Plasma NfL, GFAP and pTau181 define distinct biological axes in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is biologically heterogeneous, and blood biomarkers may reflect distinct pathological mechanisms. We investigated whether plasma neurofilament light chain (NfL), phosphorylated tau at threonine 181 (pTAU181), and glial fibrillary acidic protein (GFAP) capture complementary biological domains in amyotrophic lateral sclerosis. Plasma biomarkers were measured using a fully automated chemiluminescent immunoassay platform in patients with amyotrophic lateral sclerosis and control groups. Upper motor neuron burden was quantified using transcranial magnetic stimulation and the Penn Upper Motor Neuron Score. Lower motor neuron involvement was assessed by electromyography and Medical Research Council strength scores. Associations were tested using multivariable models adjusted for age, sex, disease progression rate, and phenotype. Latent profile analysis was applied to identify biomarker-defined subgroups. NfL levels increased with greater upper motor neuron burden across both neurophysiological and clinical measures. In contrast, pTAU181 selectively reflected lower motor neuron degeneration, particularly chronic denervation severity. GFAP levels were strongly associated with age and showed no relationship with motor neuron involvement. After adjustment for age and other covariates, higher GFAP levels were independently associated with behavioural lability. Biomarker levels did not differ across cognitive classes. Latent profile analysis identified three biologically distinct clusters characterized by selective pTAU181 elevation, progressive NfL increase, or prominent glial activation. Cluster membership independently predicted disease aggressiveness. These findings demonstrate that plasma NfL, pTAU181, and GFAP capture complementary biological processes in amyotrophic lateral sclerosis and support combined biomarker profiling for mechanistically informed patient stratification.