The mouse Optn (NM_001356487.1) coding sequencewas amplified by polymerase chain reaction (PCR). The full-length cDNA of the Optn gene was cloned into the pLVX-mOPTN-mCMV-ZsGreen lentiviral vector and sequenced for confirmation. The cloned vector and packaging components were co-infected with lentivirus-encapsulated HEK293T cells. The supernatant was collected by centrifugation after 48 h and concentrated using a concentration tube for backup. This technology was provided by Likely Biotechnology, Beijing, as shown in Supplementary Figure 2A.

Transgenic SOD1^G93A mice expressing mutant human SOD1 with a Gly93Ala substitution (B6SJL-Tg-SOD1^G93A-1Gur, No.002726) mice were kindly donated by Xiaojie Zhang of Shanghai Sixth People’s Hospital. The mice were bred in the SPF animal laboratory of Chifeng Municipal Hospital. Pure congenic versions of this mouse were nonviable, while heterozygotes exhibited pathological changes and symptoms similar to ALS. Male transgenic (Tg) mice were mated with female wild-type (Wt) B6SJL mice of the same genetic background in order to maintain and breed the obtained Tg animals.

At 30 days of age, the mice were identified using PCR to amplify mouse tail DNA and determine the mouse genotypes according to our previous report (Zhang et al., 2018), it was used to determine whether the offspring were Wt or Tg mice. Then, male mice in asymptomatic and symptomatic stages at 40 days and 90 days of age were chosen, randomizing into four groups: 40-day-old Wt mice (Wt40d), 40-day-old Tg mice (Tg40d), 90-day-old Wt mice (Wt90d), and 90-day-old Tg mice (Tg90d) (3 mice per group). The main objective of present study was to investigate alterations in Optn protein expression within the spinal cord of these mice across different stages of the disease progression.

Optn overexpression in the symptomatic phase: An Optn overexpression group (Tg-Optn) and a control group (Tg-NC) were established by randomly selecting and assigning eighteen symptomatic male SOD1^G93A mice at 90 days of age, with 9 mice in each group. The Tg-Optn group received a single-injection of 10 μl of Optn overexpression lentiviral vector (titer 5 × 10⁸/mL) into the right lateral ventricle (AP: –0.22 mm, ML: –1.0 mm, DV: –2.35 mm), while the Tg-NC group was injected with an equal volume of empty lentivirus (Chen et al., 2015). The Rotarod Test was utilized to assess the locomotor ability of the mice, alongside recording their weight changes and survival times.

Optn overexpression in the pre-symptomatic phase: 50 male Tg mice were randomly divided into Tg-Optn and Tg-NC groups of 25 mice each. Mice in the Tg-Optn group were single-injected with 10 μl of Optn-overexpressing lentiviral vector (titer 5 × 10⁸/mL) into the right lateral ventricle at 60 days of age. Mice in the Tg-NC group were injected with an equal amount of lentiviral vector (Chen et al., 2015). A total of 12 mice from each group were monitored for disease onset and survival duration, with their locomotor abilities assessed using the rotarod test, onset dates determined, and weight changes and survival times recorded. The remaining 13 mice from each group were sacrificed at 90 days of age, and samples from their lumbar segments were extracted for pathological, molecular analysis and transmission electron microscopy analysis.

The animal experiments involved were approved by the Experimental Animal Ethics Committee of Chifeng Municipal Hospital and were conducted in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals.

The body weight of each mouse was measured and recorded once every 2 days, began from the lentiviral injection until death, in order to compare the weight changes between the two groups.

For motor function assessment, the rotarod test was conducted after 7 days of training, maintaining a constant speed of 20 rpm from the age of 70 days. This test was performed once every 2 days. The mice were allowed to run on the rotarod apparatus for a maximum of 5 min and the latency to fall off the rotating rod was recorded. The onset time of disease was defined when animals fell off the rotarod three times within 5 min (Zhang et al., 2014).

As per animal ethics guidelines, the determination of the mouse’s death date involved assessing their hind limb mobility and ability to consume food. When a mouse displayed complete paralysis of the hind limbs and inability to eat, it was positioned on a flat surface with its back toward the ground. Failure to autonomously return to its normal position within 30 s marked the official time of death, prompting the required euthanasia process (Koh et al., 2007).

