Application of cGAS as target in prevention / treatment of Leigh syndrome

Abstract

The invention discloses an application of cGAS as a target in prevention / treatment of Leigh syndrome. The method comprises the following steps: constructing NDufs4; a cGAS DKO gene knockout mouse model is researched, and the result shows that by knocking out cGAS, the glial hyperplasia phenomenon weakening of the mouse with the Like's syndrome can be improved, and neuroinflammation is inhibited. The invention provides a new potential drug target and a treatment strategy for treating the Like's syndrome, and lays a foundation for researching and developing a new drug for treating the Like's syndrome.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of cGAS as a target in the prevention / treatment of Leigh syndrome. Background Technology

[0002] Leigh syndrome (LS), also known as subacute necrotizing encephalopathy or Leigh's encephalopathy, is a hereditary mitochondrial disorder that begins in infancy. It is a fatal neurodegenerative disease caused by mitochondrial dysfunction, characterized by abnormal energy metabolism, progressive neuronal death, abnormally elevated levels of reactive oxygen species (ROS), and mutations in mitochondrial DNA (mtDNA). Recent studies have found that mitochondrial damage may activate innate intracellular immune pathways (such as the cGAS-STING signaling axis) through abnormal mtDNA release, thereby driving an inflammatory phenotypic transformation between microglia and astrocytes, creating a pro-inflammatory microenvironment, and exacerbating neurodegenerative diseases. However, in Leigh syndrome, whether mitochondrial dysfunction or oxidative stress directly leads to cytoplasmic leakage of mtDNA, and how such leakage events participate in disease development through neuroinflammatory pathways, remains unclear.

[0003] Regarding the pathological mechanisms of Leigh syndrome (LS), current research reports indicate a significant increase in Iba1+ cells, characterized by microglial activation, in multiple brain regions of Leigh syndrome patients, and this increase is associated with neuronal loss. Currently, the commonly used preclinical model for Leigh syndrome is the Ndufs4 mutant mouse. The Ndufs4 gene encodes a subunit of mitochondrial complex I, whose role in humans is primarily related to cellular energy metabolism, as it participates in the mitochondrial electron transport chain (ETC), a process crucial for the production of the energy molecule adenosine triphosphate (ATP). Ndufs4-mutant iPS-brain organoids exhibit activated inflammatory pathways, and partial microglial ablation in Leigh syndrome mouse models improves survival rates and alleviates neurological deficits. Reactive oxygen species (ROS) and neuronal mitochondrial dysfunction are widely considered key factors in neurodegenerative diseases, and studies have shown that ROS and neuronal mitochondrial dysfunction lead to the accumulation of lipid droplets (LD) in glial cells. In a Drosophila model, ROS activates c-Jun-N-terminal kinase (JNK) and sterol regulatory element-binding protein (SREBP), triggering the accumulation of lipid precipitates (LD) in glial cells. This process occurs in the early or initial stages of neurodegeneration, and the accumulated lipids undergo lipid peroxidation under the influence of ROS. Targeting lipase overexpression or reducing ROS levels to decrease LD accumulation and lipid peroxidation in glial cells has been shown to significantly delay the progression of neurodegenerative diseases. In a mouse model with Ndufs4 gene mutations, similar glial LD ​​accumulation to that observed in Drosophila has been observed, suggesting that LD accumulation following mitochondrial dysfunction is an evolutionarily conserved phenomenon and may serve as a transient marker and contributing factor in the early stages of neurodegenerative diseases. Primary mitochondrial dysfunction is the underlying cause of Leigh syndrome, and related genes are primarily expressed in neurons. In Leigh syndrome (LS), central nervous system involvement is crucial, leading to motor and respiratory dysfunction, seizures, and premature death. However, only specific neuronal populations are affected. Studies have reported that inactivation of Ndufs4 in glutamatergic neurons expressing Vglut2 leads to reduced neuronal firing, brainstem inflammation, motor and respiratory dysfunction, and premature death. In contrast, deletion of Ndufs4 in GABAergic neurons causes basal ganglia inflammation without motor or respiratory involvement, but is accompanied by hypothermia and severe pre-death seizures.

