Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by loss of upper motor neurons and lower motor neurons resulting in progressive muscle atrophy and paralysis (Yang et al., 2022; Akçimen et al., 2023). The ALS phenotype varies across different ethnicities and regions. The average age of onset in ALS cohorts is around 66 years in Germany, 52 years in northern China, and 55 years in southwestern China (Rosenbohm et al., 2018; Chen et al., 2021; Wei et al., 2022). Currently, the majority of research suggests that ALS is associated with various factors such as immunity and infection, metallic elements, genetics, environmental factors, and many others (Li C. et al., 2022; Yang et al., 2022, 2023). However, the etiology and pathogenesis of ALS are highly complex and remain unknown. There are regional and racial differences in ALS patients. The research of biomarkers in cerebrospinal fluid (CSF) is one of the most active areas of ALS. Most previous studies focused on comparing the differences between healthy controls (HCs) and ALS patients, and most found that CSF IgG levels, CSF protein levels, and the quotient of CSF albumin and blood albumin (Q_ALB) in ALS patients were higher than those in controls (Gårde et al., 1971; Leonardi et al., 1984; Norris et al., 1993; Assialioui et al., 2022). However, the associations between CSF profiles and the disease phenotype of ALS are controversial. Some studies found that the CSF protein of patients with late-onset ALS was significantly higher than that of patients with early-onset ALS (Guiloff et al., 1979; Leonardi et al., 1984), while other studies found that the CSF protein of ALS patients gradually decreased with the increased age of onset (Guiloff et al., 1980; Norris et al., 1993). Some studies found that ALS patients with higher CSF IgG, CSF protein, and Q_ALB had short survival times (Guiloff et al., 1980; Li J. Y. et al., 2022; Klose et al., 2023). However, some studies did not find that CSF protein levels were associated with the survival of ALS (Norris et al., 1993; Li J. Y. et al., 2022). Besides, the associations with the disease phenotypes such as the stage of the disease or the progression rate turned out to be unapparent in the majority of the studies (Tarasiuk et al., 2012; Zhao et al., 2020). One recent study found CSF protein was significantly negatively associated with the ALS functional rating scale revised (ALSFRS-R) scores in female ALS, not in male ALS (Assialioui et al., 2022). These inconsistent findings of the CSF profiles may result from inherent limitations, including small sample size, varied applied methods, characteristics of enrolled ALS patients, ethnic differences of participants, and so on. Therefore, the relationship between CSF profiles and disease phenotypes needs further study.

As the basic laboratory examinations, CSF profiles might reflect the pathophysiological alterations along the disease course and provide insight into the pathogenesis of the disease. Therefore, in the present study, we analyzed the CSF profiles from a longitudinal cohort of ALS from Southwest China, trying to elucidate the relationship between the CSF profiles and the phenotype, severity, progression, and prognosis of the disease.

In the study, 957 ALS patients underwent CSF and serum examinations during hospitalization. Eighty-seven ALS patients were lost to follow-up and were excluded from the study (9.1%) (Supplementary Figure S1). The control group consisted of 96 patients diagnosed with primary headache (41.7%) and anxiety and depression status (58.3%). The median age of ALS patients was 57.26 (48.15–64.99) years, while that of the control group was 41.00 (26.00–52.00) years. The ALS group comprised 530 males and 340 females, whereas the control group consisted of 28 males and 68 females.

Among the included 870 ALS patients, 69.1% were classical phenotype, 77.0% were of limb onset, 23.0% were of bulbar onset, 26.2% were at stage I, 39.8% were at stage II, and 34.0% were at stage III. As of August 2023, 43.1% of the patients had taken riluzole for more than 3 months, and 70.3% of the patients had died (with a median survival time of 42.43 months). The characteristics of male ALS and female ALS patients are listed in Supplementary Table S1, and male ALS patients were found to be older and had more limb onset, higher mortality rate, and lower median survival time than female ALS patients (Supplementary Table S1).