Eight mice per group were used to extract the lumbar spinal cord. At 90 days of age, mice were anaesthetized by injection intraperitoneally with 1.25% tribromoethanol (0.2 mL/10 g body weight) and perfused transcardially with 40 mL of pre-cooled phosphate-buffered saline (PBS), then four lumbar spinal cords were extracted and stored at −80°C refrigerator for further protein cleavage. The remaining four mice continued to be perfused with 20 mL of 4% paraformaldehyde (PFA) in a fast and then slow manner. The spinal cord of lumbar segments was extracted on ice, immersed in 4% PFA, and fixed overnight. Then, tissues were immersed in 15 and 30% sucrose solutions for 24 h each to dehydrate and precipitate the sugar, respectively. Finally, the tissues were embedded using OCT and rapidly stored at −80°C refrigerator. OCT-embedded tissues were placed in a −20°C frozen sectioning machine (Leica) for serial sectioning at a thickness of 10 μm and mounted on electrostatically coated slides.

The spinal cord frozen sections were initially placed into a pre-prepared 1% toluidine blue staining solution for 10 min. They were then removed, rinsed using distilled water, and consecutively immersed in gradient alcohol dehydration solutions of 70, 80, 90, and 100% for 2 min each. Subsequently, sections were washed with xylene solution twice (5 min each time), then sealed by neutral gum and dried naturally. Finally, the sections were observed and photographed using the microscope (Olympus BX51). The unilateral anterior horn of each animal was examined by a technician who was unaware of the experimental design. A total of 20 sections, 10 μm thick, were collected at 10-slice intervals from each animal. To count MNs, each of the 4 sections of serial sections (50 per mouse) was stained with Nissl staining. The counting of MNs in the unilateral anterior horn of each slice was performed in a blinded manner based on the following criteria: (i) their location in the anterior horn region before the spinal cord’s central canal, (ii) a diameter greater than 20 μm, and (iii) the presence of distinct nuclei (Zhang et al., 2018).

TUNEL staining was utilized to specifically detect apoptosis using standard protocol. Briefly, frozen sections were first fixed with 4% PFA for 30 min and then washed three times with PBS for 10 min each. PBS containing 0.5% Triton X-100 was then added to the frozen sections and incubated for 5 min at room temperature. The sections were then washed twice with PBS and incubated in TUNEL staining solution for 60 min at 37°C, followed by three washes with PBS. Finally, the sections were sealed with an anti-fluorescence quenching solution (Solarbio, S2100) and observed under an Olympus fluorescence microscope.

Frozen lumbar spinal cord sections were rinsed three times with PBS for 5 min each, then blocked and permeabilized with 5% bovine serum albumin (BSA) in PBS containing 0.3% Triton X-100 for 1 h at room temperature. Slides were then incubated with primary antibodies Optn (1:400, Proteintech, 10837-1-AP), SMI-32(1:1000, Abcam, ab8135), Iba1 (Wako, 1:500, 019-19741), GFAP (Proteintech, 1:200, 16825-1-AP), LC3 (Proteintech, 1:200, 14600-1-AP) and P62 (Proteintech, 1:200, 18420-1-AP) at 4°C overnight. After rinsing with PBS, the sections were incubated for 90 min at room temperature in the dark with the appropriate secondary antibodies Alexa Fluor 592 (Proteintech, 1:2,000) and Alexa Fluor 488 (Cell Signaling, 1:2,000). The sections were then washed with PBS and sealed with an anti-fluorescence quenching sealer. They were then observed under the Olympus fluorescence microscope. In the ventral part of the spinal cord removed from each mouse, the number of Ibal-positive microglia and GFAP-positive astrocytes with intact cytoplasm was counted, statistical analyses were then performed.

To conduct TUNEL staining and immunofluorescence cell counting, five sections per mouse, spaced 100 μm apart, were photographed under a microscope. Each section was imaged bilaterally in the anterior horn region, capturing two fields of view per section. Subsequently, an individual blinded to the study grouping counted the number of positively stained cells in the bilateral anterior horn region. For statistical analysis, the average number of positive cells from the five sections per mouse was calculated, and the number of positive cells per mm² was determined. Additionally, the relative fluorescence density of P62 staining was analyzed using ImageJ software (Schneider et al., 2012).