[0004] Currently, there is no treatment for LS (Leukemia Spectroradiopathy), but reports indicate that administration of rapamycin to Ndufs4 mutant mice can delay the onset of neurological symptoms, reduce neuroinflammation, and prevent brain damage. Downregulation of protein kinase C (PKC) has been identified as a key factor in the therapeutic effect of rapamycin on Ndufs4 mutant mice. The use of PKC inhibitors can increase the survival rate of Ndufs4 mutant mice, delay neurological function impairment, and reduce inflammation. Acarbose, a drug that prolongs the lifespan of mice and delays normal aging, can also suppress disease symptoms and increase Ndufs4 levels. - / - Mouse survival rate. Unlike rapamycin, acarbose can restore the disease phenotype independently of the inhibition of rapamycin's mechanism target. Furthermore, the co-administration of rapamycin and acarbose further enhances Ndufs4. - / - The study also reported that prolonged hypoxic respiration can prevent or even reverse neurodegenerative diseases in Ndufs4 gene knockout (KO) mouse models. Inhalation of carbon monoxide (approximately 600 ppm in the air) reduces arterial oxygen saturation and decreases oxygen pressure in brain tissue by binding to hemoglobin, thus reducing Ndufs4... KO Prolonged survival in mice. Anemia induced by exsanguination and iron-deficiency diets reduces the number of circulating red blood cells, decreases oxygen delivery, and reduces oxygen pressure in brain tissue, thus prolonging the survival of model mice. Using the AAV-PHP.B vector, which can cross the blood-brain barrier, to restore gene expression in Ndufs4 gene knockout mouse models via systemic administration also prolonged the survival of model mice, restored metabolic parameters, improved behavioral phenotypes, and pathological phenotypes in the brain, retina, and heart.

[0005] cyclic GMP-AMP synthase (cGAS) is a key intracellular DNA sensor that plays a central role in the innate immune system. It specifically recognizes double-stranded DNA (dsDNA) located in the cytoplasm, which typically originates from genomic instability caused by viral infection, bacterial invasion, or cell damage. When cGAS binds to dsDNA, it catalyzes the synthesis of a non-classical cyclic dinucleotide—2'3'-cyclic GMP-AMP (cGAMP)—from ATP and GTP. This molecule acts as a second messenger, activating downstream STING (stimulator of interferon genes) proteins, triggering a series of signaling cascades that ultimately induce the expression of type I interferons and other pro-inflammatory cytokines, initiating antiviral and antitumor immune responses. Currently, there are no reported studies linking cGAS to Riemann syndrome. Summary of the Invention

[0006] The purpose of this invention is to address the above-mentioned problems by providing an application of cGAS as a target in the prevention / treatment of Leigh syndrome.

[0007] To achieve its objective, the present invention employs the following technical solution:

[0008] A first aspect of the present invention provides the use of cGAS as a target in screening drugs for the prevention of Leigh syndrome.

[0009] A second aspect of the invention provides the use of cGAS as a target in screening drugs for the treatment of Leigh syndrome.

[0010] Wherein, cGAS is a cyclic GMP-AMP synthase or its encoding gene.

[0011] In the above application technology solution, the drug inhibits cGAS expression.

[0012] In the above-mentioned application technology solution, the drug improves gliosis in patients with Leishman's syndrome.

[0013] In the above application technology solution, the drug inhibits the abnormal activation of the cGAS-STING pathway.

[0014] In the above-mentioned application technology solution, the drug inhibits neuroinflammation.

[0015] The beneficial effects of this invention are as follows: Research using an Ndufs4;cGAS DKO gene knockout mouse model showed that cGAS knockout can reduce glial proliferation and suppress neuroinflammation in Lechmann syndrome mice. This invention provides a novel and promising drug target and treatment strategy for Lechmann syndrome, laying the foundation for the development of new drugs to treat Lechmann syndrome. Attached Figure Description

[0016] Figure 1 This is an immunofluorescence staining image of mouse brain slices for GFAP and Iba1.

[0017] Figure 2 These are the results of weight measurements of the experimental mice.

[0018] Figure 3 These are the results of cGAMP level detection in laboratory mice.

