Introduction
Amyotrophic lateral sclerosis (ALS) is a chronic progressive neurodegenerative disease that primarily affects the upper and lower motor neurons.1 Patients often initially present with progressively worsening muscle weakness and atrophy. As the disease advances, it gradually involves the muscles responsible for swallowing, speech, and respiration. In the late stages, widespread muscle atrophy becomes pronounced, leading to dysphagia and respiratory muscle paralysis, with the majority of patients ultimately succumbing to respiratory failure.2 Research suggests that multiple mechanisms collectively contribute to the pathogenesis of ALS, including oxidative stress, excitotoxicity, mitochondrial and proteasomal dysfunction, abnormal RNA metabolism, impaired axonal transport, and neuroinflammation.3 Currently, Riluzole and Edaravone are the only drugs approved by the FDA for ALS treatment; however, they can only partially alleviate symptoms and marginally extend patient survival, without halting or reversing disease progression.4 Although emerging strategies like gene therapy offer new directions for ALS treatment,5 they remain largely exploratory, and effective therapeutic targets and interventions for the disease are still scarce.
The TAR DNA-binding Protein of 43 kDa (TDP-43) is a key pathological hallmark in various neurodegenerative diseases, including ALS.6,7 Abnormal, ubiquitinated, and phosphorylated TDP-43 inclusions are found in the affected neurons of approximately 97% of ALS patients and a significant subset of patients with Frontotemporal Lobar Degeneration (FTLD).8,9 The core pathogenic mechanisms of TDP-43 involve a loss of its nuclear function, leading to dysregulation of RNA metabolism,10 and the abnormal aggregation of its C-terminal domain in the cytoplasm, forming neurotoxic amyloid aggregates. In familial ALS, various C-terminal mutations disrupt protein homeostasis,11 causing multiple cellular functional defects and activating degradation pathways, thereby creating a vicious cycle.12
ALDOA, a member of the aldolase family, plays a crucial role in glycolysis and gluconeogenesis by reversibly catalyzing the conversion of fructose-1,6-bisphosphate to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.13 Dysregulation of its expression can mediate glycolytic dysfunction. Previous studies have indicated that enhanced glycolysis promotes the progression of neurodegenerative diseases such as Parkinson’s disease (PD).14,15 A proteomic analysis of cerebrospinal fluid from AD patients revealed a significant increase in ALDOA expression levels.16 However, the interaction between TDP-43 and ALDOA remains to be elucidated. Meanwhile, the impact of TDP-43 gene mutations on ALDOA function in the context of ALS requires further investigated.
Proteomics is a discipline focused on the study of the proteome, dedicated to the systematic analysis of protein expression levels, post-translational modification states, and protein-protein interaction networks, thereby comprehensively revealing the overall molecular mechanisms of disease pathogenesis and cellular metabolic regulation.17 This technology has been widely applied in the field of neurodegenerative disease research, playing a significant role in screening disease-related biomarkers and providing in-depth insights into pathogenic molecular mechanisms.18,19 This study comprehensively utilizes proteomic analysis and molecular biology experiments to screen proteins interacting with TDP-43 and construct their interaction network. It further validates changes in ALDOA expression levels to deeply explore the interaction between TDP-43 and ALDOA and its potential mechanism in ALS. The aim is to provide new theoretical foundations and potential therapeutic targets for ALS pathological mechanism research and clinical treatment.
Materials and Methods
Materials
The HEK-293T cell line was purchased from Procell Life Science & Technology Co., Ltd. Fetal bovine serum (FBS) was obtained from Lonsera (S711-001S, China). Lipofectamine™ 3000 Transfection Reagent was provided by Thermo Fisher Scientific (L3000015, USA). Opti-MEM™ I Reduced Serum Medium was purchased from Thermo Fisher Scientific (31985070, USA). The BCA Protein Assay Kit was acquired from Solarbio (PC0020, China).
