Application of targeted complement C4 in preparation of drug for treating autism

Abstract

The invention discloses an application of a targeted complement C4 in preparation of a medicine for treating autism. Through preparation of a Setdb1 gene defect mouse model, the research finds that the model mouse shows social disorders similar to human autism, reveals the association between complement C4 abnormal activation and autism occurrence, and points out a potential pathogenesis. Further research shows that abnormal microglial cell activation and excessive synaptic pruning caused by C4 can be improved by knocking out the C4 gene, so that electroneurographic signal transmission and social disorders related to autism are improved. In addition, in an autism animal model induced by valproic acid (VPA), after C4 is knocked out, the C4-microcolloid pathway can be improved, and the self-closing behavior can be relieved. The invention provides important experimental and theoretical basis for research and development and clinical treatment of autism treatment medicines, and has remarkable clinical application and medicine research and development values.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of a complementary C4-targeting agent in the preparation of a drug for treating autism. Background Technology

[0002] Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder characterized by impaired social communication, repetitive and stereotyped behaviors, and restricted interests. The etiology of ASD is extremely complex, involving multiple interactions of genetic, epigenetic, immune, and environmental factors. Recent research has suggested that the pathological mechanisms of ASD may be related to abnormal synaptic pruning, inappropriate neural network connectivity, and abnormal responses of the immune system during brain development.

[0003] Specifically, certain components of the complement system, such as complement protein C4, are believed to play important roles in neurodevelopment. The complement system not only functions significantly in humoral immunity and pathogen clearance, but there is also evidence that it plays a crucial role in synaptic pruning in the brain. Abnormal complement activation can lead to excessive or insufficient synaptic pruning, which is considered a potential pathological mechanism in neurodevelopmental disorders such as ASD.

[0004] Furthermore, genetic studies have shown that certain gene variants are associated with an increased risk of ASD. Among them, the expression level and variant type of the complement C4 gene are associated with brain imaging and clinical manifestations in individuals with ASD.

[0005] Current treatment for ASD primarily focuses on behavioral interventions, and there are no specific drugs that can improve the core symptoms of ASD from a pathophysiological perspective. Therefore, exploring and developing novel treatment strategies targeting the underlying molecular mechanisms of ASD, especially intervention methods based on the complement system, has become an important research direction. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing an application of targeting complement C4 in the preparation of drugs for treating autism. By targeting C4, the autism-like pathological phenotype of transgenic mice with autism is improved, providing a new molecular target for the prevention and treatment of autism.

[0007] To achieve the above objectives, the present invention provides an application of targeting complement C4 in the preparation of a drug for treating autism.

[0008] Furthermore, the active ingredient of the drug is a reagent that specifically inhibits the expression or activity of the C4 gene or protein.

[0009] Furthermore, the reagent is an antibody that specifically inhibits C4 gene expression, such as RNAi, microRNA, shRNA, siRNA, or C4b protein, or an inhibitor of C4 gene or protein activity, or a plasmid vector or adeno-associated virus vector containing the RNAi, microRNA, shRNA, or siRNA that specifically inhibits C4 gene expression.

[0010] The present invention also provides a drug for treating autism, wherein the active ingredient of the drug is a reagent that specifically inhibits the expression or activity of the C4 gene or protein.

[0011] Furthermore, the reagent is an antibody that specifically inhibits C4 gene expression, such as RNAi, microRNA, shRNA, siRNA, or C4b protein, or an inhibitor of C4 gene or protein activity, or a plasmid vector or adeno-associated virus vector containing the RNAi, microRNA, shRNA, or siRNA that specifically inhibits C4 gene expression.

[0012] Furthermore, the drug is used to improve social interaction disorders, repetitive stereotyped behaviors, abnormal neuronal discharges, and / or abnormal synaptic plasticity.

[0013] Furthermore, the therapeutic effect is achieved through at least one of the following biological mechanisms: reducing C4 levels in the prefrontal cortex; regulating the activation state of microglia and inhibiting neuroinflammatory responses; and restoring the balance of excitatory / inhibitory synaptic currents in the neuronal network.

[0014] Furthermore, it also contains combination therapy ingredients selected from one or more of nonsteroidal anti-inflammatory drugs, neurotrophic factors, and antiepileptic drugs.

