Construction method and application of ALS mouse model with mitochondrial function damage

Abstract

The invention relates to the field of biomedicine, in particular to a construction method and application of an ALS mouse model with mitochondrial function damage. A mouse model with the genotype of Romo1flox / flox ChatCre + is constructed through a Cre / loxP system, after expression of CRE recombinase in cholinergic neurons is activated by the mouse model, Romo1 genes are selectively knocked out, and amyotrophic lateral sclerosis is spontaneously induced. The ALS mouse model constructed by the invention can be used for researching the pathogenesis of amyotrophic lateral sclerosis, screening potential therapeutic drugs and estimating the curative effect and safety of the drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to a method for constructing and applying an ALS mouse model with mitochondrial function impairment. Background Art

[0002] Cholinergic neurons, as an important group of neurons using acetylcholine as a neurotransmitter, play a key role in physiological processes such as motor regulation, learning and memory. Abnormalities in their functions are closely related to the onset of various neurodegenerative diseases. Among them, amyotrophic lateral sclerosis (ALS), also known as "Lou Gehrig's disease", is a typical example. ALS is characterized by the progressive degeneration of motor neurons as the core pathological feature, with clinical manifestations of progressive muscle weakness, paralysis and multi-system dysfunction, often accompanied by cognitive and behavioral abnormalities. It is worth noting that this disease has significant clinical heterogeneity and rapid progression. The median survival time of patients after diagnosis is only 3 - 5 years, and the annual incidence of ALS shows an increasing trend. Currently, there is a lack of effective intervention means in clinical practice, and existing therapeutic drugs can only delay the progression of the disease to a limited extent. Therefore, in-depth analysis of the pathogenesis of ALS and the development of new treatment strategies have become an urgent need in the field of neurodegenerative disease research.

[0003] 95% of ALS patients are sporadic patients with unclear pathogenic causes, and 5% are familial patients. The pathogenic mechanism of ALS is still unclear, but numerous studies have shown that mitochondrial dysfunction and oxidative stress injury are common core pathophysiological phenotypes of ALS, and the two act synergistically to form a vicious cycle. Mitochondrial dysfunction: A large number of basic studies have shown that abnormal mitochondrial structure and function are early pathological events in ALS. Electron microscopy analysis shows that abnormal mitochondria are significantly accumulated in the motor neurons of ALS patients and model mice, and its functional damage is manifested as: ① decreased mitochondrial membrane potential and ATP synthesis disorder; ② reduced activity of electron transport chain complexes; ③ abnormal localization of pathological proteins (such as mutant TDP43) in mitochondria, inducing mitochondrial DNA leakage and activating the cGAS-STING-mediated apoptosis pathway. Oxidative stress injury: Systemic oxidative stress markers have been detected in clinical samples and animal models, such as lipid peroxidation damage, protein oxidation damage, oxygen free radicals, oxidized modified proteins, and stress-related protein levels. Particularly noteworthy is that the imbalance of the glutathione metabolism network in ALS patients leads to the collapse of the antioxidant defense system, forming a positive feedback loop of oxidative damage. Mitochondria, as the main source of ROS, its functional disorder and oxidative stress form a cascade amplification effect: excessive production of ROS leads to the accumulation of glutamate, triggering excitotoxicity and calcium overload, further exacerbating mitochondrial damage, and ultimately promoting the progression of the disease.

[0004] An animal model that can well simulate the symptoms of human diseases is an important tool for studying human diseases, and the same is true for amyotrophic lateral sclerosis (ALS). As a mammal with a central nervous system similar to that of humans, mice are suitable for the study of ALS diseases. ALS mouse models exhibit multiple major characteristics of ALS patients, including weakness, weight loss, muscle atrophy, body incoordination, paralysis in the late stage of the disease, early death, etc. Some mouse models exhibit cognitive decline, impaired learning and memory ability, social disorders, anxiety behaviors, etc. In addition, motor neuron death, immune inflammation, abnormal neuromuscular junctions, muscle denervation, axonal lesions, pathological inclusion bodies, etc. have also been observed in mouse models. The establishment of these mouse models can effectively verify ALS-related genes discovered by genetics, promote the analysis of the disease mechanism of ALS, and also provide tools for finding treatment methods for ALS. Although most ALS patients are sporadic patients with unclear pathogenic causes, most ALS mouse models are established based on genetic variations found in familial patients. In recent years, more and more ALS-related genes have been discovered, and corresponding animal models have also been established. These ALS mouse models based on different genetic variations exhibit similar phenotypes. Currently, the methods for establishing ALS mouse models are mainly divided into three categories: 1) gene overexpression models, 2) gene loss-of-function models, and 3) gene mutation models. The research on these ALS mouse models has deepened people's understanding of the pathogenic mechanisms of ALS-related genes. Among them, the model constructed based on the mutation of the Sod1 gene is one of the most widely used ALS mouse models so far. In 1993, Rosen et al. identified a mutation in the SOD1 gene in an fALS family, and the SOD1 gene became the first ALS-related gene. In 1994, a mutant mouse model SOD1 was constructed based on the SOD1 mutation found in patients. G93A , the SOD1 G93A model mice exhibited phenotypes of motor neuron loss, paralysis, and early death. Subsequently, many studies constructed more mutant models, such as SOD1 G37R , SOD1 G85R and so on.

[0005] The key bottleneck currently faced in ALS research lies in the insufficient pathological reproducibility of animal models: ① Most existing models are based on familial ALS pathogenic genes (such as SOD1, C9orf72, etc.), lacking sporadic case models; ② The classic SOD1 G93A model can simulate motor disorders, but cannot reproduce key clinical features such as cognitive function decline; ③ The conversion effect of treatment regimens verified by preclinical models is not good, indicating major defects in the existing model system. Particularly prominent is that, for the core pathological link of mitochondrial dysfunction, there is still a lack of an ALS animal model that specifically simulates mitochondrial damage in motor neurons, severely restricting mechanism research and drug development. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a method for constructing and applying a mouse model of amyotrophic lateral sclerosis (ALS) with mitochondrial function impairment.

[0007] The present invention adopts the following technical solutions: In a first aspect, the present invention provides a method for constructing a mouse model of ALS with mitochondrial function impairment. By using the Cre / loxP system, a mouse model with the genotype Romo1 flox / flox Chat Cre+ is constructed. After activating the expression of CRE recombinase in cholinergic neurons in the mouse model, the Romo1 gene is selectively knocked out, spontaneously inducing the occurrence of amyotrophic lateral sclerosis.

[0008] The present invention first clarifies the role of the Romo1 gene in establishing an ALS mouse model and constructs a new ALS mouse model based on mitochondrial defects. Its symptoms include: growth arrest, gradual weight loss with age; gradual decline in limb strength and eventually hindlimb paralysis; muscle fiber atrophy, which gradually worsens with age; reduced exercise endurance; memory decline; before the reduction of limb strength, myelin breakdown of sciatic nerve axons, a decrease in the number of ventral horn motor neurons in the lumbar L5 segment, and microglial hyperplasia, which increases with age. More importantly, the ALS mouse model of the present invention also has the pathological phenomenon of mitochondrial cristae structure degeneration, which not only helps us deeply understand the complexity of the ALS pathogenesis mechanism but also provides a valuable experimental platform for developing new treatment methods.