Cell Culture
All cells were cultured and preserved at the Fifth Affiliated Hospital of Sun Yat-sen University. HEK-293T cells were maintained in DMEM medium (GIBCO, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. For cell resuscitation, cryovials were retrieved from liquid nitrogen and quickly placed in a 37°C water bath with gentle shaking until thawed. After complete thawing, the cell suspension was transferred to a centrifuge tube, mixed with an appropriate amount of complete medium, and subjected to low-speed centrifugation followed by supernatant removal. The cells were then seeded in DMEM medium (GIBCO, USA) containing 10% fetal bovine serum and 1% penicillin/streptomycin, and incubated at 37°C in a 5% CO2 incubator. Cells beyond the 20th passage were excluded from the experiments.
Cell Transfection
HEK293T cells at 60% confluence were transfected with the following plasmids: Vector (GL107 pSLenti-EF1-EGFP-P2A-Puro-CMV-MCS-3×FLAG-WPRE), Flag-TDP-43 (pSLenti-EF1-EGFP-P2A-Puro-CMV-Tardbp(Tdp43)-3×FLAG-WPRE), and Flag-TDP-43 M337V (pSLenti-EF1-EGFP-P2A-Puro-CMV-Tardbp(p.M337V)-3×FLAG-WPRE). For each transfection, 3 μg of plasmid was diluted in 200 μL of Opti-MEM™ I Reduced Serum Medium and incubated for 5 minutes. Separately, 6 μL of Lipofectamine™ 3000 transfection reagent was mixed with 200 μL of Opti-MEM™ I Reduced Serum Medium and also incubated for 5 minutes. The two solutions were then combined, mixed gently, and further incubated for 5 minutes. The resulting mixture was added to the respective cell groups, and cells were harvested 48 hours post-transfection.
Sample Preparation
Proteins were extracted from the cells using a mixture of protein lysis buffer and protease inhibitors. After lysis and centrifugation, the supernatant was collected. Protein concentration was determined using the BCA Protein Assay Kit to ensure consistency across groups in subsequent experiments. Input samples were prepared as controls, and co-immunoprecipitation (IP) samples were prepared to enrich proteins interacting with TDP-43.
Mass Spectrometry Analysis and Screening
Peptides were dissolved in mobile phase A and separated using an EASY-nLC 1200 ultra-high-performance liquid chromatography (UHPLC) system. Mobile phase A consisted of aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B consisted of aqueous solution containing 0.1% formic acid and 90% acetonitrile. The liquid chromatography gradient was set as follows: 0–14.5 min, 6%–22% B; 14.5–17.5 min, 22%–34% B; 17.5–19 min, 34%–80% B; 19–20 min, 80% B, with a flow rate maintained at 700 nl/min.
After separation by the UHPLC system, the peptides were ionized via an NSI ion source and then analyzed using an Orbitrap Exploris 480 mass spectrometer. The ion source voltage was set to 2300 V, and the FAIMS compensation voltage (CV) was set to −45 V. Both the precursor ions and their secondary fragments were detected and analyzed using the high-resolution Orbitrap. The primary mass spectrometry scanning range was set to 350–1400 m/z with a resolution of 60,000; the secondary mass spectrometry scanning range started fixed at 120 m/z with a resolution of 15,000.
Data acquisition was performed using data-independent acquisition (DIA), where after full MS1 scanning, peptide ions within multiple consecutive m/z windows were fragmented in the HCD collision cell with 27% fragmentation energy, followed by sequential MS2 analysis. To improve mass spectrometry efficiency, the automatic gain control (AGC) was set to 1E6 and the maximum injection time was set to 22 ms.
Bioinformatics Analysis
The database accession numbers or protein sequences of differentially expressed proteins identified from comparative groups were compared against the STRING protein-protein interaction database. Interactions with a confidence score > 0.7 (high confidence) were extracted to construct the protein interaction network of differentially expressed proteins. The network was then visualized using the R package “visNetwork”.
Co-Immunoprecipitation Assay
First, collect the cells to be analyzed and lyse them on ice using a pre-cooled RIPA lysis buffer for 30 minutes. Then, centrifuge the lysate at 4°C and 12,000 × g for 15 minutes to remove cell debris. The resulting supernatant, referred to as Supernatant A, contains the total protein extract, and its concentration should be determined.