Total tissue protein was extracted from lumbar spinal cord of Tg-NC and Tg-Optn mice with Radio Immunoprecipitation Assay (RIPA) protein lysis buffer containing 1% phenylmethanesulfonylfluoride (PMSF) and protease inhibitor. After determining the protein concentration, 15 μg of protein was denatured at 100°C for 5 min and mixed with sample loading buffer. The denatured samples were then loaded onto sodium dodecyl sulfate-polyacrylamide gels for separation, and subsequently transferred to polyvinylidene fluoride membranes. The membranes were then incubated with primary antibodies Optn (1:2,000, Proteintech, 10837-1-AP), β-actin (1:2,000, Proteintech, 20536-1-AP), GAPDH (1:2,000, Proteintech, 60004-1-Ig), CD206 (1:2,000, Proteintech, 18704-1-AP), CD86 (1:1000, Santa Cruz, sc-28347), OPA1 (1:2,000, Proteintech, 27733-1-AP), Mfn1 (1:2,000, Proteintech, 13798-1-AP), Mfn2 (1:2,000, Proteintech, 12186-1-AP), Drp1(1:2,000, Proteintech, 12957-1-AP), Mff (1:2,000, Proteintech, 17090-1-AP), PINK1 (1:2,000, Proteintech, 23274-1-AP), Parkin (1:1,000, Proteintech, 14060-1-AP), TOM40 (1:1,000, Santa Cruz, sc-365467), TIM23 (1:1,000, Santa Cruz, sc-514463), Cytochrome c (1:2,000, Proteintech, 10993-1-AP), Bcl-2 (1:2,000, Proteintech, 68103-1-Ig) and Bax (1:10,000, Proteintech, 50599-2-Ig) at 4°C overnight, after blocking with 5% non-fat milk powder for 1 h at room temperature and three 10-min washes in tris buffered saline with tween-20 (TBST). The membranes were then incubated with HRP-conjugated goat anti-mouse IgG or goat anti-rabbit IgG secondary antibodies for 1.5 h at room temperature. Finally, the membranes were washed three times and imaged using a BIORAD chemiluminescence imager, using the Enhancement of Luminescence Kit (Proteintech, PK10003) as the substrate for visualization.

Total RNA was extracted from spinal cord samples of Tg-NC and Tg-Optn mice (n = 3 in each group) using the TransZol Up (Transgene, ET111-01-V2). Subsequently, The extracted RNA was synthesized to cDNA using the EasyScript^® First-Strand cDNA kit (Transgene, AE301-02). For the quantification of gene expression, real-time PCR was performed with the TransStart^® Top Green qPCR SuperMix (Transgene, AQ131-01), and the results were monitored by the BIO-RAD real-time PCR system. The primer sequences were provided upon request as summarized in Table 1. The relative expression levels of each primer sequences mRNA were analyzed by the 2^–ΔΔCt algorithm normalizing to GAPDH and relative to the Tg-NC groups.

