Abstract
BACKGROUND: 5q-associated spinal muscular atrophy (SMA) is a monogenic disease causing progressive alpha motor neuron degeneration, muscle atrophy, and weakness. Intrathecal therapy with the antisense oligonucleotide nusinersen modifies the disease course. However, biomarkers for understanding underlying molecular pathomechanisms and monitoring therapy are not yet known. METHODS: A total of 130 cerebrospinal fluid (CSF) samples from 24 adult patients with SMA type 2 or 3 were collected over 3.5 years, and CSF proteome was analyzed using mass spectrometry (MS). By applying two complementary MS protein quantification methods, label-free quantification (LFQ) and tandem mass tag (TMT) isotopic labeling, specific protein patterns reflecting changes in the CSF in response to nusinersen therapy were identified. These results were combined with cellular and metabolic profiles. RESULTS: Nusinersen therapy led to a median motor function improvement of 2.2 Hammersmith Functional Motor Scale-Expanded points after 10 months and 2.6 points after 34 months. CSF macrophages increased in number and showed an altered morphology. Albumin quotient (qAlb), glucose, and lactate concentrations were inversely correlated with clinical improvement. MS analysis of CSF identified 1,674 (TMT) and 441 (LFQ) proteins. Protein profiles reflected reduced inhibition of "nervous system development" and "axogenesis" pathways under therapy. In addition, clinical improvement was associated with upregulation of the interacting proteins α-dystroglycan and beta-1,4-glucuronyltransferase 1, reduction of complement factors, negative correlation in immunoglobulin- and B cell-related pathways, and reduction of cellular mediators such as lymphocytes. CONCLUSION: The present multi-proteomic analysis contributes to the understanding of the molecular mechanisms underlying nusinersen's therapeutic effects and offers potential biomarkers for monitoring treatment response in SMA.