Next, divide Supernatant A equally into two portions: one for the experimental group, to which a specific antibody against the target protein is added, and the other for the negative control group, to which the same amount of a non-specific immunoglobulin from the same species or IgG is added. Incubate the mixtures overnight at 4°C with slow agitation to allow sufficient formation of immune complexes between the antibodies and the target protein.
The following day, add an appropriate amount of Protein A/G magnetic beads, pre-washed with lysis buffer, to each reaction system. Continue incubation at 4°C with slow agitation for 2–4 hours to efficiently capture the antibody-target protein complexes by the magnetic beads.
After incubation, place the samples on a magnetic stand and discard the supernatant. Wash the precipitated magnetic beads 3–4 times with pre-cooled lysis buffer to thoroughly remove non-specifically bound proteins.
Finally, add an appropriate volume of 1× SDS-PAGE loading buffer to the magnetic bead pellet, heat the mixture at boiling temperature for 5–10 minutes, and then collect the supernatant by centrifugation for subsequent Western Blotting analysis.
Western Blot
SDS-PAGE gels with concentrations ranging from 6% to 12% were prepared. Samples were loaded at 30 μg of protein per well. Electrophoresis was performed at a constant voltage of 100 V to separate the proteins, which were then transferred to a PVDF membrane under a constant current of 400 mA. The membrane was subsequently blocked with TBST containing 5% skim milk at room temperature for 1 hour.
After blocking, the membrane was incubated overnight at 4 °C with the following primary antibodies: anti-ALDOA (1:1000, 11,217-1-AP, Proteintech, China), anti-TDP-43 (1:1000, 10,782-2-AP, Proteintech, China), and anti-Tubulin (1:5000, 11,224-1-AP, Proteintech, China). The membrane was then washed three times with TBST to remove unbound primary antibodies.
Next, corresponding species-specific HRP-conjugated secondary antibodies (1:5000) were applied and incubated at room temperature for 1 hour. After incubation, the membrane was washed again with TBST. Finally, specific protein bands were detected using a chemiluminescence imaging system, and band intensity was quantified to compare the expression levels of target proteins across different samples.
Immunofluorescence
After removing the culture medium, HEK293T cells were gently washed twice with room temperature PBS buffer for 5 seconds each. Then, 4% neutral formaldehyde fixative was added to cover the cells and incubated at room temperature for 15 minutes. After fixation, the fixative was discarded, and the cells were washed three times with pre-chilled PBS buffer (4°C) for 5 minutes each. Subsequently, the cells were permeabilized with Triton X-100 on ice for 10 minutes.
After permeabilization, the samples were covered with 5% goat serum and blocked at room temperature for 1 hour. The blocking solution was then removed, and the samples were incubated overnight at 4°C with the following primary antibodies: anti-ALDOA (1:200 dilution, Cat# ab252953, Abcam, USA) and anti-TDP-43 (1:200 dilution, Cat# 10782-2-AP, Proteintech, Wuhan, China).
The next day, the primary antibodies were discarded, and species-specific HRP-conjugated secondary antibodies were applied to cover the samples. Incubation was carried out at room temperature for 2 hours in the dark. The samples were then washed three times with PBS buffer. Finally, an anti-fade mounting medium was applied, and the images were observed and captured under a fluorescence microscope.
Statistical Analysis
Data from in vivo experiments were analyzed using GraphPad Prism 10.3.1 and the statsmodels Python package (v0.13.0). An unpaired t-test was employed for comparisons. All data presented as mean ± SD. Statistical significance was set at P < 0.05.
Results
Construction of TDP-43 Wild-Type and TDP-43 Mutant Cell Models
Forty-eight hours after plasmid transfection into HEK293T cells from each group, transfection efficiency was observed under a fluorescence microscope. The results showed a high fluorescence expression rate in each group, indicating successful plasmid transfection (Figure 1A). Silver staining results revealed clear protein bands in the silver-stained profiles of all samples, demonstrating good integrity and effective separation of the protein samples. Under equal protein concentrations, differences in protein expression patterns were observed among the groups, suggesting that plasmid transfection led to alterations in the protein expression profiles (Figure 1B).