[0015] Compared with existing technologies, this invention has the following beneficial effects: Experiments conducted in Setdb1 conditional knockdown (Het) or knockout (cKO) models and VPA-induced autism mouse models showed that C4 was significantly increased in these models. C4 gene knockout significantly inhibited the abnormal microglial activation of pruning synapses caused by abnormal C4 upregulation, while significantly improving synaptic structure and function in the mouse models. Furthermore, C4 gene knockout also significantly improved repetitive and social behaviors in mice. Therefore, this invention provides a potential drug target and research direction for the prevention and treatment of ASD. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a diagram showing the results of C4 immunofluorescence staining.

[0018] Figure 2 The image shows the results of IBA1 / CD68 / PSD95 immunofluorescence staining.

[0019] Figure 3 This is a diagram showing the results of Syn / PSD95 immunofluorescence staining.

[0020] Figure 4 This is a graph showing the results of an electrophysiological experiment.

[0021] Figure 5 The figure shows the results of the open field experiment.

[0022] Figure 6 This is a diagram showing the results of a three-box social experiment.

[0023] Figure 7 For Ctrl, VPA and VPA-C4 - / - Figures showing the results of immunofluorescence staining, electrophysiological, open field, and three-box social experiments for mice in each group. Detailed Implementation

[0024] The present invention will now be described in detail with reference to the accompanying drawings. Unless otherwise specified, the features of the following embodiments and implementations can be combined with each other.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified; the WT mice used in the following examples were purchased from Shanghai SLAC Laboratory Animal Co., Ltd., the hGFAP-Cre mice were from Jackson Laboratory, and the Setdb1 mice were from the Jackson Laboratory. flox / flox The mice were from Jiang Yan's laboratory, C4 - / - The mice were from Hu Weiguo's laboratory, and the animal experiments have been approved by the Ethics Committee of Zhejiang University. Example

[0026] (I) Experimental Methods: 1. Construction of autism mouse models and autism C4 knockout or knockdown mouse models Through hybridization Setdb1 flox / flox Or Setdb1 flox / +Conditional knockout (cKO) or knockdown (Het) mouse strains were constructed using mice and hGFAP-Cre mice. Control mice (Ctrl) were same-sex littermates without the Cre transgene. In constructing the VPA autism model, wild-type C57BL / 6 (WT) mice were intraperitoneally injected with VPA on day 12.5 of gestation, while WT mice injected with saline served as controls (Ctrl). In constructing the reverse C4 knockout mouse model, cKO or Het was further combined with C4... - / - cKO-C4 was obtained through hybridization - / - and Het-C4 - / - C4 - / - Mice obtained VPA-C4 by intraperitoneal injection of VPA on day 12.5 of gestation. - / - Mice. As for the C4 knockdown mouse model, cKO-shSCR and cKO-shC4 mice were obtained by injecting AAV-hSYN-shSCR or AAV-hSYN-shC4 into the brains of P7 cKO mice.

[0027] 2. Immunofluorescence staining and statistical analysis First, set Ctrl, Het, cKO, VPA, and C4. - / - Het-C4 - / - cKO-C4 - / - VPA-C4 - / - Frozen sections of brain tissue from cKO-shSCR and cKO-shC4 models were baked in a 55°C oven for 1 hour. Subsequently, the sections were washed three times with PBS for 5 minutes each time. Afterward, the sections were blocked with blocking buffer for 30 minutes. After removing the blocking buffer, primary antibodies of IBA1 / CD68 / PSD95 or PSD95 / Syn or C4 were added, and the sections were incubated overnight at 4°C. The next day, the sections were washed three more times with PBS for 5 minutes each time. Secondary antibodies were then added and the sections were incubated at room temperature for 1 hour, followed by three more washes with PBS. After washing, the sections were mounted using mounting medium.

[0028] Image acquisition was performed using a confocal laser microscope (LEICA SP8 or Olympus FV3000). For statistical analysis of microglia, the signals of IBA1, CD68, and PSD95 were reconstructed and counted using the Surface function in Imaris software. Statistical analysis was then performed to obtain relevant data.

[0029] 3. Open field experiment Motor abilities, including movement and spontaneous activity, were assessed by observing the behavior of mice freely exploring an open area (50 cm wide × 50 cm long × 40 cm high). The time spent in the central area by two-month-old mice during the 30-minute experiment was recorded using a video tracking system (Smart 3.0).

[0030] 4. The Three-Box Social Experiment Autism-like behaviors were assessed using a three-box setup of equal size. In the social behavior test, two empty cages were placed in the side boxes. An age- and sex-matched unfamiliar mouse (Unknown Mouse 1, S1) was placed in one cage, while a novel object was placed in the other. The mice were allowed to freely explore the three boxes for 10 minutes. Sniffing time on S1 or the novel object was recorded using behavioral software (Smart 3.0).