[0019] Figure 4 This is the result of detecting the mRNA expression level of a key regulatory factor in the type I interferon signaling pathway. Detailed Implementation

[0020] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0021] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0022] Main reagent sources:

[0023]

[0024] The primer sequences for PCR detection are as follows:

[0025]

[0026] Example 1

[0027] I. Laboratory Mice

[0028] Ndufs4 knockout mice: These mice were constructed by knocking out the Ndufs4 gene in C57BL / 6J background mice. The mice were purchased from The Jackson Laboratory (Bartholomew's Harbor, Maine, USA).

[0029] DKO mice: Ndufs4 + / - Mice and cGAS - / - Mice were crossbred in the C57BL / 6J background to obtain a double gene knockout (DKO) model. Ndufs4 & cGAS double knockout mutant mice (C57BL / 6) were housed in SPF-grade feed, five mice per cage. They were fed a standard diet (21% protein, 4.5% fat, 4% fiber, 1.404 kcal / g), with no restrictions on water or food intake. The ambient temperature was maintained at a constant 22℃, with a 12-hour light cycle (light from 6:00 to 18:00, darkness from 18:00 to 6:00 the next day). Reproduction was carried out by mating 8-week-old sexually mature males with 8-week-old sexually mature females at a 1:2 ratio. Approximately one week after birth, the paws of the offspring were clipped for numbering and genotyping. Offspring were separated from their mothers three weeks after birth, and experimental use began at 60 days of age.

[0030] Wild-type control group: C57BL / 6J mice that are homologous to the experimental group and are age and sex matched.

[0031] P60 mice (i.e., mice 60 days after birth) were perfused to obtain brain tissue. The brain was longitudinally divided into left and right parts along the sagittal direction (anteroposterior direction). One part was embedded in OCT for frozen sectioning, and the other part was separated into brain regions, flash-frozen in liquid nitrogen, and stored at -80°C for subsequent experiments.

[0032] II. Immunofluorescence staining of GFAP and Iba1

[0033] Immunofluorescence staining of brain sections from DKO mice and Ndufs4 knockout mice with GFAP and Iba1 was performed, and the results are as follows: Figure 1 As shown, Iba1 tagged all microglia, while high CD68 expression indicated that microglia were in an active phagocytic state. In Ndufs4 knockout mice, Iba1 staining showed a significant increase in microglia density, and CD68 staining showed punctate signals and was highly co-localized with Iba1-positive cells. These CD68-positive granules filled the cytoplasm of hypertrophic microglia, indicating that a large number of microglia were actively performing phagocytosis and digesting ingested substances. In DKO mice, the number of Iba1-positive cells was significantly reduced, and the CD68 signal intensity was significantly weakened. In a few slightly morphologically altered microglia, only weak and sparse CD68 spots were observed, indicating a significant decrease in phagocytic activity. This demonstrates that DKO treatment effectively inhibited the functional activation of microglia.

[0034] III. Ndufs4 - / - cGAS - / - Weight loss has recovered somewhat

[0035] On day 60, the body weight of all experimental mice was measured, which corresponds to the stage when the disease phenotype of Ndufs4 knockout mice was fully manifested. Figure 2 The results showed that DKO mice were heavier than Ndufs4 knockout mice, indicating that cGAS KO intervention effectively improved the overall nutritional status and weight maintenance ability of mice during the critical period of the disease.

[0036] IV. Analysis of cGAS KO Mechanism

[0037] 1. cGAMP level detection

[0038] Rinse the tissue with pre-chilled PBS (0.01M, pH=7.4), weigh it, and then mince it. Add the minced tissue and the corresponding volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1g of tissue sample to 9mL of PBS; the specific volume can be adjusted according to experimental needs and recorded. Adding a protease inhibitor to the PBS is recommended) to a glass homogenizer and homogenize thoroughly on ice. To further lyse the tissue cells, the homogenate can be sonicated or subjected to repeated freeze-thaw cycles. Finally, centrifuge the homogenate at 5000×g for 5-10 minutes and collect the supernatant for analysis.

