Introduction

Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease characterized by progressive degeneration of motor neurons in the cerebral motor cortex, brainstem and corticospinal tracts1. It is one of the most common types of Motor neuron disease (MND), others include Progressive bulbar paralysis (PBP), Progressive muscular atrophy (PMA) and Primary lateral sclerosis (PLS)1,2. The main clinical manifestation of ALS is the involvement of both upper and lower motor neurons. In recent years, some studies have shown that the serum NFL levels of ALS patients are significantly higher than those of normal subjects; especially in the early stage of ALS, the increase in NFL levels is more pronounced, which may be related to the faster axonal degeneration and degeneration of neurons in the early stage of the disease3. It has also been shown that serum GFAP levels are significantly elevated in ALS patients compared to healthy individuals, especially when expressed differently in different subtypes of motor neuron disease4.

Neurofilaments (NFs) are important components of nerve fibers and were first found in neuronal cells. NFs are widely distributed in the cytoplasm of neuronal cells under electron microscopy, with a diameter of about 10 nm, and are in the form of thin filaments. NFs localized in the dendrites and the periphery of nucleus are curved in morphology, while those in the axon are in a straight and parallel arrangement. Neurofilament proteins not only maintain the morphology and diameter of axons, but also play an important role in maintaining cellular morphology and regulating axoplasmic transport, as well as increasing the speed of nerve signaling in myelinated nerve fibers. Neurofilament light chain (NFL) is the main component of neurofilament protein, a marker of axonal damage.

Glial fibrillary acidic protein (GFAP) is the main intermediate filament protein of mature astrocytes, which are the most numerous glial cells in the central nervous system, and has the function of regulating the stability of cytoskeletal structure, maintaining the mechanical potential of glial cells, and maintaining energy metabolism, synaptic activity, and integrity of the blood-brain barrier5,6. It has been found that the proliferation of astrocytes in ALS occurs in the cerebral cortex and spinal cord and that astrocytes are involved in non-cell-autonomous processes leading to neurodegeneration, that astrocytes exert toxicity on healthy motor neurons in vitro, which leads to axonal damage, and that higher concentrations of GFAP have been detected in the peripheral blood of ALS7,8.

Objects of study and methods

Objects of study

Based on international ALS diagnostic criteria and treatment guidelines9, Patients with confirmed ALS admitted to the General Hospital of Ningxia Medical University from February 2023 to May 2024 were selected. There were 12 patients with ALS, 6 males and 6 females, average of (56.75 ± 9.99); 12 cases of healthy medical examiners, 7 males and 5 females, average of (55.58 ± 5.63). All the study subjects were excluded from the history of serious and infectious diseases of other systems and there was no statistically significant difference between the two groups in terms of age and gender (P > 0.05).

Methods

For all study subjects, 5 ml of early morning elbow venous blood was taken, centrifuged at 3000 RPM (centrifugation radius 10 cm) for 15 min, and the supernatant (serum) was collected and cryopreserved at -80 °C for testing. Serum NFL and GFAP levels were detected by Simoa using a fully automated Simoa SR-X platform (Tianjin, China), which was operated in strict accordance with the Human NFL and GFAP Immunoarray Assay Kit Procedure (Quanterix, Massachusetts, USA). The Human NFL and GFAP Single Molecule Immunoarray Assay Kit (Target No. 104074) was purchased from Quanterix. Standards, internal references and samples were assayed on a single run basis and the NFL and GFAP concentrations of unknown samples were determined by standard curves.

Statistical methods

All data were analyzed using SPSS27.0 statistical software, and the measurement information was expressed as (mean ± standard deviation), t-test or corrected t-test was used for comparison between groups, and one-way analysis (F-test) was used for multi-group comparison, and Pearson’s correlation analysis was performed between serum NFL and GFAP levels and disease duration of patients in the ALS group. P < 0.05 was taken as the difference was statistically significant. Using PASS15 software, post hoc efficacy calculations were performed for the mean difference and standard deviation of NfL between the two groups showing a minimum sample size of 7 cases and a sample size of 12 cases in our study.

Results

Serum NFL and GFAP levels

Serum NFL and GFAP levels in ALS patients were 81.49 ± 47.06 pg/mL and 104.42 ± 37.31 pg/mL, respectively, significantly higher than those in healthy controls (9.21 ± 3.05 pg/mL and 57.71 ± 11.64 pg/mL; P < 0.001).

Relationship between serum NFL and GFAP levels and disease course in ALS patients

Serum NFL and GFAP levels in ALS patients were correlated with the duration of the disease as respectively (r = 0.746, P = 0.005; r = 0.668, P = 0.018) (Figs. 1, 2).

Fig. 1
Fig. 1The alternative text for this image may have been generated using AI.
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Relationship between serum NFL levels and disease duration in patients with ALS.

Fig. 2
Fig. 2The alternative text for this image may have been generated using AI.
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Relationship between serum GFAP levels and disease duration in patients with ALS.

