We used an untargeted mass spectrometric approach, tandem mass tag (TMT) proteomics, for the identification of biomarker signatures in genetic frontotemporal dementia (FTD). A total of 238 cerebrospinal fluid (CSF) samples from the GENetic FTD Initiative (GENFI) were analysed, including 107 presymptomatic (44 C9orf72, 38 GRN, 25 MAPT) and 55 symptomatic (27 C9orf72, 17 GRN, 11 MAPT) mutation carriers as well as 76 mutation-negative controls (‘non-carriers’). We found both shared and specific proteomic alterations in each genetic form of FTD. Among the proteins significantly altered in symptomatic mutation carriers compared to non-carriers, we found that YWHAZ, YWHAG, UCHL1, NPTXR, NPTX2 and FABP3 were changed across all three genetic forms, as well as in patients with Alzheimer’s disease from previously published datasets. We observed differential changes in lysosomal proteins among symptomatic mutation carriers with marked abundance decreases in MAPT carriers. Further, we identified mutation-associated proteomic changes already evident in presymptomatic mutation carriers. Weighted gene co-expression network analysis combined with gene ontology annotation revealed clusters of proteins enriched in neurodegeneration and glial responses, as well as synapse-, or lysosome-related proteins indicating that these are the central biological processes affected in genetic FTD. These clusters correlated with measures of disease severity and predicted cognitive decline. This study revealed distinct proteomic changes in the CSF of patients with genetic FTD, providing insights into the pathological processes involved in the disease. Additionally, we identified proteins that warrant further exploration as diagnostic and prognostic biomarker candidates, which could improve clinical management and therapeutic development for FTD.

Participants and sample collection

Participants were recruited from the GENFI study, which includes individuals with a diagnosis of FTD due to a pathogenic mutation in MAPT, GRN, or C9orf72 (symptomatic mutation carriers), at-risk first-degree relatives (presymptomatic mutation carriers), and non-carriers (mutation-negative first-degree relatives from the same families). Demographics of the cohort are described in Table 1.

Participants were assessed using a standardised history and examination and were classified as symptomatic if they met consensus diagnostic criteria (51, 52). The CDR Dementia Staging Instrument with National Alzheimer Coordinating Centre Frontotemporal Lobar Degeneration component (CDR® plus NACC FTLD) was used to assess disease severity, and the CDR® plus NACC FTLD sum of boxes (SOB) was used for quantitative analyses in this paper. Participants underwent Volumetric T1-weighted MRI scans. More details on clinical evaluation and imaging can be found in Supplementary Methods.

CSF collection and sample preparation

CSF was collected in polypropylene tubes through a lumbar puncture and centrifuged to remove insoluble material and cells. Supernatants were aliquoted and stored at -80 °C within 2 hours after collection. CSF samples (25 µL) were reduced by the addition of Tris(2)-carboxyethylphosphine (TCEP) in sodium deoxycholate (DOC), and triethylammonium bicarbonate (TEAB) to a final concentration of 5 mM TCEP (1% DOC, 100 mM TEAB). Following incubation at 55 °C for one hour, samples were equilibrated to room temperature (RT). Carbamidomethylation was performed by adding iodoacetamide to a concentration of 10 mM and subsequently incubating the reaction mixture in the dark for 30 min at RT. Trypsin (100 µg per vial; Promega) was dissolved in resuspension buffer (Promega) and 1.5 µg were added for overnight digestion at 37 °C. The following day, TMTpro reagents (TMT 18plex, Thermo Fisher, 5 mg) were dissolved in 200 µL acetonitrile (ACN) having been equilibrated to RT. Samples were randomised across TMT sets and TMT labelling was performed by adding 10 µL of TMT reagent to each sample. Per set, a global internal standard (GIS; pool of all cohort samples) was included as the last TMT channel (135N) for reference and normalisation. The reaction mixture was incubated for one hour under constant agitation and afterwards the labelling process was quenched by the addition of hydroxylamine to a final concentration of 0.2% (v/v). Following an incubation period of 30 min, samples were combined into 18-plex sets and subsequently acidified with 0.5 M hydrochloric acid to precipitate DOC as well as diluted with 0.1% trifluoroacetic acid (TFA). To remove DOC, TMT sets were centrifuged at 4000*g for 15 min at 4 °C and the resulting supernatant was subjected to desalting by solid phase extraction (SPE). Desalting was performed on reversed-phase C18 cartridges (Sep-Pak C18 light) with a vacuum manifold. The columns were first washed with 2*1000 µL 0.1% TFA in 80% ACN and then equilibrated with 2*1000 µL 0.1% TFA. After sample loading, the column was again washed twice with 1000 µL 0.1% TFA and finally peptides were eluted with 0.1% TFA, 80% ACN. The eluate was split into three aliquots of equal volume, dried by vacuum centrifugation, and stored at -20 °C.

Plasma NfL and other CSF marker measurements are detailed in Supplementary Methods.

Offline high-pH reverse phase HPLC sample fractionation

Offline high-pH HPLC fractionation was performed on an UltiMate™ 3000 Nano LC system. Each TMT set aliquot was dissolved in 22 µL of 2.5 mM NH₄OH of which 20 µL were injected to be separated on an XBridge BEH C18 column (pore size: 130 Å, inner diameter: 4.6 mm). Peptide elution was accomplished using the following gradient: Buffer B was increased from 1% to 45% over a 65-minute period (flow rate of 100 µL/min), while Buffer C was maintained at 10% (Buffer A: H₂O, Buffer B: 84% ACN, Buffer C: 25 mM NH₄OH). Resulting fractions were collected circling over two rows in a 96-well microtiter plate at 1 min intervals, yielding 24 concatenated fractions. Subsequent column cleaning was performed at 90% B and 10% C for 10 minutes followed by an equilibration at 1% B and 10% C for 10 minutes. All fractions were subjected to vacuum centrifugation and stored dry at -20 °C until subsequent LC-MS analysis.

Liquid chromatography-mass spectrometry (LC-MS)

Fractions were dissolved in 50 µL 0.05% TFA, 0.1% bovine serum albumin (loading buffer) and loaded on a nano-LC (Ultimate RSLC Nano, Thermo Scientific) equipped with a C18 trap column (PepMap Acclaim 300 µm mm * 5 mm, Thermo Scientific) and C18 separation column (PepMap Acclaim 75 µm * 500 mm, Thermo Scientific), connected to an Orbitrap Fusion^TM Lumos^TM Tribrid^TM mass spectrometer (Thermo Scientific), fitted with an Easy Spray Source and a high-field asymmetric waveform ion mobility spectrometry (FAIMS) unit for spatial ion separation. Peptides were separated according to the following gradient: 5 min, 4% B; 6 min, 10% B; 74 min, 40% B; 75 min, 100% B (Buffer A: 0.1% FA; Buffer B: 84% ACN, 0.1% FA). In the positive ion mode, alternating MS/MS cycles (cycle time = 1.5 s) were performed at compensation voltages (CV) of CV=-70 V, CV = -50 V. A full Orbitrap MS scan was recorded with the parameters specified as follows: R = 120 k, AGC target = 100%, max injection time = 50 ms. The full MS scan was then followed by data dependent Orbitrap MS/MS scans set to the following parameters: R = 50 k, AGC target = 200%, max. injection time = 120 ms, isolation window = 0.7 m/z, activation type = HCD.