[0009] In certain embodiments, a mouse model with the genotype Romo1 flox / flox Chat Cre+ is constructed through the Chat-Cre / loxP system.

[0010] In certain embodiments, the construction method includes the following steps: Hybridize Romo1 flox / flox genotype mice with Chat-Cre mice, and screen to obtain first-generation Romo1 LoxP / + Chat cre / + genotype mice. Hybridize these genotype mice, and screen in the second-generation to obtain cholinergic-specific Romo1 flox / flox Chat Cre+ mice with the Romo1 gene knocked out specifically.

[0011] In certain embodiments, cholinergic-specific Romo1 flox / flox Chat Cre+Before the cholinergic-specific knockout of Romo1 in mice, it also includes a PCR verification step.

[0012] In certain embodiments, the primer sequences for the PCR verification are shown in SEQ ID NO.1-6.

[0013] In a second aspect, the present invention provides the application of the ALS mouse model constructed by the method for constructing an ALS mouse model with mitochondrial function impairment in studying the pathogenesis of amyotrophic lateral sclerosis.

[0014] In a third aspect, the present invention provides the application of the ALS mouse model constructed by the method for constructing an ALS mouse model with mitochondrial function impairment in screening drugs for treating amyotrophic lateral sclerosis.

[0015] The present invention has the following beneficial effects: The Romo1-cKO mouse model created by the present invention has three major breakthrough values: ① Pathological reproducibility breakthrough: Compared with the traditional SOD1 G93A model, this model not only reproduces the phenotypes of motor disorders and progressive paralysis, but also simulates clinical multidimensional phenotypes such as cognitive decline (the Y-maze index decreased by 24.2%). ② Mechanism specificity breakthrough: As the first ALS model with a mitochondrial protein-coding gene defect, its pathological progression shows a strict temporal correlation with the degeneration of mitochondrial cristae structure, providing a pure model for studying mitochondrial-specific mechanisms. ③ Translational application breakthrough: This model can be applied to study the pathogenesis of ALS, screen potential treatment strategies, and evaluate the efficacy and safety of drugs, especially for the screening and efficacy evaluation of mitochondria-targeted drugs, helping to predict preclinical research. Description of the Drawings

[0016] Figure 1 Construction strategy for Romo1 cholinergic neuron-specific knockout mice. A. Flow chart of obtaining Romo1 flox / flox mice by mating Romo1 flox / flox mice with Chat-Cre mice for two generations to obtain Romo1 + / Chat-Cre flox / flox mice. B. DNA electrophoresis result map for genotype identification of mice. Upper panel: Lanes M, 1, and 2 are DNA molecular weight standard samples, Romo1 flox / + , Romo1

[0017] Figure 2Appearance photos of Romo1-cKO mice of all ages and their cage-mate WT mice. Upper panel: The appearance of the cage-mate WT mice is normal; Lower panel: In Romo1-cKO mice, the hind limbs gradually atrophy and become paralyzed with age, and muscle atrophy also progresses to the forelimbs.

[0018] Figure 3 Curves of body weight changes of Romo1-cKO and WT mice with age. A. Curves of body weight changes of male Romo1-cKO group and cage-mate WT mice with age. In the Romo1-cKO group, N = 19; in the WT group, N = 13. B. Curves of body weight changes of female Romo1-cKO group and cage-mate WT mice with age. In the Romo1-cKO group, N = 19; in the WT group, N = 14. * p < 0.05 compared with WT mice; *** p < 0.001 compared with WT mice.

[0019] Figure 4 Metabolic cage tests of 2-month-old WT mice and Romo1-cKO mice. A. O2 consumption rates of WT mice and Romo1-cKO mice. B. Respiratory exchange ratio (RER) of WT mice and Romo1-cKO mice, RER = VCO2 / VO2. C. Heat production of WT mice and Romo1-cKO mice. Heat = (3.815 + 1.232 * RER) * VO2. All experimental mice are male, N = 6 in the Romo1-cKO group; N = 5 in the WT group. L: Daytime; D: Nighttime.

[0020] Figure 5 Metabolic cage tests of 4-month-old WT mice and Romo1-cKO mice. A. O2 consumption rates of WT mice and Romo1-cKO mice. B. Respiratory exchange ratio RER = VCO2 / VO2 of WT mice and Romo1-cKO mice. C. Heat production Heat = (3.815 + 1.232 * RER) * VO2 of WT mice and Romo1-cKO mice. D. Food intake of WT mice and Romo1-cKO mice. All experimental mice are male, N = 9 in the Romo1-cKO group; N = 6 in the WT group. * p < 0.05 compared with WT mice; *** p < 0.001 compared with WT mice. L: Daytime; D: Nighttime.

[0021] Figure 6Wheel running duration curves of Romo1-cKO and WT mice. A. Wheel running duration curves of male Romo1-cKO mice and cage-mate WT mice of each age group. N = 13 in the Romo1-cKO group; N = 7 in the WT group. B. Wheel running duration curves of female Romo1-cKO mice and cage-mate WT mice of each age group. N = 11 in the Romo1-cKO group; N = 8 in the WT group. * p < 0.05 compared with WT mice; *** p < 0.001 compared with WT mice.

[0022] Figure 7 Limb grip strength of Romo1-cKO mice as a function of age. A. Limb grip strength curves of male Romo1-cKO mice and cage-mate WT mice as a function of age. N = 13 in the Romo1-cKO group; N = 7 in the WT group. B. Limb grip strength curves of female Romo1-cKO mice and cage-mate WT mice as a function of age. N = 11 in the Romo1-cKO group; N = 8 in the WT group. * p < 0.05 compared with WT mice; ** p < 0.01 compared with WT mice; *** p < 0.001 compared with WT mice.

[0023] Figure 8 Changes in short-term spatial memory ability of Romo1-cKO mice at 6 and 12 weeks of age. A. Total number of actual exploration arms of male mice at 6 and 12 weeks of age within 5 minutes. B. Percentage of correct alternation number / maximum alternation number of male mice at 6 and 12 weeks of age within 5 minutes compared with the corresponding indexes of WT mice. For 6-week-old mice, N = 3 in the Romo1-cKO group and N = 5 in the WT group; for 12-week-old mice, N = 11 in the Romo1-cKO group and N = 7 in the WT group. C. Total number of actual exploration arms of female mice at 6 and 12 weeks of age within 5 minutes. D. Percentage of correct alternation number / maximum alternation number of female mice at 6 and 12 weeks of age within 5 minutes compared with the corresponding indexes of WT mice. For 6-week-old mice, N = 11 in the Romo1-cKO group and N = 8 in the WT group; for 12-week-old mice, N = 10 in the Romo1-cKO group and N = 8 in the WT group. * p < 0.05 compared with WT mice.

[0024] Figure 9 Survival statistics of Romo1-cKO mice. A. Survival curves of non-breeding Romo1-cKO mice and cage-mate WT mice. All mice are male, N = 32 in the Romo1-cKO group; N = 27 in the WT group. B. Survival curves of breeding Romo1-cKO mice and cage-mate WT female mice. N = 17 in the Romo1-cKO group; N = 17 in the WT group.