Discussion

ALS is a rare, disabling, and fatal neurodegenerative disease involving upper motor neurons in the corticospinal tracts and lower motor neurons originating in the brainstem nuclei and ventral roots of the spinal cord9,10. The onset of ALS is insidious, the course of the disease progresses rapidly11,12, and the pathogenesis of ALS is still unclear. Domestic and foreign scholars have produced numerous articles studying serum biomarkers in ALS patients, among which NFL and GFAP are potential rising stars of serum biomarkers in ALS patients. NFL is neuron-specific structural backbone proteins significantly expressed especially in motor neurons. In the context of nerve injury, NFL and GFAP serve as biomarkers for different cell types: NFL is released in response to neuronal axonal damage, whereas GFAP is released in response to astrocyte activation or damage. Our study applied the Simoa method to detect serum NFL and GFAP levels in ALS patients with the aim of investigating the correlation between serum NFL and GFAP levels and ALS.

NFL has the role of stabilizing neuronal cell structure and can maintain nerve fiber axon diameter and regulate nerve impulse conduction velocity. Compared with neurofilament medium chain and neurofilament heavy chain, NFL can realize self-assembly and is an important component of neurofilament triad protein. It has been found that partial knockout of the NFL gene in model mice resulted in a significant reduction in neuronal axon diameter, weakened axon regeneration, and a subsequent reduction in nerve conduction velocity. NFL not only has an important role in the maintenance of neuronal axonal function, but also participates in the regulation of synaptic plasticity. It has been found that transgenic mice knocked out of the NFL exhibit impaired spatial memory, whereas mice deficient in the neurofilament heavy chain do not continue to maintain the long duration enhancement process13. However, neurodegenerative diseases are closely related to neurofilament proteins, abnormal transport and assembly of neurofilament protein subunits can affect neuronal axonal function, and neuronal axoplasmic transport dysfunction is one of the major pathological manifestations of neurodegenerative diseases12. Numerous studies have found that serum NFL levels are significantly elevated in patients with ALS14,15,16,17,18,19,20,21,22, and that this elevation occurs in the early stages of the disease16,22, and that dysfunction of axoplasmic transport due to abnormal NFL levels may also be one of the pathogenic mechanisms of neurodegenerative diseases23.

GFAP is a specific astrocyte biomarker protein that is significantly expressed especially in neurodegenerative diseases, and it has been suggested that the pathogenesis of ALS may be due to astrocyte activation leading to a significant increase in peripheral blood levels of GFAP4, and that serum levels of NFL and GFAP are elevated in response to axonal injury to the central nervous system24. Previous studies have found that serum NFL levels and GFAP levels are significantly elevated in ALS patients and correlate with the course of the disease4,24, and serum NFL and GFAP levels are expected to be serologic biomarkers for predicting the onset and progression of ALS disease. In recent years, with the advancement of detection technology, the use of Simoa technology to detect the levels of NFL and GFAP in serum has high sensitivity, providing a new technical means for the early diagnosis of ALS and the prediction of disease progression. The results of a Swiss multicenter longitudinal study suggest that serum GFAP is a prognostic biomarker for future studies of multiple sclerosis (MS) and has complementary potential with NFL, and that GFAP and NFL may serve as useful biomarkers for studying disease progression in MS25. MS and ALS are both neurodegenerative diseases and our study used Simoa technique to detect serum NFL and GFAP levels and analyze their correlation with ALS patients. We observed that serum NFL and GFAP levels were significantly higher in the ALS group than in the HC group (P < 0.001) and were positively correlated with the duration of ALS disease (P < 0.05).

The differences between our study and previous literature on NfL and GFAP levels in ALS patients may stem from the following factors: first, differences in cohort characteristics, such as racial differences, disease duration, site of onset, and phenotypic heterogeneity, can significantly affect biomarker concentrations. Second, cerebrospinal fluid, plasma, and serum were used in previous studies, whereas serum was used in our study, which may have contributed to interstudy variability due to differences in sample processing. Further, Simoa or Elisa was used to detect the levels of NFL and GFAP in previous studies, whereas our study used Simoa, which has high sensitivity and specificity. Finally, statistical methods (including sample size and correction for covariates such as age) can also affect the results.

However, our study has some limitations; firstly, ALS is a rare disease and the small number of patients included in this study makes the results restrictive; secondly, this study lacks disease controls, such as Parkinson’s disease, multiple sclerosis, peripheral neuropathies, frontotemporal dementia, progressive supranuclear palsy, and other neurodegenerative diseases, which would allow us to make a detailed evaluation of serum NFL and GFAP diagnosticity in detail, and follow-up studies must include relevant disease control groups to conclusively determine the specificity of these biomarkers for ALS; third, we lacked an assessment of the relationship between serum NFL levels and GFAP levels and the extent of upper and lower motor neuron disease; and fourth, we lacked a staging of the disease course of ALS, an ALSFRS-R score, and a rate of disease progression, which hinders the use of the reliable assessment of the utility of biomarkers in monitoring disease progression, and in future studies, the above indicators of ALS disease will be collected in detail. However, the application of single molecule array (Simoa) technology to detect serum NFL levels and GFAP levels in ALS patients and healthy population in our study provides feasibility and lays the foundation for further research. We should do further prospective longitudinal studies with more ALS patients and proper disease control to confirm the findings in this study.

Conclusions

In this study, we investigated the diagnostic value of serum NFL and GFAP levels in the ALS population and their clinical significance using Simoa technology. The results suggest that serum NFL and GFAP levels may be potential biomarkers for the diagnosis of ALS and are positively correlated with disease progression. However, its diagnostic specificity awaits further studies that include disease controls.