[0039] Remove the required strips from the aluminum foil bag after equilibration at room temperature for 60 minutes. Seal the remaining strips in a resealable bag and return them to 4°C. Set up standard and sample wells, adding 50 μL of different concentrations of standard to each well. Add 50 μL of the test sample to each sample well; do not add any to the blank wells. Add 50 mL of biotin-labeled antibody to each well, seal the reaction wells with sealing film, and incubate at 37°C in a water bath or incubator for 30 minutes; do not add any to the standard or blank wells. Discard the liquid, pat dry on absorbent paper, add 350 μL of washing buffer to each well, let stand for 1 minute, discard the washing buffer, pat dry on absorbent paper, and repeat this washing process 5 times. Except for the blank wells, add 100 mL of horseradish peroxidase (HRP)-labeled detection antibody to each standard and sample well, seal the reaction wells with sealing film, and incubate at 37°C in a water bath or incubator for 30 minutes. Discard the liquid, blot dry on absorbent paper, add 350 μL of washing buffer to each well, let stand for 1 min, discard the washing buffer, blot dry on absorbent paper, and repeat this washing process 5 times. Add 50 mL each of substrate A and B to each well, and incubate at 37°C in the dark for 15 min. Add 50 μL of stop solution to each well, and measure the OD value of each well at 450 nm within 15 min.

[0040] To investigate the underlying mechanism, we examined the levels of the second messenger 2'3'-cGAMP (a key activator of the cGAS-STING pathway) in brain tissue. In Ndufs4 knockout mice, significantly elevated cGAMP levels were observed. Figure 3 This suggests that mitochondrial dysfunction (due to Ndufs4 deficiency) may trigger mitochondrial DNA leakage into the cytoplasm, thereby activating the cGAS-STING innate immune pathway. This provides a key upstream initiation signal for previously observed severe neuroinflammation. In DKO mice, elevated cGAMP levels were significantly decreased ( Figure 3 The levels returned to near normal. This result demonstrates that DKO intervention can effectively inhibit the abnormal activation of the cGAS-STING pathway.

[0041] 2. qPCR detection of key regulatory factors and downstream effector genes in the type I interferon signaling pathway.

[0042] RNA extraction and reverse transcription were performed on tissues.

[0043] qPCR quantitative PCR detection:

[0044] First, the cDNA stock solution obtained from reverse transcription was diluted 7-fold with sterile water. This experiment used Novizan (China) ChamQ Universal SYBR qPCR Master Mix reagent. The prepared reaction system was centrifuged and mixed. The reaction program was set as follows: 95℃ for 30 s, (95℃ for 15 s, 60℃ for 30 s) for 40 cycles. Melting curve: 95℃ for 15 s, 60℃ for 1 min, 95℃ for 15 s. The obtained CT values ​​were... The method was used to perform relative gene expression analysis.

[0045] qPCR was used to detect key regulatory factors and downstream effector genes in the type I interferon signaling pathway to verify the functional output of the cGAS-STING pathway after activation. The results are as follows: Figure 4 As shown, in Ndufs4 knockout mice, the mRNA expression levels of all tested genes (Irf3, Irf7, Usp18, Oasl, Isg15, Cxcl10, Ccl5) ​​were significantly upregulated. The upregulation of Irf3 / Irf7 (a key transcription factor) confirms the initiation of the interferon response.

[0046] Strong induction of Usp18, Oasl, and Isg15 (classic interferon-stimulated genes, ISGs) clearly indicates sustained and effective activation of type I interferon signaling. High expression of Cxcl10 and Ccl5 (chemokines) provides a direct molecular basis for the recruitment and activation of microglia / immune cells, consistent with previous immunostaining results. In DKO mice, the abnormally high expression of these genes was reduced.

Claims

1. Application of cGAS as a target in screening drugs for the prevention of Leigh syndrome.

2. Application of cGAS as a target in screening drugs for the treatment of Leigh syndrome.

3. The application according to claim 1 or 2, characterized in that: The cGAS is a cyclic GMP-AMP synthase or its encoding gene.

4. The application according to claim 1 or 2, characterized in that: The drug inhibits cGAS expression.

5. The application according to claim 1 or 2, characterized in that: The drug improves gliosis in patients with Riesler syndrome.

6. The application according to claim 1 or 2, characterized in that: The drug inhibits the abnormal activation of the cGAS-STING pathway.

7. The application according to claim 1 or 2, characterized in that: The drug inhibits neuroinflammation.

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