired t-test, t = 4.747, P = 0.005). CFC memory retrieval test results showed that overexpression of NPTX1 increased freezing levels in aged mice, indicating enhanced memory formation ability. Figure 4F, Two-tailed unpaired t-test, t = 2.253, P = 0.040). Open field tests in mice revealed that overexpression of NPTX1 in the brain did not affect motor function (4G, Two-tailed unpaired t-test, t = 0.364, P = 0.720; 4H, Two-tailed unpaired t-test, t = 0.204, P = 0.840), indicating that changes in freezing levels were not caused by alterations in mouse activity.

[0047] 5. Intraperitoneal injection of retegabin improves age-related neuronal activation impairment and scene-related fear memory formation disorders.

[0048] The ability of NPTX1 to inhibit neuronal hyperexcitability depends on the regulation of the membrane expression of its interacting protein, Kv7.2. Retigabine has been used clinically as an antiepileptic drug. To investigate whether activating Kv7.2 potassium channels could also reverse age-related neuronal activation impairment and scene-based fear memory disorder, we administered Retigabine intraperitoneally half an hour before CFC training. One hour after training, brain tissue was perfused and harvested, followed by c-Fos staining to detect neuronal activation. Figure 5 A-5B). The results showed that pharmacological activation of the NPTX1 interacting protein Kv7.2 also enhanced DG neuron activation (A-5B). Figure 5 C, Two-tailed unpaired t-test, t = 2.727, P = 0.030). Consistent with NPTX1 overexpression, CFC behavioral tests also showed that pharmacological activation of Kv7.2 improved memory formation impairment in aged mice. Figure 5 E, Two-tailed unpaired t-test, t = 3.364, P = 0.002. However, intraperitoneal injection of retigabine did not affect the motor function of mice (5F, Two-tailed unpaired t-test, t = 0.525, P = 0.604; 5G, Two-tailed unpaired t-test, t = 0.937, P = 0.357).

[0049] In summary, the use of the AAV gene delivery system to specifically replenish NPTX1 in the DG brain region or the use of pharmacological methods to stimulate its interacting protein Kv7.2 can successfully improve age-related neuronal activation impairment and thus improve memory impairment. This suggests that targeting the NPTX1-Kv7.2 signaling axis has important clinical significance and good application prospects for the treatment of age-induced memory impairment.

Claims

1. Use of a biomarker NPTX1 in the manufacture of a medicament for treating memory impairment caused by aging.

2. Use according to claim 1, characterized in that, The NPTX1 improves the formation of contextual fear memory caused by aging through Kv7.2 protein.

3. Use according to claim 1, characterized in that, The formation of contextual fear memory caused by impaired neuronal activation and impaired formation is improved by supplementing NPTX1.

4. Use according to claim 2, characterized in that, The medicament comprises a Kv7.2 protein agonist.

5. Use according to claim 4, characterized in that, The Kv7.2 protein agonist is retigabine, or any formulation form of a pharmaceutically acceptable salt, solvate or prodrug thereof.

6. Use according to one of claims 1 to 5, characterized in that, The medicament is in the form of an injection by intraperitoneal injection.

Images