[0025] Figure 10Changes in tibial length and weights of various types of muscles in 12-month-old Romo1-cKO mice. A. Hindlimb tibia images of WT mice and Romo1-cKO mice. Scale bar length: 1 cm. B. Statistical analysis chart of hindlimb tibial length in mice. N = 6 / group. C. Representative diagram of muscle dissection. Quad, quadriceps femoris; EDL, extensor digitorum longus; TA, tibialis anterior; GST, gastrocnemius; Sol, soleus. D. Statistical chart of the weights of various muscles in mice. N = 4 / group. ns indicates no significant difference compared with WT mice; ** p < 0.01 compared with WT mice; *** p < 0.001 compared with WT mice.

[0026] Figure 11 Longitudinal section HE staining of various types of muscles in the hindlimbs of 12-month-old mice. Compared with WT mice, the gastrocnemius, quadriceps femoris, tibialis anterior, and extensor digitorum longus of Romo1-cKO mice showed extensive atrophy and hyperplasia of muscle cell nuclei. Left column: WT mice; right column: Romo1-cKO mice. Scale bar: 50 μm.

[0027] Figure 12 Cross-section HE staining of the tibialis anterior muscle in 10-week-old and 24-week-old mice. Red arrow marks: nuclear centering. Scale bar length: 20 μm.

[0028] Figure 13 Immunofluorescence staining results of neuromuscular junctions in the hindlimb tibialis anterior muscle of Romo1-cKO mice and cage-mate WT mice. A. Typical intact neuromuscular junction structure in the tibialis anterior muscle of WT mice. B. Neuromuscular junction structure in the tibialis anterior muscle of Romo1-cKO mice. Green arrow indicates intact neuromuscular junction structure; red arrow indicates neuromuscular junction structure with presynaptic nerve terminal loss. C. Neuromuscular junction structure in the tibialis anterior muscle of Romo1-cKO mice with presynaptic nerve terminal loss and diffuse arrangement of postsynaptic acetylcholine receptors. First column: immunofluorescence staining of anti-synaptophysin and anti-neurofilament protein; second column: α-bungarotoxin (BTX) conjugated with tetramethylrhodamine labeling acetylcholine receptors; third column: double staining of neuromuscular junction structure. Scale bar length: 20 μm.

[0029] Figure 14 Changes in nerve innervation of the hindlimb tibialis anterior muscle of Romo1-cKO mice and cage-mate WT mice with age. A. Typical images of nerve innervation in the hindlimb tibialis anterior muscle of mice at each age. Green: immunofluorescence staining of anti-synaptophysin and anti-neurofilament protein; red: α-bungarotoxin (BTX) conjugated with tetramethylrhodamine labeling acetylcholine receptors. Scale bar length: 20 μm. B. Statistical chart of the proportion of intact neuromuscular junction structures in the tibialis anterior muscle of 6-week-old, 8-week-old, and 24-week-old mice. N = 3 mice / group. *** p < 0.001 compared with WT mice.

[0030] Figure 15 Electron microscopy results and statistical analysis of sciatic nerve axons in Romo1-cKO and WT mice of different ages. A. Electron micrographs of cross-sections of the sciatic nerve in 8-week-old and 16-week-old mice. Scale bar: 50 μm. B. Electron micrographs of axon cross-sections in WT and Romo1-cKO mice at 8 and 16 weeks of age. Scale bar: 500 nm. C. Statistical analysis of the number of axons in the sciatic nerve of 8-week-old and 16-week-old mice. At 8 weeks of age: N = 3 in the WT group and N = 4 in the Romo1-cKO group; at 16 weeks of age: N = 5 in the WT group and N = 5 in the Romo1-cKO group. D. Statistical chart of the proportion of degenerated axons in the sciatic nerve of mice of different ages. *p < 0.05 compared with WT mice.

[0031] Figure 16 Changes in the number of motor neurons in Romo1-cKO mice with age. A. Immunohistochemical images of motor neurons in the lumbar L5 layer of 48-week-old mice. Dashed line: the ventral horn region of the spinal cord used for counting the number of motor neurons. Scale bars: 200 μm (upper panel) and 10 μm (lower panel). B. Statistical chart of the number of motor neurons in 6-, 8-, 24-, and 48-week-old mice. At 6 weeks of age: N = 7 in the WT group and N = 13 in the Romo1-cKO group; at 8 weeks of age: N = 7 in the WT group and N = 13 in the Romo1-cKO group; at 24 weeks of age: N = 7 in the WT group and N = 13 in the Romo1-cKO group; at 48 weeks of age: N = 7 in the WT group and N = 13 in the Romo1-cKO group. **p < 0.01 compared with WT mice; ***p < 0.001 compared with WT mice.

[0032] Figure 17 Changes in the number of large neurons in the ventral horn of the lumbar L5 layer in Romo1-cKO mice with age. A. Nissl staining images of large neurons in the ventral horn of the lumbar L5 spinal cord in 8-, 16-, 24-, and 48-week-old mice. Scale bar: 100 μm. B. Statistical chart of the number of large neurons in the ventral horn of the lumbar L5 spinal cord in 8-, 24-, and 48-week-old mice. At 8 weeks of age: N = 15 in the WT group and N = 25 in the Romo1-cKO group; at 24 weeks of age: N = 19 in the WT group and N = 17 in the Romo1-cKO group; at 48 weeks of age: N = 4 in the WT group and N = 4 in the Romo1-cKO group. *p < 0.05 compared with WT mice.

[0033] Figure 18Microgliosis in the lumbar L5 layer of 8-week-old Romo1-cKO mice. A. Immunofluorescence results of glial cells in the lumbar L5 layer of mice. Green fluorescence: anti-microglial specific protein IBA1. Scale bar: 200 μm (upper panel), 50 μm (lower panel). B. Statistical analysis of the percentage of the total area of glial cells in the spinal cord cross-section of the lumbar L5 layer of mice compared with the corresponding indicators of WT mice. N = 3 in the WT group, N = 4 in the Romo1-cKO group. *p < 0.05 compared with WT mice.

[0034] Figure 19 Electron micrographs of the lumbar L5 layer of Romo1-cKO and WT mice. A. Electron micrograph of the cross-section of the lumbar L5 layer of 16-week-old Romo1-cKO mice. Left panel: Local electron micrographs of non-cholinergic neurons (1) and cholinergic neurons (2), scale bar: 2 μm; Right panel: Magnified views of fields 1 and 2, scale bar: 500 nm. B. Statistical chart of mitochondrial area in 6-week-old and 16-week-old mice. N = 230 - 260 mitochondria / group from 6 motor neurons of 3 mice / group. C. Distribution chart of mitochondrial curvature in 6-week-old mice. N = 3 motor neurons / group from 3 mice / group. **p < 0.01 compared with WT mice.

[0035] Figure 20 Mitochondrial structural changes in motor neurons of the lumbar spinal cord L5 layer of 6-week-old Romo1-cKO mice. A. Electron micrograph of the mitochondrial cross-section in motor neurons of the lumbar spinal cord L5 layer of 6-week-old mice. Red arrow indicates: Residual strip-like cristae in motor neuron mitochondria. B. Statistical chart of mitochondrial cristae width in motor neurons of the lumbar spinal cord L5 layer of 6-week-old mice. N = 30 mitochondria / group from 3 mice / group. ***p < 0.001 compared with the WT group.