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Figure 1 Successful plasmid transfection in HEK293T cells and qualified protein samples. (A) Fluorescence microscopy images showing transfection efficiency in each group of cells (200×, n=3); (B) Silver staining results (n=3).
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Proteomic Analysis Reveals an Interaction Between ALDOA and TDP-43
Based on the mass spectrometry results, we successfully identified a specific peptide sequence of the ALDOA protein using affinity purification coupled with mass spectrometry. In the MS/MS spectrum of this peptide, we observed continuously distributed b-ions (b2–b4, b8–b11) and y-ions (y3, y7–y12) fragment signals. The most abundant fragment ions included y10⁺ (m/z 1049.51), y11⁺ (m/z 907.44), and y12⁺ (m/z 1162.6), while characteristic ions such as b3⁺ (m/z 284.2) and b4⁺ (m/z 355.23) were also detected in the low-mass region. These complementary ion series provided comprehensive fragment coverage, confirming that the peptide sequence is GAA DESGSK, corresponding to residues 120–128 of the ALDOA protein, which confirms the definite expression of ALDOA in the sample (Figure 2A).
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Figure 2 Proteomic analysis reveals an interaction between ALDOA and TDP-43. (A) Mass spectrometry identification of a specific peptide derived from ALDOA; (B) Protein-protein interaction (PPI) network. TARDBP (TDP-43) and its interaction partner ALDOA are highlighted by red circles.
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By comparing results with the STRING protein-protein interaction database and extracting interactions with a confidence score > 0.7 (high confidence), differential protein interaction relationships were obtained. Using Cytoscape software, a differential protein interaction network including ALDOA was constructed. The network analysis revealed that ALDOA occupies a central position and exhibits an interaction with TDP-43 (Figure 2B).
Validation of the Interaction and Co-Localization Between TDP-43 and ALDOA in HEK293T Cells
In this study, the expression and localization of TDP-43 and ALDOA within cells were detected by immunofluorescence assays. Following plasmid transfection, TDP-43 was localized to both the nucleus and cytoplasm, whereas ALDOA was predominantly nuclear. Consequently, the co-localization of the two proteins was primarily observed in the nucleus (Figure 3A and B). Furthermore, to further validate the interaction between the two proteins, we performed co-immunoprecipitation experiments. The results indicated that no ALDOA signal was detected in the Vector control group, whereas distinct ALDOA bands were observed in both the wild-type and mutant TDP-43 groups (Figure 3C), suggesting that ALDOA interacts directly or indirectly with both wild-type and mutant TDP-43.
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Figure 3 Interaction between ALDOA and TDP-43 validated by immunofluorescence and co-immunoprecipitation. (A) Immunofluorescence images showing co-localization of ALDOA (green) and TDP-43 (red) in each group of cells; Bar = 50 μm. (B) Visual representation of fluorescence co-localization from (A). (C) Western blot results of co-immunoprecipitation assays.
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TDP-43 Gene Mutation Leads to Upregulation of ALDOA Expression in HEK293T Cells
Western blot results further confirmed that TDP-43 protein bands were effectively detected in both the Vector control group and the mutant TDP-43 transfection group, with the TDP-43 protein expression level in the mutant TDP-43 transfection group being significantly higher than that in the Vector control group, indicating successful plasmid transfection (Figure 4A and B, P < 0.0001). Concurrently, at the protein level, ALDOA expression was significantly upregulated in the mutant TDP-43 transfection group compared to the Vector group (Figure 4A and C, P = 0.0157). Quantitative real-time PCR results showed that ALDOA mRNA expression was significantly higher in the mutant TDP-43 transfection group than in the Vector group (Figure 4A and D, P = 0.0117).
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Figure 4 Expression of ALDOA is up-regulated in the TDP-43M337V group. (A) Representative Western blot images of TDP-43 and ALDOA protein bands in each group. (B and C) Quantitative analysis of band intensities shown in (A). (D) mRNA expression levels of ALDOA in each group. Data were analyzed by unpaired t‑test (n=3). *P < 0.05, ***P < 0.001 compared to the control group.