[0031] 5. Electrophysiological experiments Mice of different genotypes and underwent treatments until 2.5 months of age. After anesthesia with isoflurane, they were rapidly decapitated. The brains were immediately removed and placed in a solution containing ice-cold anatomical artificial cerebrospinal fluid (ACSF), composed of 64 mM NaCl, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 0.5 mM CaCl₂, 10 mM MgSO₄, 26 mM NaHCO₃, 10 mM D-glucose, and 120 mM sucrose (pH 7.4), with an osmotic pressure of approximately 310 mOsm, under conditions of 95% O₂ and 5% CO₂. Brain tissue was sectioned into 400 μm thick sections using a Leica VT1200S vibratory microtome. Sections were incubated in ACSF containing 120 mM NaCl, 3.5 mM KCl, 1.25 mM NaH₂PO₄, 1.3 mM MgSO₄, 2.5 mM CaCl₂, 10 mM D-glucose, and 26 mM NaHCO₃ (pH 7.4, approximately 300 mOsm) at 32 °C for 30 min under 95% O₂ and 5% CO₂ conditions, followed by a 30 min recovery period at room temperature. For recording, sections were transferred to an immersion recording chamber and incubated at 2–3 ml / min. –1 The rate of synaptic currents was measured using ACSF perfusion. Spontaneous excitatory postsynaptic currents (sEPSCs) and spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded from the prefrontal cortex using ACSF-filled extracellular glass microelectrodes (4–6 MΩ). Synaptic current data were analyzed using MiniAnalysis software.

[0032] 6. Statistical Analysis All statistical analyses with specific sample sizes are shown in the corresponding graphs, with each point representing a biological replicate (neurons for electrophysiology, mice for others). Normality was tested using the Shapiro-Wilk test. Homogeneity of variance was tested using the Levene test. Data meeting both criteria were analyzed using one-way ANOVA. Figure 1 and Figure 7 The analysis was performed using a two-way ANOVA with Bonferroni correction. Figures 2-6 All statistical analyses were performed using GraphPadPrism version 9.5, and results are expressed as mean ± standard error (SEM). The null hypothesis was rejected when the p-value was greater than or equal to 0.05. The symbols *, **, ***, ****, and ns represent p<0.05, p<0.01, p<0.001, p<0.0001, and p>0.05, respectively.

[0033] Experimental results 1. C4 immunofluorescence staining was used to detect C4 expression in the prefrontal cortex (PFC) of mice in the Ctrl, Het, and cKO groups after one month. The results showed that C4 expression was significantly increased in Het and cKO mice compared to Ctrl mice, indicating that Setdb1 gene deficiency leads to abnormal upregulation of C4 expression in the PFC region. Figure 1 As shown in ab.

[0034] 2. IBA1 / CD68 / PSD95 immunofluorescence staining to detect Ctrl, Het, cKO, and C4 levels after 21 days. - / - Het-C4 - / - cKO-C4 - / - The study also measured the volume of microglia in the PFC region of mice in the cKO-shSCR and cKO-shC4 groups after 37 days, the volume of CD68 within microglia, and the volume of PSD95 signal phagocytosed by each microglia. The results showed that microglia in Het and cKO mice after C4 knockout returned to normal, with reduced phagocytic synapses, similar to Ctrl mice. This indicates that C4 knockout can completely restore the abnormally activated and over-pruned synapses in the microglia of Het and cKO mice. Figure 2 As shown in the ad section. Simultaneously, knocking down C4 using shRNA constructed via AAV also improves microglia phagocytic synapses, as... Figure 2 As shown in ei.

[0035] 3. Syn / PSD95 immunofluorescence staining was used to detect Ctrl, Het, cKO, and C4 levels after 21 days. - / - Het-C4 - / - and cKO-C4 - / -The number of synapses in the PFC region of mice in each group. The experimental results showed that the number of synapses in Het and cKO mice after C4 knockout returned to normal, no different from that in Ctrl mice. This indicates that C4 knockout can completely restore the synapse reduction caused by abnormal microglial activation and over-pruning of synapses in Het and cKO mice. Figure 3 As shown in ab.

[0036] 4. Electrophysiological testing of Ctrl, Het, cKO, and C4 over 2.5 months. - / - Het-C4 - / - and cKO-C4 - / - sEPSCs and sIPSCs in the PFC region of mice in each group. Experimental results showed that, compared with the control group, the frequencies of sEPSCs and sIPSCs in Het and cKO mice were significantly reduced, and these frequencies were completely restored after C4 knockout. Figure 4 As shown in ac.