[0036] Figure 21 Mitochondrial structural changes in motor neurons of the lumbar spinal cord L5 layer of 16-week-old Romo1-cKO mice. A. Electron micrograph of the mitochondrial cross-section in motor neurons of the lumbar spinal cord L5 layer of 16-week-old mice, scale bar: 500 nm. B. Electron micrograph of abnormal mitochondrial morphology in motor neurons of 16-week-old Romo1-cKO mice, scale bar: 200 nm.

[0037] Figure 22 Classification analysis of mitochondrial structures in motor neurons of the lumbar L5 layer of Romo1-cKO and WT mice. A. Electron micrographs of four types of mitochondria. Scale bar: 100 nm. B. Cross-sectional areas of four types of mitochondria (μm 2)Statistical chart. N = 80 mitochondria / group from 6 mice. C. Distribution diagram of the proportions of 4 types of mitochondria in lumbar L5 layer motor neurons of 6-week-old and 16-week-old mice. N = 3-5 cells / group from 3 mice. * p < 0.05 for comparison between groups; *** p < 0.001 for comparison between groups. Percentage of the 4 types of mitochondria in the total number of mitochondria (%)

[0038] Figure 23 Electron microscopic structure of the endoplasmic reticulum in lumbar L5 layer motor neurons of 6-week-old and 16-week-old WT and Romo1-cKO mice. Scale bar: 500 nm. Specific embodiments

[0039] To make the objectives, technical solutions and advantages of the present disclosure more clear and understandable, the following further elaborates on the present disclosure in detail with reference to specific embodiments and the accompanying drawings.

[0040] It should be noted that unless otherwise defined, the technical terms or scientific terms used in the embodiments of the present disclosure should have the ordinary meanings understood by those of ordinary skill in the art to which the present disclosure pertains.

[0041] In the context of the present invention, Romo1 refers to Reactive Oxygen Species Modulator 1, which is a mitochondrial membrane protein. The key role of Romo1 protein in various diseases, especially its extensive involvement in cancer, makes it a potential therapeutic target. The Romo1 protein is also associated with some non-cancer diseases, such as premature ovarian failure 19 and optic nerve atrophy, etc. However, the function of Romo1 protein in affecting the development of amyotrophic lateral sclerosis has not been reported yet.

[0042] To expand the application of Romo1 protein, the present invention provides a method for constructing an ALS mouse model with mitochondrial function impairment. Through the Cre / loxP system, a mouse model with the genotype Romo1 flox / flox Chat Cre+ is constructed. After activating the expression of CRE recombinase in cholinergic neurons in the mouse model, the Romo1 gene is selectively knocked out, spontaneously inducing the occurrence of ALS.

[0043] To better achieve specific gene knockout in cholinergic neurons, the above selective knockout of the Romo1 gene is specifically achieved through the Chat-Cre / loxP system, that is, the expression of Cre recombinase is regulated by the choline acetyltransferase (CHAT) promoter, obtaining a mouse model with the genotype Romo1 flox / flox

[0044] The experimental methods used in the following examples are all conventional methods unless otherwise specified.​

[0045] Materials, reagents, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0046] Example 1: Construction of mice with specific knockout of Romo1 gene in cholinergic neurons 1. Experimental method The Romo1 flox / flox genotype (strain name: C57BL / 6Smoc-Romo1 em1（flox）Smoc ) mice were crossed with Chat-Cre (strain name: B6;129S6-Chattm2(cre)Lowl / J, catalog number: 006410) mice purchased from The Jackson Laboratory (JAX) to obtain the first-generation Romo1 LoxP / + Chat cre / + , and the heterozygous offspring of this genotype were crossed to obtain Romo1 flox / flox Chat Cre+ mice with specific knockout of Romo1 in cholinergic neurons.

[0047] Genotyping of mice: Mouse genomic DNA was extracted using the alkaline lysis method. A small piece of mouse tail tip, no more than 0.2 cm, was cut. When cutting multiple mouse tails, pay attention to wiping the scissors with alcohol after each cut to prevent gene cross-contamination. The mouse tail was placed in a 1.5 mL EP tube, and 200 μL of 50 mM NaOH was added, followed by incubation in a metal bath at 100 °C for 20 minutes; then, 17 μL of 1 M Tris-HCl (pH = 8.0) was added to each tube for neutralization. The extracted mouse tail genes can be stored at 4 °C for later use.

[0048] Genotyping of mouse tail genes was performed using PCR technology: By designing primers for the target gene (mostly 19 - 25 bp in length), the target gene fragment was specifically amplified, and gene fragments of different lengths were separated by agarose gel electrophoresis. The genotype was determined by observing whether there was a band of the size of the target gene fragment. It was necessary to identify the Cre gene and the Romo1 flox gene. Primers were designed for the sequence upstream of the vector at the insertion site of the target gene and the sequence upstream of the target gene, and a common reverse primer was designed for the vector fragment downstream of the insertion site, which could achieve simultaneous detection of the target gene and the WT gene to determine the homozygous state of the gene. The primer sequences used (Table 1) and the PCR system (Table 2) are as follows.

[0049] Table 1 Primer sequences for mouse gene identification Table 2 PCR system and conditions 2. Experimental results Using the LoxP / Cre recombinase system, Romo1 LoxP-site modified mice were crossed with Chat-Cre transgenic mice that specifically express CRE recombinase in cholinergic neurons to obtain the first-generation Romo1 flox / + / Chat-Cre + mice. Then, male and female first-generation mice were mated to obtain cholinergic neuron-specific knockout mice (Romo1-cKO) with the genotype Romo1 flox / flox / Chat-Cre + (Figure A). Figure 1 ).

[0050] The proportions of each genotype of the offspring produced by mating Romo1 flox / + / Chat-Cre + mice were counted. It was found that the proportion of Romo1 flox / flox / Chat-Cre mice was close to 1 / 4, which conforms to Mendel's law of inheritance, indicating that cholinergic neuron-specific knockout of Romo1 does not affect mouse embryonic development (Table 3).

[0051] Table 3 Statistics of the Proportions of Each Genotype of the Offspring Produced by Mating Romo1 flox / + / Chat-Cre + Syngeneic Mice Example 2: ALS Symptoms of Romo1-cKO Mice 1. Experimental Methods The following experiments were performed on the Romo1-cKO mice constructed in Example 1 of the present invention: (1) Continuously observe the growth of Romo1-cKO and control group mice (WT).

[0052] (2) Continuously record the changes in the body weight of the mice.

[0053] (3) Using an Oxymax / CLAMS metabolic cage, record the metabolic indices of WT mice and Romo1-cKO mice at 2 months of age (before onset) and 4 months of age (after onset) within 48 hours, including food intake, water intake, total oxygen consumption, total carbon dioxide production, respiratory exchange ratio, and heat production.

[0054] (4) Measure the changes in the motor coordination ability and fatigue resistance ability of the mice under strenuous exercise conditions using a rotarod test.

[0055] (5) Record the changes in the limb grip strength of the mice using a mouse grip strength meter to detect the muscle degeneration status of the mice.

[0056] (6)The spatial memory abilities of 6-week-old and 12-week-old Romo1-cKO mice and their cage-mate WT mice were detected using a Y-maze.

[0057] (7)The survival of Romo1-cKO mice and WT mice was tracked.