We compared the CSF profiles between the 870 ALS patients and 96 control subjects, and found that CSF microprotein, albumin, IgG, and Q_ALB in ALS patients were higher than those in the control group, but CSF IgG_index was lower than that in the control group (Table 1), although the difference was small. In ALS patients, the proportions above the median of the CSF microprotein, IgG, albumin, and Q_ALB were significantly higher than those in controls (CSF microprotein: 56.9% vs. 13.5%; IgG: 53.6% vs. 26.0%; albumin: 53.5% vs. 19.8%; Q_ALB: 53.7% vs. 11.5%). In ALS patients, the proportion of the CSF microprotein exceeding the upper limit of the normal range was 32.6%, IgG was 26.1%, and albumin was 14.6%. Besides, we found that the difference in CSF microprotein, IgG, albumin, and Q_ALB remained significantly higher between ALS and controls in both male and female subgroups (Supplementary Tables S2, S3). However, the differences in the CSF IgG_index between female ALS and female control subjects were insignificant (Supplementary Table S3). Taking into account the significant differences in age and sex between ALS and control participants, propensity score matching was conducted based on age and sex, resulting in the selection of 69 ALS participants and 69 control participants in a 1:1 matching ratio. After matching, there were no significant differences in age and sex between the groups. However, the levels of CSF microprotein, IgG, albumin, and Q_ALB in the ALS group remained significantly higher than those in the control group, and the CSF IgG_index remained significantly lower than that in the control group, which were consistent with the results of the unmatched group comparisons (Table 1). In addition, in the subgroup analyses, we found that CSF microprotein, albumin, IgG, and Q_ALB were higher in male than in female participants (Supplementary Tables S4, S5).

The correlation analyses found that CSF IgG was correlated with survival status (r = −0.075, p = 0.028; Table 2), that is, the higher the CSF IgG, the higher the risk of death from ALS. CSF IgG_index was negatively correlated with ALSFRS-R (r = −0.067, p = 0.048; Table 2). CSF IgG, albumin, and Q_ALB were correlated with disease duration (CSF IgG: r = −0.105, p = 0.002; CSF albumin: r = −0.146, p < 0.001; Q_ALB: r = 0.155 p < 0.001; Table 2). Considering the influence of sex in CSF profiles, we stratified ALS patients according to sex and found that in male ALS patients, CSF IgG and albumin were correlated with survival status, and CSF IgG_index was negatively correlated with ALSFRS-R (r = −0.109, p = 0.012, Table 2), but these associations did not exist in female ALS. Besides, we found that the associations of CSF IgG, albumin, Q_ALB, and disease duration were found only in female ALS, but not in male ALS (Table 2).

In addition, we classified ALS into tertiles based on ALSFRS-R scores to analyze the correlation between CSF IgG_index and disease severity. We found that CSF IgG_index had significant differences between tertiles. Moderate ALS patients had higher CSF IgG_index compared to mild ALS patients (0.504 vs. 0.490, p = 0.006; Figure 1).

In order to verify whether the relationship between CSF IgG_index and ALSFRS-R was affected by other factors, multiple regression analysis was applied, and the results showed that CSF IgG_index was weakly correlated with ALSFRS-R scores in ALS patients after adjusting age of onset, sex, BMI, disease stage, site of onset, disease duration, and riluzole use (F = 30.624, p < 0.001; CSF IgG_index: β = −0.062, p = 0.041; Table 3). In the subgroup analyses, these correlations still existed in male ALS patients (β = −0.095, p = 0.015; Supplementary Table S6), but not in female ALS patients (β = −0.014, p = 0.784; Supplementary Table S7).

Since a significant correlation between CSF IgG and survival status was identified in ALS patients, we conducted further subgroup analysis based on the normal range of CSF IgG, and found that the survival time was shorter and survival rate were lower in the subgroup with CSF IgG exceeding the upper limit of the normal range (p < 0.001; Figure 2). In order to adjust the effects of other factors, including age of onset, disease course, disease stage, and site of onset, Cox survival analyses were performed. CSF IgG exceeding the upper limit of the normal range was significantly associated with the increased mortality risk in ALS patients [HR = 1.219 (1.010–1.470), p = 0.039; Figure 3]. Besides, ALS patients in stages II and III might be associated with increased mortality risk compared to those in stage I [stage II: HR = 1.689 (1.374–2.076), p < 0.001; stage III: HR = 2.100 (1.688–2.611), p < 0.001; Figure 3]. Older age of onset might be associated with increased mortality risk [age of onset: HR = 1.034 (1.027–1.042), p < 0.001; Figure 3], while longer disease duration and limb-onset compared to other onset regions may be associated with lower mortality risk [disease duration: HR = 0.947 (0.939–0.955), p < 0.001; site of onset: HR = 0.771 (0.639–0.931), p = 0.007; Figure 3].

Sensitivity analysis: First, we compared the levels of CSF profiles between the lost to follow-up ALS patients and ALS patients included in the analysis and found no significant differences between ALS patients who were lost to follow-up and ALS patients who completed follow-up (Supplementary Table S8). Secondly, we included the loss to follow-up ALS patients in the sensitivity analysis and found that it did not affect the results of the aforementioned analysis.

The study found a significant association between CSF IgG and survival status in ALS patients. The survival time was shorter and survival rates were lower in the ALS with CSF IgG exceeding the upper limit of the normal range. CSF IgG_index may negatively correlate with ALSFRS-R scores, especially in male ALS.