Fresh tissues were collected from the L4-5 lumbar spinal cord of Tg-NC and Tg-Optn mice within 1–3 min and sized to 1 mm³. Samples were then transferred to Eppendorf tubes containing fresh electron microscope fixative and stored at 4°C. The tissues were then washed 3 times for 15 min each with 0.1 M PBS (phosphate-buffered, pH 7.4). The samples were initially fixed with 1% OsO4 in 0.1 M PB (pH 7.4) for 2 h at room temperature, followed by post-fixation in dark. Subsequently, the tissues were rinsed in 0.1 M PB (pH 7.4) three times for 15 min each to eliminate the OsO4. Following this, the tissues underwent sequential dehydration in increasing concentrations of ethanol (30, 50, 70, 80, 95%, and two rounds of 100%) for 20 min each. Finally, 100% acetone was applied twice for 15 min in each round to complete the dehydration process. The tissue samples underwent a series of steps for resin infiltration and embedding. Initially, samples were treated with a mixture of Acetone and EMBed 812 in a 1:1 ratio for 2–4 h at 37°C, followed by another round of treatment with Acetone and EMBed 812 in a 1:2 ratio overnight at 37°C. Subsequently, the samples were subjected to pure EMBed 812 for 5–8 h at 37°C. Once the infiltration process was completed, the pure EMBed 812 was poured into the embedding plates, and the tissues were inserted into the pure EMBed 812, followed by an overnight incubation at 37°C. The embedding plates containing the resin and samples were then transferred to a 65°C oven for polymerization, which lasted for more than 48 h. Subsequently, the resin blocks were removed from the embedding plates and set aside at room temperature for further processing. The resin blocks were then sliced into thin sections of 60–80 nm on an ultramicrotome, and the tissues were carefully transferred onto 150-mesh cuprum grids with formvar film. The samples were subsequently stained with a 2% uranium acetate saturated alcohol solution in the dark for 8 min, followed by rinsing in 70% ethanol three times and then in ultra-pure water three times. Additionally, a 2.6% lead citrate solution was used for staining to avoid CO₂-induced artifacts, followed by rinsing with ultra-pure water three times. After being dried with filter paper, the cuprum grids were placed in a grid box and left to dry overnight at room temperature. Finally, the cuprum grids were examined under a TEM, and images were captured for analysis. In the electron microscopic analysis, two groups of ALS model mice, Tg-NC and Tg- Optn, were employed, with each group consisting of five mice. From the spinal cord samples of each mouse, fifteen electron microscope images, including those of MNs, were randomly selected for examination. A blinded evaluator, who was unaware of the experimental groupings, conducted the analysis by counting and measuring the number and maximum diameter of mitochondria in each image. The average values derived from these measurements were subsequently used for statistical analysis. All TEM techniques in this experiment were provided by Wuhan Servicebio Technology Co., Ltd.

The data were presented as Mean ± SD. Statistical analyses were performed using one-way or two-way ANOVA to a normal distribution. Non-parametric tests were performed for non-normally distributed data by using SPSS 24.0. In addition, the time to onset and survival data were statistically examined using Kaplan-Meier survival analysis. Graphs of the results were generated using GraphPad Prism 8. Statistical significance was set at a P< 0.05.

Western blotting was used to determine Optn protein expression in the lumbar spinal cords. Our data indicated that the Optn protein in spinal cords of 40-day SOD1^G93A mice was approximately 50% higher than that of age-matched Wt mice. However, after the disease onset (at 90 days), Optn expression decreased significantly and was about 60% lower than Wt mice (Figures 1A,B). To identify the type of cells in which Optn is mainly expressed, lumbar spinal cord sections from SOD1^G93A mice were immunofluorescently stained with antibodies against Optn. Consistent with the Western blotting results, immunofluorescence staining revealed similar dynamics of Optn expression in the lumbar spinal cords of SOD1^G93A mice and Optn predominantly stained on neurons (Figure 1C and Supplementary Figure S1). This aligns with previous studies that demonstrate Optn’s specific localization in neurons and its close association with the pathological processes of various neurodegenerative diseases (Mori et al., 2012; Korac et al., 2013; Nakamura et al., 2014; Wise and Cannon, 2016; Wen et al., 2024). These findings suggest that pathology-related changes in Optn levels may be involved in neurodegeneration during ALS progression.

In order to determine the effect of the Optn overexpression in the spinal cord, Optn protein levels were assessed by immunoblotting at 1, 2, 3, and 4 weeks after Optn lentivirus injection in Wt mice. Our findings demonstrated a significant increase in Optn expression in the spinal cord after 1, 2, 3, and 4 weeks of injection compared to control group. Moreover, the Optn protein levels remained consistent throughout this 4-week period (Supplementary Figures S2B,C). We also confirmed a significant increase in Optn expression in the spinal cord of SOD1^G93A mice after 4 weeks of lentiviral infection by western blot and fluorescence photomicrography (Supplementary Figures S2D–F).