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Discussion
Amyotrophic lateral sclerosis (ALS) is a highly debilitating motor neuron disease for which no effective treatment is currently available.4 The pathogenesis of ALS involves multiple mechanisms, including oxidative stress, mitochondrial and proteasomal dysfunction, abnormal RNA metabolism, altered synaptic function, disrupted axonal transport, and neuroinflammation.20 Previous studies have identified the pathological roles of certain brain proteins in ALS progression, such as TDP-4321 and FUS,22 whose mutations or dysfunctions can trigger various neurodegenerative diseases.23,24
TDP-43, encoded by the TARDBP gene, is a highly conserved nuclear protein primarily localized in the nucleus. It plays a critical role in regulating RNA transcription, alternative splicing, and the processing of miRNAs and lncRNAs, thereby maintaining cellular RNA homeostasis.25 Its central role in neurodegenerative diseases was first established in 2006, when two independent research groups simultaneously identified TDP-43 as the primary component of neuronal inclusions in patients with sporadic amyotrophic lateral sclerosis (sALS) and frontotemporal lobar degeneration (FTLD).26 Subsequent studies confirmed that the pathological aggregation of TDP-43 serves as a key biochemical hallmark of ALS.27 In specific ALS subtypes, neuronal cytoplasmic inclusions formed by ubiquitinated and phosphorylated C-terminal TDP-43 fragments represent a characteristic neuropathological feature.28,29 Notably, approximately 4% of familial ALS cases are directly linked to mutations in the TARDBP gene itself.30 These pathogenic mutations (including M337V,31 A382T,32 G298S33 and Q331K34) are predominantly clustered within the C-terminal glycine-rich domain of the TDP-43 protein. Among them, M337V, as one of the most frequent pathogenic mutations, plays a critical role in ALS pathogenesis: this mutation significantly enhances the abnormal aggregation propensity of TDP-43, disrupts its normal nucleocytoplasmic localization, impairs liquid-liquid phase separation equilibrium, and induces a cytotoxic gain-of-function in the cytoplasm. Consequently, these alterations compromise TDP-43’s ability to regulate RNA metabolism, ultimately leading to motor neuron degeneration and driving the progression of ALS.35
ALDOA is a key enzyme in the glycolytic pathway. Its high expression enhances glycolytic flux primarily by elevating its catalytic efficiency, accelerating the conversion of glucose to pyruvate, thereby increasing lactate production and facilitating rapid ATP generation. Studies have demonstrated that ALDOA promotes disease progression in malignancies such as hepatocellular carcinoma (HCC) by enhancing glycolysis.36 Furthermore, ALDOA has been implicated in neurodegenerative diseases. Research indicates that ALDOA and pyruvate kinase (PKM) are specifically upregulated in the cerebrospinal fluid (CSF) of Alzheimer’s disease (AD) patients.16 In sporadic Creutzfeldt-Jakob disease (sCJD), ALDOA expression is also specifically elevated and closely associated with prion protein (PrPSc) deposition and disease progression. The underlying mechanism may involve abnormal prion proteins interfering with the activity of ALDOA and other glycolytic enzymes, disrupting energy metabolism homeostasis in the brain.37 Studies utilizing TDP-43 cellular models carrying familial ALS mutations (A315T, M337V and S379P), specifically a triple mutant (3×-TDP-43) model, revealed that phosphorylated TDP-43 aggregates cause autophagy dysfunction and subsequently disrupt the expression of key glycolytic molecules, including ALDOA. This suggests that TDP-43 pathology may contribute to ALS pathogenesis by perturbing energy metabolism homeostasis.38 However, the specific biological functions and molecular mechanisms of ALDOA in ALS remain incompletely elucidated. This study identified and experimentally validated an interaction between ALS biomarker protein TDP-43 and ALDOA, providing new directions for further exploration of ALDOA’s role in ALS pathogenesis.