The current study found approximately one-third of ALS patients had higher CSF IgG levels. A previous study found IgG isolated from ALS patients increased the mobility of primary astrocyte endosomes and lysosomes, suggesting that it may be involved in the endocytosis/autophagy pathway (Stenovec et al., 2011). A single intraperitoneal injection of serum IgG from ALS patients induced a selective increase in the excitatory amino acids aspartate and glutamate levels in the CSF of rats (La Bella et al., 1997). Furthermore, some studies showed that IgG isolated from ALS patients can bind to CD16 receptors on microglia or lymphocytes and immune synapses between microglia and neurons (Edri-Brami et al., 2012, 2015), and can activate the oxidative stress response of microglia and release inflammatory factors (Milošević et al., 2017). Therefore, CSF IgG in ALS patients may accelerate the mortality risk in ALS by activating microglia to release oxidative stress. However, future basic research is still needed to elucidate the role of increased CSF IgG.

IgG_index = Q_IgG/Q_ALB(Q_IgG = CSF IgG/s-IgG, Q_ALB = Alb_CSF/Alb_S). In our study, we found that in mild ALS, the CSF IgG_index was lower compared to the control group. We considered this may be due to the significant increase in Q_ALB in the ALS group compared to the control group. Therefore, as Q_ALB serves as the divisor in the formula (IgG_index = Q_IgG/Q_ALB), its significant increase may lead to a decrease in the quotient. However, the difference in Q_ALB between mild ALS and moderate ALS was not significant. In this situation, CSF IgG_index may better reflect changes in Q_IgG, as consistent with the finding of the negative association between CSF IgG_index and ALSFRS-R scores in ALS patients. That is, the more severe the ALS patient, the higher the CSF IgG_index may be, indicating potentially more intrathecal IgG synthesis. Therefore, as an accessible CSF biomarker, CSF IgG_index can only roughly reflect the situation of intrathecal synthesis, and compared to the most reliable indicator of intrathecal oligoclonal protein synthesis, OB, it definitely has its limitations.

Q_ALB is recognized as an effective marker to evaluate the permeability of the BBB (Reiber and Peter, 2001). The current finding of the significant increase in Q_ALB of ALS patients was consistent with some studies (Wu et al., 2020; Alarcan et al., 2023). Some researchers believed that the disruption of BBB was an early event in ALS, and most studies suggested that BBB disruption in neurodegenerative diseases mediates the invasion of immune cells from the blood, resulting in neurodegeneration (Aragón-González et al., 2022; Beaman et al., 2022). However, Sweeney et al. suggested that decreased CSF reabsorption and/or production may elevate Q_ALB, leading to potential false-positive results in reflecting BBB breakdown (Sweeney et al., 2018). Therefore, further research is necessary to investigate the generation and absorption of CSF in ALS patients, as well as the extent of BBB disruption, for a comprehensive analysis of the role of the immune system in the occurrence and progression of ALS.

In the study, we found that CSF microprotein, IgG, albumin, and Q_ALB in male ALS patients were higher than those in female ALS patients, and the association between IgG_index and ALSFRS-R was found in male ALS patients, not in females. The male-to-female ratio of sporadic ALS is 1.3–1.5 (Logroscino et al., 2010). Hormones may play a protective role in the lower prevalence of ALS in females. The protective effect of female steroids may be due to their ability to prevent cell death and reduce the inflammatory component of the disease by acting directly on receptors expressed by motor neurons and muscle cells (Zhang et al., 2005). Therefore, sex differences should be paid attention to in the future.

There are some limitations in the current study. First of all, although the research findings of this study were significant, the associations were relatively diverse, and this study only represents the situation of the local cohort; therefore, further validation is required through longitudinal multi-center studies. Secondly, it was not combined with other inflammatory and immune-related biomarkers in blood and CSF to analyze its correlation and its role in the disease progression and prognosis of ALS. Third, the study was observational only, and studies on the mechanism of changes of CSF IgG involved in the pathophysiology of ALS are needed in the future.

In the current study, we found that the higher CFS IgG was associated with increased mortality risk of ALS. Additionally, CSF IgG_index may be associated with the severity of ALS, especially in male ALS.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

The studies involving humans were approved by the ethical standards committee on human experimentation at West China Hospital. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

JF: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Writing – original draft. XL: Conceptualization, Data curation, Formal analysis, Investigation, Software, Writing – original draft. QW: Conceptualization, Data curation, Investigation, Writing – original draft. XC: Conceptualization, Data curation, Investigation, Writing – original draft. HS: Conceptualization, Funding acquisition, Supervision, Writing – review & editing.