Moreover, our findings revealed that the time of weight loss in Tg-Optn mice was notably later than that in Tg-NC mice, as indicated by the lower decrease rate curve for Tg-Optn mice compared to Tg-NC mice (Figure 2A). Furthermore, Tg-Optn mice exhibited a longer mean stick-turning time than Tg-NC mice at the same age (Figure 2B). Additionally, Optn overexpression significantly postponed disease onset by approximately 9.7% (106.00 ± 3.91 days vs. 96.67 ± 3.55 days, P < 0.01) at the pre-symptomatic stage (Figures 2C,D) and extended survival by about 7.2% (124.83 ± 7.88 days vs. 116.00 ± 7.82 days, P < 0.05) of SOD1^G93A transgenic mice (Figure 2E), but failed to extend the duration of the disease (Figure 2F).

Before that, we also increased the expression of Optn in symptomatic (90-day-old) SOD1^G93A mice. However, subsequent statistical analysis revealed that Optn overexpression had no significant effect on delaying the time of disease onset, reducing weight loss or prolonging survival at 90 days of age (Supplementary Figures S3A–D). These results strongly indicate that Optn maybe beneficial in improving ALS condition before the disease onset.

As we know, an important pathological feature of ALS is the degeneration of MNs (Pasinelli and Brown, 2006). Thus, by counting large lumbar MNs within the anterior horn lamina IX using Nissl staining at 90 days of age, we found that MNs increased by 46.8% (P < 0.01) in the Tg-Optn group (575 ± 43) compared to the Tg-NC group (358 ± 63) in the L4-5 segments of SOD1^G93A mice (Figures 3A,B).

Apoptosis has been implicated as the main mechanism of neuronal death in ALS. Therefore, we then examined the effect of Optn overexpression on apoptosis by TUNEL staining. Our data reveled a significant decrease in the number of TUNEL-positive cells in the Tg-Optn group compared to the Tg-NC group (Figures 3C,D, P < 0.001). Additionally, Optn overexpression was found to regulate the expression of apoptosis-related factors Bcl-2 and Bax, resulting in a 30% decrease of the pro-apoptotic protein Bax and a 20% increase in the anti-apoptotic protein Bcl-2 (Figures 3E–G). Moreover, we found that cytochrome C (Cyt C) protein expression levels were significantly reduced in lumbar spinal cord of SOD1^G93A mice after Optn overexpression in the pre-symptomatic phase (Figures 3H,I).

The inflammation of the CNS is a critical feature of ALS neuropathology, and NF-κB plays a cardinal role in cellular inflammatory and immune response processes. In our experiments, no statistically significant difference was achieved in the number of microglia (Figures 4A,B). However, Optn overexpression resulted in microglial activation as evidenced by larger microglial cytoplasm and shorter synapses compared to the Tg-NC group (Figures 4A,C). Conventionally, microglial activation could be distinguished into an anti-inflammatory M2 phenotype expressing CD206 and a pro-inflammatory M1 phenotype expressing CD86 (Kwon and Koh, 2020). The Western Blot results of our present study indicated a significant increase in CD206 expression and a non-significant trend of higher levels of CD86 (Figures 4D–F). Meanwhile, we observed that Optn overexpression led to a significant increase in the mRNA levels of the anti-inflammatory cytokines IL-10 and TGF-β in the pre-symptomatic phase. Conversely, there was no statistically significant change in the mRNA levels of the pro-inflammatory cytokines IL-1β and TNF-αin the pre-symptomatic phase. This suggests that Optn overexpression may selectively enhance anti-inflammatory pathways without obviously affecting pro-inflammatory cytokine expression between Tg-NC and Tg-Optn groups (Figures 4G–K). Moreover, there was a significant reduction in the astrocytes counts in the Tg-Optn group (Figures 4L,M). Meanwhile, the average area of astrocyte soma increased significantly in the Tg-Optn group (Figure 4N). These findings suggest Optn overexpression might have an anti-inflammatory effect in the pre-symptomatic phase.