[0058] 2. Experimental results (1)Muscle atrophy occurred in Romo1-cKO mice Romo1-cKO mice showed no obvious symptoms in the first three months. However, when they reached 4 months of age, a phenotype of muscle atrophy appeared in the hind limbs of the mice, and the mice were listless. When the mice reached 6 months of age, their hind limb motor function was almost completely lost, and their movement mainly relied on the forelimbs. By 12 months of age, their forelimb support ability was further lost, and the mice crawled on the ground ( Figure 2 ). While their cage-mate WT mice did not show any abnormal behaviors and phenotypes at each time point ( Figure 2 ). This phenotype of Romo1-cKO mice is very similar to the phenotype of ALS disease, suggesting that this mouse model may be used as a mouse model of ALS.

[0059] (2)Growth retardation occurred in Romo1-cKO mice There was no significant difference between male Romo1-cKO mice and their cage-mate WT mice in the first 10 weeks. However, starting from 10 weeks, Romo1-cKO mice showed growth retardation, their body weight stopped increasing, and continued to decrease with age ( Figure 3 A). Female Romo1-cKO mice showed a similar growth retardation phenomenon as male mice, that is, the body weight of Romo1-cKO female mice reached a peak at 14 weeks and then gradually decreased with age ( Figure 3 B).

[0060] (3)The metabolic pattern of Romo1-cKO mice changed The results of metabolic cage measurements showed that there was no significant difference in the oxygen consumption and heat production per hour of 2-month-old Romo1-cKO mice compared with their cage-mate WT mice at night ( Figure 4 A, B). The respiration exchange ratio (RER) can reflect the use of metabolic substrates by mice. When the respiration exchange ratio is 0.7, it indicates that the main oxidative metabolic substrate is fat, and when the respiration exchange ratio is 1, it indicates that the main oxidative metabolic substrate is carbohydrate. The measurement results indicated that the respiration exchange ratios of Romo1-cKO and WT mice were both between 0.7 and 1, indicating that both types of metabolic substrates were involved in mouse metabolism. The respiration exchange ratios of Romo1-cKO mice and WT mice fluctuated with the circadian rhythm, with the respiration exchange ratio rising at night and falling during the day ( Figure 4B), which may be related to the fact that mice are nocturnal, ingest food, and preferentially use carbohydrates, while during the day, mice are in a resting state and food intake is greatly reduced, resulting in more fat burning.

[0061] 4-month-old mice already showed obvious muscle atrophy and decreased motor ability. Analyzing the metabolic cage data of 4-month-old Romo1-cKO mice and WT mice, the results showed that there were no significant changes in the oxygen consumption and respiratory exchange rate of Romo1-cKO mice, but the heat production per hour decreased significantly, and the food intake also decreased significantly ( Figure 5 ), which may be related to the decrease in mouse body weight and motor ability.

[0062] (4)The motor coordination and fatigue tolerance of Romo1-cKO mice are reduced At 1-2 months of age, the performance of male Romo1-cKO mice in the rotarod test was similar to that of WT mice in the same cage. However, at 3 months of age, the movement duration on the rotarod decreased significantly, indicating a reduction in their motor coordination and fatigue tolerance. Moreover, as they aged, their motor ability further declined, and by 5 months of age, they were unable to perform running wheel exercise ( Figure 6 A). While the WT mice in the same cage maintained a stable high running wheel duration ( Figure 6 A). Similarly, the motor coordination and fatigue tolerance of female Romo1-cKO mice also gradually declined with age ( Figure 6 B).

[0063] (5)The limb grip strength of Romo1-cKO mice gradually weakens with age The results of the tensile test showed that the limb tensile strength of male Romo1-cKO mice gradually increased with age before 8 weeks old, but gradually decreased with age after 8 weeks old. While the WT mice in the same cage showed that the tensile strength gradually increased with age before 8 weeks old, reached the highest value at 8 weeks old, and then remained at the highest tensile strength level until 20 weeks old ( Figure 7 A). Similar to the tensile strength change of male mice, the tensile strength of female mice reached the maximum value at 8 weeks old, and then showed a decrease in tensile strength with age ( Figure 7 B). The experimental results showed that although 10-week-old Romo1-cKO mice did not show obvious movement disorders ( Figure 6 ) and weight loss ( Figure 3 ), the explosive power of their limb muscles had already decreased significantly at this time.

[0064] (6)The short-term spatial memory ability of Romo1-cKO mice decreases The results showed that compared with the WT mice in the same cage, there was no significant difference in the correct alternation ratio (%) and the total number of arm entries of male Romo1-cKO mice at 6 weeks of age, indicating that the short-term memory ability of Romo1-cKO mice at this age was not changed. At 12 weeks of age, compared with the WT mice in the same cage, there was no significant difference in the total number of arm entries of male Romo1-cKO mice, but their correct alternation ratio decreased significantly, suggesting a decline in their short-term memory ability ( Figure 8 A, B). Similarly, the short-term memory ability of female Romo1-cKO mice remained unchanged at 6 weeks of age, but showed a downward trend at 12 weeks of age ( Figure 8 C, D). These results suggest that the cholinergic neurons in the brains of Romo1-cKO mice were functionally damaged at 12 weeks of age, resulting in a decline in the short-term spatial memory ability of the mice.

[0065] (7)The lifespan of Romo1-cKO mice was shortened The lifespan of Romo1-cKO mice was significantly shortened, with a median survival period of 386 days. By the statistical time of 710 days, the survival rate of Romo1-cKO mice had dropped to 0. In contrast, the survival rate of the WT mice in the same cage was as high as 84% at 710 days ( Figure 9 A).

[0066] Mating behavior severely accelerated the disease process of Romo1-cKO mice. Female and male Romo1-cKO mice at 2 months of age could both complete mating behavior. Female Romo1-cKO mice could successfully reproduce and nurse their offspring, but both mating and nursing behaviors significantly accelerated their disease process, causing the mice to become paralyzed earlier and die earlier. As Figure 9 shown in B, the median survival period of Romo1-cKO mice involved in mating was only 163 days, significantly lower than the median survival period of the aforementioned non-mated Romo1-cKO mice (386 days). By the statistical cut-off time of 203 days, the survival rate of Romo1-cKO mice dropped to 0, while the survival rate of their WT littermates in the same cage was 100%.

[0067] These results showed that specific knockout of Romo1 in cholinergic neurons significantly shortened the lifespan of mice, and related body stress stimuli such as reproductive behavior accelerated the disease process of Romo1-cKO mice.

[0068] In summary, Romo1-cKO mice exhibited multiple symptoms similar to those of ALS patients, including reduced limb strength, decreased motor coordination, reduced fatigue tolerance, and weight loss at a young age (10-12 weeks old). Obvious hindlimb movement abnormalities appeared at 4 months old, and with age, these symptoms gradually worsened, eventually leading to paralysis and death. Moreover, both female and male Romo1-cKO mice showed similar symptoms. These results indicate that Romo1-cKO mice can be used as a new mouse model of ALS with mitochondrial localization protein deficiency.