When ALDOA is highly expressed, it enhances glycolytic flux. The primary mechanism lies in the increased expression level of ALDOA directly elevating its catalytic efficiency, accelerating the conversion of glucose to pyruvate, which consequently leads to increased lactate production and rapid ATP generation. Under certain pathological conditions, the glycolytic process can be aberrantly activated and contribute to disease progression, as seen in cancers,39 Alzheimer’s disease,40 and Parkinson’s disease.41 Notably, disrupted glycolysis has been demonstrated to participate in various pathological processes such as cellular apoptosis42 and inflammatory responses,43 suggesting that abnormalities in this pathway may represent a common mechanism underlying multiple neurological disorders.44 In the pathogenesis of ALS, such coordinated metabolic mechanisms are disrupted, and metabolic dysregulation becomes a key factor driving disease progression. The interaction between TDP-43 and ALDOA and its alterations observed in this study may precisely represent one manifestation of glycolytic metabolic disruption in ALS.
This study integrated proteomics and molecular experiments by transfecting HEK293T cells with Flag-Vector, Flag-TDP-43, and Flag-TDP-43 M337V plasmids, respectively, to further investigate cellular mechanisms and related molecular targets. Based on the proteomics findings, we performed co-immunoprecipitation (co-IP) validation, which demonstrated that both wild-type TDP-43 and mutant TDP-43 M337V interact with the ALDOA protein. Furthermore, immunofluorescence assays confirmed the co-localization of ALDOA with both wild-type and mutant TDP-43 M337V in HEK293T cells. Subsequently, we examined ALDOA expression in the Vector group and the mutant TDP-43 M337V group. Both RT-qPCR and Western blot analyses revealed that compared to the Vector group, ALDOA mRNA and protein levels were elevated in the mutant TDP-43 M337V group, suggesting that TDP-43 mutation may upregulate ALDOA expression, thereby influencing the glycolytic pathway and contributing to the pathological process of ALS.
In summary, this study conducted an in-depth investigation of the proteome in cells transfected with ALS-associated wild-type and mutant TDP-43 plasmids, leading to the identification of ALDOA as an interacting partner. Differential expression levels of ALDOA were observed across various TDP-43 experimental groups. These findings establish a foundation for further exploration of the molecular mechanisms through which TDP-43-interacting protein ALDOA contributes to ALS pathogenesis. ALDOA and key proteins within its interactome may emerge as potential therapeutic targets for neurodegenerative diseases, suggesting that modulating the functions of these interacting proteins could offer novel strategic approaches for treatment.
Limitations of the Study
This study, through an in-depth investigation of the interaction between TDP-43 and ALDOA, reveals its potential role in the pathogenesis of ALS. The findings not only enhance the understanding of the pathophysiological processes of ALS but also provide a theoretical foundation for developing novel therapeutic strategies targeting this disease. However, this study has certain limitations, such as a relatively small sample size and the need for further optimization of experimental conditions. Future research should expand the sample size, delve deeper into the specific molecular mechanisms of the TDP-43-ALDOA interaction, and evaluate its feasibility and effectiveness as a therapeutic target. Moreover, utilizing preclinical models to further validate the translational potential of these findings could offer new hope for ALS patients.
Conclusion
This study confirms an interaction between TDP-43 and ALDOA, and demonstrates that the TDP-43 M337V mutation significantly promotes the upregulation of ALDOA expression. These results suggest that ALDOA, as a key glycolytic enzyme, may participate in TDP-43-mediated ALS pathogenesis by influencing cellular energy metabolism processes. The findings provide new experimental evidence for a deeper understanding of the molecular pathological mechanisms of ALS and also identify a potential target for therapeutic strategies aimed at intervening in metabolic pathways.
Funding
This study was supported by the Central Government Guidance Funds for Local Science and Technology Development (ZYYD2024ZY05) and the Xinjiang Medical University Young Top Talent Training Program (XYD2024Q08), National Natural Science Foundation of China (Grant No. 82171433), and Natural Science Foundation of Hunan Province of China (Grant No.2022JJ30918).
Disclosure
The authors report no conflicts of interest for this work.
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