Mitochondria, being vital organelles, plays a crucial role in providing cells with the energy necessary for life activities (Duann and Lin, 2017). These abnormalities encompass structural deformities, pathological swelling, excessive fission, dysregulated mitophagy, and a compromised mitochondrial membrane potential, ultimately disrupting cellular bioenergetics and neuronal homeostasis (Guo et al., 2024). Transporting proteins across the inner and outer mitochondrial membranes is crucial for maintaining mitochondrial function. This transportation process is facilitated by a series of protein complexes, including TOM complex in the outer membrane and the TIM23 complex in the inner membrane (Wiedemann and Pfanner, 2017). TOM and TIM are membrane protein complexes, with Tom40 serving as the core channel component of the TOM complex and Tim23 fulfilling a similar role in the TIM complex. Variations in their expression levels affect mitochondrial quality. Mitochondrial dysfunction plays a critical role in the onset and progression of ALS, and previous research, including TEM studies, has linked SOD1 mutations to a cascade of mitochondrial abnormalities. Specifically, in VSC4.1 cells expressing the SOD1^G93A mutation, increased mitochondrial ROS production leads to a decline in mitochondrial membrane potential, further impairing crucial mitochondrial functions such as the operational integrity of the TIM23 complex. Here, we observed an approximately 1-fold increase in the levels of the outer membrane transporter protein TOM40 and the inner membrane transporter protein TIM23 following Optn overexpression (Figures 5A–C). In addition, Optn overexpression was associated with fewer mitochondria undergoing swollen vacuolization in the MN soma and axon, according to TEM analysis (Figures 5D,E). Additionally, the anterior horn MNs in mice from the Tg-Optn group showed that the number of normal mitochondria increased by approximately 16.1% (11.33 ± 1.52 vs. 14.17 ± 1.52, P < 0.05) compared to those in the Tg-NC group (Figure 5F), while more giant and swollen mitochondria were found in the MNs soma of the Tg-NC group, and the average maximum diameter of the mitochondria decreased by approximately 41% in the Tg-Optn group (1.17 ± 0.15 μm vs. 0.67 ± 0.11 μm, P < 0.05) (Figure 5G). All these results indicated that Optn overexpression could improve mitochondrial quality in SOD1^G93A mice.

The maintenance of mitochondrial health and function is achieved through the mitochondrial quality control system including mitochondrial dynamics and mitophagy. Mitochondrial fusion and mitochondrial fission are the primary pathway to regulate the mitochondria morphology and mitochondrial dynamics. Previous studies have demonstrated that SOD1 mutations lead to reduced expression of mitochondrial fusion-associated proteins, specifically optic atrophic protein 1 (OPA1), Mitofusin 1 (Mfn1), and Mitofusin 2 (Mfn2) (Ferri et al., 2010). Furthermore, these mutations impaired autophagic flux, resulting in compromised degradation and subsequent intracellular accumulation of P62 (Zhang et al., 2014). Our data showed that Optn overexpression resulted in a significant up-regulation of mitochondrial fusion-associated proteins in SOD1^G93A mice, i.e., OPA1, Mfn1, and Mfn2 (Figures 6A–D). Meanwhile, the expression of mitochondrial division-related proteins Dynamin-related protein 1 (Drp1) and Mitochondrial fission factor (Mff) were also significantly elevated (Figures 6A,E,F), These results suggest that Optn may regulate mitochondrial dynamics by controlling fusion and division.

Mitophagy, the selective autophagy of dysfunctional mitochondria from the cytoplasm, is another important pathway for maintaining mitochondrial quality (Heinemeyer et al., 2019; Akabane et al., 2023). The PINK1/Parkin signaling is a key pathway in mitophagy regulation, in response to mitochondrial damage. PINK1 can phosphorylate and recruit Parkin to initiate autophagy via autophagy receptors such as OPTN (Lazarou et al., 2015). In the present study, we observed a significant increase in the protein expression levels of PINK1 and Parkin in the Tg-Optn group compared to the Tg-NC group (Figures 6A,G,H). Furthermore, the double immunostaining for LC3, P62, and Optn illustrated a marked increase in LC3 puncta and a decrease in P62 density in the Optn-positive cells of the Tg-Optn group in contrast to the Tg-NC group (Figures 6I–L). These findings indicate that Optn has the potential to stimulate mitophagy in SOD1^G93A mice.