[0069] Example 3 Pathological Mechanism of ALS-like Disorders in Romo1-cKO Mice 1. Experimental Methods To explore the pathological mechanism of Romo1-cKO mice showing ALS-like symptoms, in view of the phenotype of gradually lost motor ability and eventually paralysis in Romo1-cKO mice, the effects of ROMO1 deficiency on the morphology and structure of the hindlimb muscles and motor neurons (cholinergic neurons) controlling the hindlimb muscles in mice were analyzed. Considering that female and male Romo1-cKO mice showed similar symptoms, subsequent studies were all carried out in male mice. The experiments included:

[0070] (1) Measuring the tibia length of the hind legs of mice. Analyzing the changes in different types of muscle fibers in Romo1-cKO mice; isolating various muscles of 12-month-old mice and performing staining; measuring muscle weight; performing HE staining on various types of muscles to analyze the morphological changes of muscle cells.

[0071] (2) Detecting the nerve innervation of the tibialis anterior muscle in mice by immunofluorescence. Using anti-synaptophysin antibody and anti-neurofilament-L antibody to label the presynaptic synaptic vesicles and the cytoskeleton of axon terminals of neurons respectively, and using α-bungarotoxin (a-BTX) conjugated with tetramethylrhodamine that binds to acetylcholine receptors to label the acetylcholine receptors of muscles. Imaging was performed using a laser confocal microscope.

[0072] (3) To explore the etiology of muscle denervation in Romo1-cKO mice, the changes in sciatic nerve axons were further analyzed. Isolating the sciatic nerve of mice, preparing semi-thin sections, and imaging the cross-section of the intact sciatic nerve using a focused ion beam scanning electron microscope.

[0073] (4) Using ChAT antibody to specifically label motor neurons, and further analyzing the changes in the number of motor neurons in the ventral horn of the lumbar L5 spinal cord of Romo1-cKO and WT mice at different ages.

[0074] (5) Label microglia in the spinal cord with an antibody against IBA1 (ionized calcium binding adapter molecule 1) that is specifically expressed in microglia, and analyze the gliosis in the spinal cord of Romo1-cKO mice.

[0075] 2. Experimental results (1) Romo1-cKO mice showed progressive muscle atrophy There was no significant difference in the length of the tibia between Romo1-cKO mice and WT mice in the same cage ( Figure 10 A, B), indicating that Romo1-cKO mice did not have tibial hypoplasia. Mouse skeletal muscle is divided according to the function of muscle fibers, and can be divided into slow-twitch, fatigue-resistant slow muscle (type I) and fast-twitch fast muscle (type II), and type II muscle fibers are further divided into fatigue-resistant type IIa muscle fibers and fatigue-prone type IIb muscle fibers. Various muscles of 12-month-old mice were isolated and stained, including soleus (Sol), which is mainly composed of type I muscle fibers, gastrocnemius (GST) and extensor digitorum longus (EDL), which are mainly composed of type IIa muscle fibers, tibialis anterior (TA), which is mainly composed of type IIb muscle fibers, and mixed quadriceps (Quad) ( Figure 10 C). Measuring the muscle weight, it was found that the weights of various types of muscles in 12-month-old Romo1-cKO mice were significantly lower than those of WT mice in the same cage ( Figure 10 D).

[0076] Subsequently, HE staining was performed on various types of muscles to analyze the morphological changes of muscle cells. The results showed that compared with WT mice, the morphological changes of the above-mentioned various types of muscle cells in Romo1-cKO mice were obvious, manifested as hyperplasia of muscle fiber nuclei and arranged in a string-like pattern, and large-area atrophy of muscle fibers ( Figure 11 ). Since no obvious inflammatory cell infiltration was observed in the HE results ( Figure 11 ), it indicated that muscle fiber atrophy was not caused by myogenic necrosis.

[0077] Since similar lesions occurred in various types of muscles, the tibialis anterior muscle was used as a representative in subsequent studies to further study the morphological changes of muscles in mice of different ages. The HE staining results showed that in 10-week-old Romo1-cKO mice at the early stage of the disease, the cross-section of muscle fibers was similar to that of WT mice, while at 24 weeks of age, the disease worsened, and the muscle fiber area of Romo1-cKO mice was significantly reduced, and the nuclei of some muscle cells were centralized ( Figure 12(red arrow in Fig. 0). This result indicates that specific deletion of ROMO1 in cholinergic neurons causes progressive muscle atrophy.

[0078] (2)Denervation occurs in Romo1-cKO mouse muscles before atrophy In WT mice, most of the observed neuromuscular junction structures are typically innervated ( Figure 13 in Fig. A), that is, the presynaptic membrane labeled with anti-synaptic vesicle protein antibody and the postsynaptic membrane labeled with α-bungarotoxin show good co-localization ( Figure 13 in Fig. A). In Romo1-cKO mice, however, three types of neuromuscular junction structures were observed, namely 1) intact neuromuscular junction structures with co-localization of the presynaptic membrane and acetylcholine receptors on the muscle ( Figure 13 yellow arrow in Fig. B); 2) loss of presynaptic nerve terminal structures, with no significant change in the structure of acetylcholine receptors on the muscle, but a smaller area ( Figure 13 red arrow in Fig. B); 3) loss of presynaptic nerve terminal structures and no typical structure, dispersion, or even disappearance of the arrangement of acetylcholine receptors on the muscle ( Figure 13 in Fig. C). These three different neuromuscular junction structures indicate the occurrence of muscle denervation in Romo1-cKO mice.

[0079] Subsequently, the muscle denervation situation in Romo1-cKO mice of different ages was counted. The results showed that WT mice of all ages showed typical intact neuromuscular junction structures ( Figure 14 ), indicating good nerve innervation of the muscle; while obvious muscle denervation occurred in Romo1-cKO mice, and with increasing age, the degree of muscle denervation became more and more severe ( Figure 14 ), specifically manifested as: at 6 weeks of age, no nerve denervation occurred in Romo1-cKO mice ( Figure 14 in Fig. B); while obvious denervation occurred at 8 weeks of age, and about 59.6% of the muscles showed denervation ( Figure 14 in Fig. B), and this disease occurred earlier than muscle atrophy; at 24 weeks, muscle denervation further aggravated, and only 3.6% of the neuromuscular junctions had nerve innervation ( Figure 14 in Fig. B), and a significant reduction in acetylcholine receptors on the postsynaptic muscle membrane labeled with BTX could be seen ( Figure 14 in Fig. A), which was consistent with the muscle atrophy phenomenon.

[0080] (3)Axonal degeneration and myelin breakdown of the sciatic nerve occur in Romo1-cKO mice It was found that there was no significant difference in the cross-sectional area of the sciatic nerve between 8-week-old Romo1-cKO mice and WT mice. However, by 16 weeks of age, the cross-sectional area of the sciatic nerve in Romo1-cKO mice decreased significantly ( Figure 15 in A). Further analysis of the number and area of axons revealed that at 8 weeks of age, there was no significant change in the total number of axons in the sciatic nerve of Romo1-cKO mice compared with WT mice. However, by 16 weeks of age, the total number of axons decreased significantly, approximately 79% of that in WT mice ( Figure 15 in C). Importantly, although there was no significant change in the total number of axons in the sciatic nerve of 8-week-old Romo1-cKO mice, myelin breakdown began to occur in the axons ( Figure 15 in B). It was found that 0.8% of the axons showed myelin breakdown, twice that in WT mice ( Figure 15 in D). By 16 weeks of age, the number of axons with myelin breakdown further increased, accounting for 4.9%, which was significantly higher than that in WT mice ( Figure 15 in D), and some myelin structures had completely degenerated ( Figure 15 in B). Such axonal changes have also been reported in ALS patients, further indicating that this Romo1-cKO mouse model can be used as a new animal model for ALS.

