Personalized neuroinflammatory status in neurodegenerative diseases by a panel of 20 cerebrospinal fluid markers

Neuroinflammation plays a critical role in the pathogenesis and progression of Alzheimer"s Disease (AD) and other neurodegenerative disorders. We developed and validated a sensitive and accurate multiprotein mass spectrometry assay for the absolute (molar) quantification of 20 key neuroinflammation markers in human CSF, and employed the method to study the inflammatory status along the AD continuum in the cohorts of subjective cognitive decline (SCD), mild cognitive impairment (MCI) and AD dementia, as well as corticobasal degeneration (CBD), a rare progressive neurodegenerative disorder.

The panel comprised apolipoproteins: ApoA1, ApoD, ApoJ as well as ApoE with all of its three isoforms (E2, E3 and E4), the strongest genetic risk factor for AD. It also included the markers of reactive astrogliosis (GFAP, YKL-40 and complement proteins: C1q, C3, C4) and microglia activation (TREM2). Besides, it contained proinflammatory cytokines (MIF, osteopontin and alpha-1-antichymotrypsin) and the two TAM receptors (Axl and Tyro3). Altogether, the quantified marker proteins spanned 10,000-fold molar concentrations ranging from 20 pM (GFAP) to 200 nM (ApoD) and were detected in a single LC-MS/MS run with excellent precision (median CV <6%) and inter-peptide agreement (typical r >0.98).

We applied the developed panel to the cohort of 109 patients (SCD=24; MCI=42; AD=43). We investigated the association of panel proteins with other CSF clinical markers (Aβ42, p-Tau and t-Tau) using the ATN classification system, as well as with the mini-mental state examination (MMSE) scores. Notably, four proteins (GFAP, osteopontin, YKL-40 and MIF) showed a significantly higher CSF molar concentrations in AD (and two proteins in MCI) vs. our controls (SCD). Among them, osteopontin yielded the best diagnostic performance in the discrimination of AD from controls (AUC 0.81). Next, by investigating whether a minimal combination of biomarkers could discriminate AD from controls with higher accuracies, we identified a panel of 5 proteins (GFAP, osteopontin, MIF, C1q and C3) that showed a considerably higher accuracy than any marker alone (AUC 0.91). Finally, we compared the CSF neuroinflammatory markers for AD (n=35) and Aβ-negative CBD patients (n=37). Interestingly, the results showed no discrimination between these two groups, thus suggesting a similar neuroinflammation profile shared by these two tauopathies. In the end, we propose that the developed multiprotein panel will contribute to personalized diagnostics by monitoring inflammatory signatures of CSF in the broad spectrum of neurodegenerative diseases.