[0037] 5. The open field test was used to assess repetitive behaviors in experimental animals. Results showed that, compared to the control group, Het and cKO mice spent significantly less time in the center and exhibited more pronounced field-circling behavior. C4 knockout completely restored these behaviors. Figure 5 ).

[0038] 6. The three-box socialization test is used to assess the social abilities of experimental animals. Normal mice prefer to stay in boxes containing other mice for socialization. The results showed that, compared to the control group, Het and cKO mice showed no significant difference between mouse and object boxes, indicating no interest in mouse socialization. C4 knockout completely restored this behavior, and like Ctrl mice, they preferred the mouse box side. Figure 6 ).

[0039] 7. For Ctrl, VPA, and VPA-C4 - / - Mice in each group underwent IBA1 / CD68 / PSD95 and Syn / PSD95 immunofluorescence staining, electrophysiological testing, open field tests, and three-box social interaction tests. The results showed that in C4 knockout VPA mice, microglia returned to normal, phagocytic synapses decreased, synapse number returned to normal, sEPSCs and sIPSCs frequencies returned to normal, stereotyped repetitions were restored, and social interaction also returned to normal, all indistinguishable from Ctrl mice. This indicates that C4 knockout can completely restore abnormal neuronal firing caused by abnormal microglial activation and over-pruned synapses in VPA mice and improve autistic-like behavior. Figure 7 As shown in am.

[0040] Based on the above experimental results, inhibiting the expression or activity of the C4 gene or protein can improve synapse formation in autistic mouse models, enhance neuronal plasticity and functional connectivity, improve autism-like behavior in mice, and alleviate core autism symptoms such as social communication impairment and repetitive stereotyped behaviors.

[0041] In Example 1, AAV-hSYN-shSCR or AAV-hSYN-shC4 was injected as an example to inhibit the expression or activity of the C4 gene or protein. Those skilled in the art should understand that the reagents can be RNAi, microRNA, shRNA, siRNA, antibodies against C4b protein that specifically inhibit C4 gene expression, inhibitors of C4 gene or protein activity, or plasmid vectors or adeno-associated virus vectors containing the aforementioned RNAi, microRNA, shRNA, or siRNA that specifically inhibits C4 gene expression. Repeating the above experiments will yield the same experimental results, which will not be elaborated here.

[0042] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

[0043] The above embodiments are only used to illustrate the design concept and features of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. The protection scope of the present invention is not limited to the above embodiments. Therefore, all equivalent changes or modifications made based on the principles and design ideas disclosed in the present invention are within the protection scope of the present invention.

Claims

1. Application of targeting complement C4 in the preparation of drugs for the treatment of autism.

2. The application as described in claim 1, characterized in that, The active ingredient of the drug is a reagent that specifically inhibits the expression or activity of the C4 gene or protein.

3. The application as described in claim 2, characterized in that, The reagent is an antibody that specifically inhibits C4 gene expression, such as RNAi, microRNA, shRNA, siRNA, or C4b protein, or an inhibitor of C4 gene or protein activity, or a plasmid vector or adeno-associated virus vector containing the RNAi, microRNA, shRNA, or siRNA that specifically inhibits C4 gene expression.

4. A drug for treating autism, characterized in that, The active ingredient of the drug is a reagent that specifically inhibits the expression or activity of the C4 gene or protein.

5. The drug as described in claim 4, characterized in that, The reagent is an antibody that specifically inhibits C4 gene expression, such as RNAi, microRNA, shRNA, siRNA, or C4b protein, or an inhibitor of C4 gene or protein activity, or a plasmid vector or adeno-associated virus vector containing the RNAi, microRNA, shRNA, or siRNA that specifically inhibits C4 gene expression.

6. The drug as described in claim 4, characterized in that, The drug is used to improve social interaction disorders, repetitive stereotyped behaviors, abnormal neuronal discharges, and / or abnormal synaptic plasticity.

7. The drug as described in claim 4, characterized in that, The therapeutic effect is achieved through at least one of the following biological mechanisms: reducing C4 levels in the prefrontal cortex; regulating the activation state of microglia and inhibiting neuroinflammatory responses; and restoring the balance of excitatory / inhibitory synaptic currents in the neuronal network.

8. The drug according to claim 4, characterized in that, It also contains combination therapy ingredients selected from one or more of nonsteroidal anti-inflammatory drugs, neurotrophic factors, and antiepileptic drugs.

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