[0081] (4)Progressive death of motor neurons in Romo1-cKO mice The lumbar spinal cord at the L5 layer of the same position of each mouse was embedded and sectioned transversely. The typical butterfly-shaped gray matter and peripheral white matter structures could be observed. A group of larger CHAT-positive neurons ( Figure 16 in A) were aggregated at the ventral horn of the gray matter, which were motor neurons.

[0082] Analysis of the number of motor neurons in the ventral horn of the L5 layer of the spinal cord in mice of different ages ( Figure 16 the dotted box in A) showed that at 6 weeks of age, Romo1-cKO mice had the same number of motor neurons as WT mice ( Figure 16 in B); at 8 weeks of age, the number of motor neurons in Romo1-cKO mice decreased significantly compared with WT mice; with age, the number of motor neurons in 24-week-old and 48-week-old Romo1-cKO mice further decreased ( Figure 17 in B). These results indicate that motor neuron death began to occur in Romo1-cKO mice at 8 weeks of age, and the number of motor neuron deaths increased with age.

[0083] To exclude the influence of changes in CHAT expression levels on the counting of CHAT-positive motor neurons, the number of large neurons in the ventral horn of the lumbar L5 spinal cord of mice was further analyzed and counted using Nissl staining. Nissl staining can indicate all neurons, and the large neurons in the ventral horn of the lumbar L5 spinal cord are motor neurons. The results showed that compared with WT mice, the number of large neurons in the ventral horn of the lumbar L5 spinal cord of Romo1-cKO mice began to decrease at 8 weeks of age, and the number of large neurons became fewer and fewer with increasing age ( Figure 17 ). The results of this experiment were consistent with the results of CHAT staining, further verifying that motor neurons in Romo1-cKO mice gradually died with increasing age.

[0084] (5)Microgliosis in the spinal cord of Romo1-cKO mice At 8 weeks of age, the total area occupied by microglia in the lumbar L5 spinal cord of Romo1-cKO mice increased by approximately 60% compared with WT mice ( Figure 18 B), and it was obvious that Romo1-cKO mice had more microglial processes ( Figure 18 A). These results indicate that the knockout of Romo1 causes the death of motor neurons and also leads to the proliferation of microglia.

[0085] The above results indicate that the ALS-like symptoms of gradually declining motor ability and ultimately paralysis and death exhibited by Romo1-cKO mice are due to muscle denervation caused by the degeneration and death of motor neuron axons.

[0086] Example 4: Pathological mechanism of mitochondrial cristae structure degeneration in Romo1-cKO mice 1. Experimental methods (1)Electron microscopy sample collection 1) Rinse the blood of mice with approximately 20 mL of 0.1 M PB (R23128, Yuanye).

[0087] 2) Perfuse with a solution containing 1.25% GA in 2% PFA (prepared with 0.1 M PB) for 30 mL over 15 minutes.

[0088] 3) Place the mouse in the prone position, fix the limbs, and sequentially remove the sciatic nerve, spinal cord, and brain tissue. Place the removed biological tissue in a fixative containing 2.5% GA in 4% PFA (prepared with 0.1 M PB), fix at room temperature for 1 - 2 hours, and then fix at 4°C for 24 hours.

[0089] (2)Electron microscopy sample preparation 1) Wash the fixed samples with 0.1 M PB 3 times, 20 minutes each time.

[0090] 2) Transfer the sample to a solution containing 2% OsO4 and 2.5 M formamide (diluted with 0.1 M PB), and soak it at room temperature for 2 hours.

[0091] 3) Treat the sample with a 3% ferrocyanide solution (diluted with 0.1 M PB), and soak it at room temperature for 2 hours.

[0092] 4) Treat the sample with 2% OsO4 (diluted with 0.1 M PB) at room temperature for 2 hours.

[0093] 5) Wash the sample with 0.1 M PB three times, 20 minutes each time.

[0094] 6) Treat the sample with an aqueous solution of 320 mM pyrogallol at pH 4.1 at room temperature for 1.5 hours.

[0095] 7) Wash with ultrapure water three times, 20 minutes each time.

[0096] 8) Treat the sample with 2% OsO4 (diluted with ultrapure water) at room temperature for 1.5 hours.

[0097] 9) Wash with ultrapure water three times, 20 minutes each time.

[0098] 10) Treat with 2% uranyl acetate, soak overnight at 4°C.

[0099] 11) The next day, transfer to a 50°C oven and treat for 30 minutes.

[0100] 12) Wash with ultrapure water three times, 20 minutes each time.

[0101] 13) Dehydration: The sample is dehydrated through 50%, 70%, 80%, 90% and 100% ethanol, 1:1 ethanol:acetone, and acetone in three gradient steps. Soak for 12 minutes each time.

[0102] 14) Epoxy resin infiltration and fixation.

[0103] 15) Embedding: Place in a 60°C oven for 24 hours.

[0104] (3) Electron microscopy imaging Electron microscopy images are all collected using a focused ion beam scanning electron microscope Crossbeam550 (ZEISS).

[0105] 2. Experimental results Electron microscopy results showed that at 6 weeks of age, there were no significant changes in the morphology and size of motor neurons, nor in the mitochondrial density within neurons, between Romo1-cKO mice and WT mice. At 16 weeks of age, although there were no significant changes in the size of motor neurons and mitochondrial density in WT mice compared to those at 6 weeks of age, the motor neurons in Romo1-cKO mice showed varying degrees of abnormalities. These included abnormal accumulation of mitochondria in a corner of some motor neurons, atrophy of some neuronal cell bodies, and in some neurons, almost no mitochondria could be seen.

[0106] Further analysis of the changes in the structure of individual mitochondria revealed that all mitochondria in the motor neurons of Romo1-cKO mice showed abnormal cristae structures ( Figure 19 A in, enlarged area 2), in sharp contrast to the normal mitochondrial cristae structures in adjacent non-cholinergic neurons ( Figure 19 A in, enlarged area 1). This result also strongly demonstrated that the identified motor neurons were cholinergic neurons with Romo1 knockout, indicating that the ALS-like phenotype in mice caused by ROMO1 deficiency was mainly due to abnormal function of cholinergic neurons.

[0107] Therefore, the mitochondrial areas in the motor neurons of Romo1-cKO and WT mice at different ages were statistically analyzed. It was found that compared with WT mice, the mitochondrial area in Romo1-cKO mice at 6 weeks of age had a tendency to increase ( Figure 19 B in), while at 16 weeks of age, the mitochondrial area in Romo1-cKO mice decreased significantly ( Figure 19 B in). Further, the mitochondrial curvature, that is, the length-width ratio, of 6-week-old mice was statistically analyzed. The closer this value is to 1, the more circular the mitochondrial morphology is, and the closer the value is to 0, the more filamentous the mitochondria are. Mitochondria were classified into three categories according to the curvature ( Figure 19 C in), and the proportions of mitochondria with the three types of curvatures were statistically analyzed. It was found that the proportion of mitochondria with high curvature in the motor neurons of 6-week-old Romo1-cKO mice increased, indicating that although Romo1 knockout did not cause a significant change in the size of motor neuron mitochondria at 6 weeks of age, it had already caused a morphological change of swelling and rounding ( Figure 19 C in).

