Study on Serum LDL Promoting Microglial Activation and Exacerbating Demyelination in Neuromyelitis Optica Spectrum Disorder

Neuromyelitis Optica Spectrum Disorder (NMOSD) is an autoimmune inflammatory demyelinating disease of the central nervous system (CNS), often accompanied by disruption of the blood-brain barrier (BBB). Dysfunction of lipid metabolism in microglia is considered to be closely related to the neuropathology of NMOSD. However, current evidence on the role of circulating lipids in CNS demyelination, cellular metabolism, and microglial function is still limited. This study aims to reveal the functional relevance of serum low-density lipoprotein (LDL) in NMOSD and its potential mechanisms.

Research Background and Significance

NMOSD is an inflammatory demyelinating disease mediated by autoimmune regulation, characterized by recurrent attacks that often lead to severe sequelae such as vision loss and limb weakness. About 80% of NMOSD patients have aquaporin-4 immunoglobulin G antibodies (AQP4-IgG) in their serum, which bind to AQP4 on astrocytes, triggering classical complement cascade reactions, leading to microglial activation and subsequent demyelination. BBB disruption can lead to leakage of AQP4-IgG and cytokines, which is considered a key step in the development of NMOSD demyelination. Under normal conditions, brain and peripheral lipid metabolism are independent, but when the BBB is disrupted, lipid components in peripheral blood can penetrate into the CNS. Previous studies have shown that triglyceride (TG) and LDL levels in peripheral blood of NMOSD patients are significantly elevated, but research on the impact of these penetrating lipids on the pathogenesis of NMOSD is still insufficient.

Research Source

This article was written by Man Chen, Yun-Hui Chu, Wen-Xiang Yu, Yun-Fan You, Yue Tang, Xiao-Wei Pang, Hang Zhang, Ke Shang, Gang Deng, Luo-Qi Zhou, Sheng Yang, Wei Wang, Jun Xiao, Dai-Shi Tian, and Chuan Qin, and published in “Neuroscience Bulletin”. The authors are from the Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, and Hubei Key Laboratory of Neurological Injury and Functional Reconstruction, Hainan General Hospital, and Hainan Medical College Affiliated Hospital.

Research Methods

Experimental Procedures and Participants

The study was approved by the Ethics Committee of Tongji Hospital, Huazhong University of Science and Technology. Newly diagnosed and untreated AQP4-IgG positive NMOSD patients were recruited, their serum samples were collected, and serum neurofilament light (NFL) was measured using electrochemiluminescence immunoassay. Animal experiments used C57BL/6 wild-type (WT) mice, establishing an NMOSD model by injecting AQP4-IgG and human complement (HC) into their striatum, and analyzed using methods including balance beam test, brain tissue frozen sections, and various staining methods (such as HE staining, Oil Red O staining, Luxol Fast Blue staining).

Main Experimental Steps

1. Participants and Sample Collection: Collect clinical data including gender, age, Expanded Disability Status Scale (EDSS) scores, and serum lipid levels.
2. Serum NFL Measurement: Using electrochemiluminescence immunoassay.
3. AQP4-IgG Purification: Using G-agarose glycoprotein to purify IgG and adjust its concentration.
4. Mouse NMOSD Model Establishment: Injecting AQP4-IgG and HC into mouse striatum.
5. Neurological Function Assessment: Using MNSS scale and balance beam test.
6. BBB Integrity Detection: Through immunofluorescence staining and Western Blot to detect ZO-1 and Occludin proteins.
7. Effects of LDL on Microglia and Demyelination: Through direct LDL injection and intervention using Evolocumab (PCSK9 inhibitor).

Data Analysis Methods

Statistical analysis used Pearson χ2 test, Mann-Whitney U test, Spearman correlation analysis, one-way ANOVA, Bonferroni multiple comparisons, and generalized linear models, using SPSS and GraphPad Prism software for data processing.

Research Results

Association of Serum LDL with Neuronal Damage in NMOSD

By analyzing patients’ serum lipid levels, the study found that total cholesterol (TC) and LDL levels in NMOSD patients were significantly higher than those in the control group, and serum NFL levels were also significantly elevated. Further analysis showed that serum LDL was positively correlated with NFL (r=0.347, p=0.033) and ΔEDSS (r=0.348, p=0.032), indicating that serum LDL levels are associated with NMOSD disease progression.

BBB Disruption in Mouse NMOSD Model

Immunofluorescence staining revealed that after AQP4-IgG and HC injection, AQP4 was reduced in the lesion area, microglia were activated, astrocyte reactivity was enhanced, and demyelination was evident, accompanied by inflammatory cell infiltration and extensive lipid deposition. BBB integrity was assessed by Western Blot and immunofluorescence staining for ZO-1 and Occludin proteins, which were found to be significantly reduced, indicating BBB damage in the NMOSD model.

LDL Injection Exacerbates Demyelination and Microglial Activation

Experiments showed that compared to the PBS group, mice injected with LDL had higher MNSS scores and took longer to cross the balance beam. Immunofluorescence staining showed larger lesion areas after LDL injection, with significantly aggregated and morphologically altered activated microglia, characterized by increased classical activation microglial markers and decreased alternative activation microglial markers.

Therapeutic Effect of Evolocumab

As a PCSK9 inhibitor, Evolocumab reduced serum LDL levels, significantly decreased demyelinated lesion areas and microglial activation, and improved neurological function and motor coordination in mice.

Effects of LDL on Microglial Activation and Glucose-Lipid Metabolism

Microglia treated with LDL accumulated more lipid droplets and myelin debris, showed increased expression of glycolysis genes and decreased expression of oxidative phosphorylation genes, accompanied by upregulation of inflammatory genes and downregulation of remyelination genes. In co-culture with oligodendrocyte precursor cells (OPCs), LDL-treated microglia inhibited OPC maturation, hindering myelin regeneration.

Conclusions and Significance

This study reveals the association between serum LDL and disease severity in NMOSD patients. Further confirmation in the mouse NMOSD model shows that LDL penetrates the damaged BBB into the CNS and activates microglia, leading to microglial metabolic dysregulation and exacerbation of demyelination. The study suggests that therapies aimed at reducing circulating LDL may be an effective strategy for alleviating acute demyelinating damage in NMOSD.

Highlights of the study include revealing the association between circulating lipids and CNS demyelination and cellular metabolism. Through a series of meticulous experimental procedures and data analysis, it strongly supports the key role of LDL in the pathophysiology of NMOSD, providing new directions for future therapeutic strategies.