[0108] Furthermore, an in-depth analysis was carried out on how the mitochondrial cristae structure in the motor neurons of Romo1-cKO mice changed at different ages. The results showed that as early as 6 weeks of age, the mitochondrial cristae structure in the motor neurons of Romo1-cKO mice had changed significantly, manifested as all the mitochondrial cristae changing from lamellar to circular ( Figure 20 A in), and only a small amount of lamellar cristae structure could be observed in extremely few mitochondria ( Figure 20The red arrow in A marks the lamellar cristae). The width of the mitochondrial cristae was measured, and the results showed that the width of the mitochondrial cristae in the motor neurons of 6-week-old Romo1-cKO mice was significantly increased compared with that of WT mice ( Figure 20 In B), indicating that the mitochondrial cristae were swollen, which was consistent with the swelling and rounding of the mitochondria ( Figure 19 In C). Therefore, it was speculated that the circular mitochondrial cristae structure in the motor neurons of Romo1-cKO mice might be caused by the swelling of the mitochondrial cristae, and the swelling of the cristae further caused the swelling of the mitochondria. It should be noted that this abnormal mitochondrial structure was also found in sporadic ALS patients, further indicating that the Romo1-cKO mouse model could be used as a new animal model of ALS.

[0109] Further analysis of the changes in the mitochondrial structure of the motor neurons of 16-week-old Romo1-cKO mice found that the cristae structure was further damaged and the number of cristae was significantly reduced ( Figure 21 In A), and importantly, there were various abnormal morphologies in the mitochondrial cristae structure, such as the observation of a multi-ringed mitochondrial structure similar to an onion ring ( Figure 21 In B1), and many abnormal mitochondria in the intermediate state of forming this onion-ring-like mitochondrial structure were also observed. Some onion-ring-like structures were forming inside the mitochondria, and a small number of swollen cristae structures could be observed outside the onion-like structure ( Figure 21 In B2 and B3). There were also swollen cristae inside some onion-like mitochondria ( Figure 21 In B4 and B5). And some onion-like mitochondria were surrounded by curved mitochondria, showing a tendency to engulf the onion-like mitochondria ( Figure 21 In B6). Based on these different mitochondrial morphologies, it was speculated that this onion-ring-like mitochondrial structure might be formed by the fusion of the inner mitochondrial cristae or the fusion of degenerated mitochondria.

[0110] Based on the above strong changes in the mitochondrial cristae structure, the mitochondria in the motor neurons of WT and Romo1-cKO mice were further classified into 4 categories according to the morphology of the cristae, namely: 1) Type I mitochondria: The mitochondrial cristae showed a typical lamellar structure; 2) Type II mitochondria: The mitochondrial cristae showed a circular structure; 3) Type III mitochondria: The mitochondrial cristae showed a lamellar structure, but the number was significantly reduced; 4) Type IV mitochondria: The mitochondrial cristae showed an onion-ring-like structure ( Figure 22 In A). The areas of the four types of mitochondria were measured respectively, and it was found that the areas of Type II and Type III mitochondria were significantly larger than that of Type I mitochondria, while the area of Type IV mitochondria was the smallest ( Figure 22 In B). Further statistics on the proportions of the four types of mitochondria in the motor neurons of WT and Romo1-cKO mice showed that at 6 weeks of age, Type I mitochondria were the main type in the motor neurons of WT mice, accounting for about 99% ( Figure 22C); while the mitochondria in the motor neurons of Romo1-cKO mice are mainly class II mitochondria, accounting for about 89%, only 3% are class I mitochondria, and 8% are class III mitochondria ( Figure 22 C). By 16 weeks of age, the motor neurons of WT mice still mainly have typical class I mitochondria, accounting for 99% ( Figure 22 C), while the mitochondria in the motor neurons of Romo1-cKO mice are mainly class III and class IV mitochondria, accounting for 40% and 58% respectively, and no longer contain typical class I mitochondria ( Figure 22 C). This trend of the change in mitochondrial cristae structure with age in Romo1-cKO motor neurons suggests that class II mitochondria are the early mitochondrial abnormalities caused by the deletion of ROMO1, and as mitochondrial function is further impaired, the mitochondrial structure further transforms into class III and class IV. At the same time, at 16 weeks of age, partial atrophy of the motor neuron cell bodies and complete disappearance of mitochondria can also be observed in Romo1-cKO mice, suggesting that the mitochondria in these ROMO1-deficient neurons may have been completely damaged.

[0111] In addition, the electron microscopy results show that in addition to the abnormal mitochondrial structure in the motor neurons of Romo1-cKO mice, the morphological structure of the endoplasmic reticulum also shows obvious abnormalities. Such as Figure 23 shown, compared with the WT mice of the same age, the endoplasmic reticulum in the motor neurons of 6-week-old Romo1-cKO mice shows mild irregular swelling, and at 16 weeks of age, the endoplasmic reticulum structure is further damaged, the endoplasmic reticulum presents fragmentation, and almost no typical endoplasmic reticulum structure can be observed in the neurons. It is speculated that the abnormal endoplasmic reticulum structure may be a secondary effect of the disorder of mitochondrial structure and function caused by the deletion of ROMO1.

[0112] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

Claims

1. A method for constructing an ALS mouse model with mitochondrial function impairment, characterized in that, By using the Cre / loxP system, a mouse model with the genotype Romo1 flox / flox Chat Cre+ was constructed. After activating the expression of CRE recombinase in cholinergic neurons, the Romo1 gene was selectively knocked out in this mouse model, spontaneously inducing the occurrence of amyotrophic lateral sclerosis.

2. The construction method of an ALS mouse model with mitochondrial function impairment according to claim 1, wherein, Construction of a mouse model with the genotype Romo1 by the Chat-Cre / loxP system flox / flox Chat Cre+ of 3. The method for constructing an ALS mouse model with mitochondrial function impairment according to claim 2, wherein, It includes the following steps: Cross Romo1 flox / flox genotype mice with Chat-Cre mice and screen for the first-generation Romo1 LoxP / + Chat cre / + genotype mice. Cross these genotype mice and screen for mice with the genotype Romo1 flox / flox Chat Cre+ in the second-generation, which are cholinergic-specific knockout Romo1 mice.

4. The method for constructing an ALS mouse model with mitochondrial function impairment according to claim 3, characterized in that, Screening in the second filial generation to obtain Romo1 with a genotype flox / flox Chat Cre+ Before obtaining cholinergic-specific knockout Romo1 mice, it also includes a PCR verification step.

5. The method for constructing an ALS mouse model with mitochondrial function impairment according to claim 4, characterized in that, The primer sequences for PCR verification are shown in SEQ ID NO.1 to 6.

6. Application of the ALS mouse model constructed by the method for constructing an ALS mouse model with mitochondrial function impairment according to any one of claims 1 to 5 in the study of the pathogenesis of amyotrophic lateral sclerosis.

7. Application of the ALS mouse model constructed by the method for constructing an ALS mouse model with mitochondrial function impairment according to any one of claims 1 to 5 in the screening of drugs for treating amyotrophic lateral sclerosis.

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