Frontotemporal dementia (FTD) is a neurodegenerative disorder, characterized by progressive deficits in behavior, language, and cognition associated pathologically with atrophy of the frontal and temporal lobes. Abundantly expressed in central nervous system and involved in neurodegeneration, gangliosides (GGs) exhibit an elevated structural complexity, their expression being strongly correlated to certain neurological disease.
In view of the tight relationship between GGs and FTD, the FTDBioMark project proposes the development of a superior bioanalytical platform based on MS and collision induced dissociation (CID) for GG biomarker discovery in blood serum of patients diagnosed with FTD. The novelty of FTDBioMark consists in: i) implementation of the MS and CID MS/MS for comparative investigation of GG mixtures extracted from normal and diseased (FTD) human serum; ii) approaching FTD from glycolipidomics perspective; iii) discovery of blood-based biomarkers for FTD based on comparative studies and iv) the implementation in neurological studies as a method of early diagnosis of FTD.
Identifying this new type of FTD specific biomarkers in blood opens new directions for development and application of innovative methods for high risk population screening with a particular focus on elderly population that will enhance the early and accurate diagnosis of FTD in body fluids with the possibility of incipient pharmacological and nonpharmacological management of patients with FTD.
The main objective of this research project is to identify specific GGs associated to FTD through innovative glycoscreening methods, using high-performance and high-resolution mass spectrometry and to determine their expression in blood serum.
To accomplish this final objective it is necessary to achieve the intermediate goals of the project encompassing: i) the extraction, purification, screening and structural analysis of GGs specific for healthy control and FTD serum; ii) comparative assays to establish the set of biomarkers characteristics for FTD; iii) the ultimate goal of this research is to conceive a sturdy method able to correlate FTD with GG molecules in the blood through minimally invasive procedures applied to the patients.
The method will represent the basis for clinical validation and trials and subsequent introduction for routine investigations in elderly and risk population. The scientific objectives of FTDBioMark targets i) the implementation of the MS and CID MS/MS approach for comparative investigation of GG mixtures extracted from normal and diseased (FTD) human serum; ii) discovery of blood-based biomarkers for FTD based on comparative studies and iii) the implementation in neurological studies as a method of early diagnosis of frontotemporal degeneration.
Stage 1: Identification of inclusion lot, patient and healthy donor blood sampling, extraction and purification of gangliosides. Semiquantitative analysis on the individual GG fraction concentration by high performance thin layer chromatography (HPTLC) (January-December 2025)
Stage 2: Development of MS and MS/MS for screening and sequencing of GGs derived from sera of FTD patients and healthy individuals, of different ages. Construction of a database with the identified species and proposed biomarkers (January-December 2026)
R1. Patient and donor inclusion
A representative inclusion batch was established, comprising patients and healthy donors selected according to predefined clinical and demographic criteria. All participants provided informed consent, and peripheral blood samples were collected under standardized conditions to ensure sample integrity and comparability.
R2. Extraction and purification of gangliosides
Gangliosides were successfully extracted from collected blood samples using validated lipid extraction procedures. The purification process was optimized to eliminate non-ganglioside lipid contaminants, resulting in high-purity ganglioside fractions suitable for downstream analytical characterization. Detailed extraction and purification protocols were established.
R3. Separation and visualization of ganglioside classes
Thin-layer chromatography (TLC) enabled the clear separation of distinct ganglioside classes. The resulting chromatographic profiles exhibited consistent resolution across samples, with observable variations between patient and control groups. TLC plates containing separated gangliosides were prepared for subsequent analyses.
R4. Quantification of ganglioside profiles
Densitometric scanning of TLC plates allowed for the quantification of individual ganglioside species. The measurements demonstrated reliable reproducibility and revealed group-specific differences in ganglioside abundance and distribution.
R5. Preliminary mass spectrometry screening
Preliminary mass spectrometric (MS) analyses of ganglioside fractions isolated from TLC plates confirmed the molecular identity and purity of the compounds. The obtained MS spectra displayed characteristic ion patterns consistent with known ganglioside species, providing initial molecular fingerprints for further detailed studies.
Executive summary – Stage I
During Stage I of the project, all planned activities were fully completed, achieving the proposed objectives and establishing a solid foundation for subsequent scientific developments. The main focus of this stage was the optimization and standardization of experimental procedures required for the analysis of serum gangliosides and the assessment of their potential as biomarkers for the differential diagnosis of frontotemporal dementia (FTD). Representative cohorts of patients with FTD and healthy donors were established, selected according to current clinical and neuroimaging criteria. Peripheral blood samples were collected and processed under rigorous conditions, ensuring the integrity and comparability of the biological data. In parallel, standardized protocols for the extraction and purification of gangliosides were developed, optimized, and validated, enabling the acquisition of high-purity lipid fractions suitable for chromatographic and spectrometric analyses. The HPTLC analyses performed allowed clear separation of the main ganglioside classes (GM3, GM2, GD3, GM1, GD1a, GD1b, GT1b) and generated reproducible profiles, highlighting specific differences between FTD patients and healthy controls. Densitometric quantification confirmed the presence of relevant quantitative variations between the two groups, consistent with lipid metabolism alterations reported in neurodegenerative pathologies. The isolated fractions were subsequently subjected to preliminary screening by mass spectrometry, which confirmed the molecular identity and purity of the compounds, providing characteristic ionic fingerprints required for in-depth structural analyses. These results support the feasibility of using MS and HPTLC techniques for profiling serum gangliosides and demonstrate their potential to generate molecular signatures useful in FTD diagnosis. Based on the obtained data, an initial database of ganglioside species considered candidate biomarkers was established and will be expanded in the next stage of the project. Throughout this stage, the scientific activity of the team was reflected in the publication of four articles in ISI-indexed journals, one manuscript currently under review, and the presentation of 11 contributions at prestigious international conferences, thereby enhancing the visibility and impact of the achieved results. In conclusion, Stage I of the project was successfully completed, with all technical, analytical, and dissemination activities carried out according to plan. The results obtained confirm the validity of the proposed approach and open new research directions by strengthening the basis for advanced mapping of ganglioside profiles and the identification of specific biomarkers for frontotemporal dementia.
Cognitive and Scientific Impact
Over recent decades, significant scientific discoveries have deepened our understanding of the pathophysiological mechanisms occurring in the human brain, largely due to the progress achieved across the various “–OMICS” disciplines. The continuously increasing incidence of neurodegenerative diseases within the dementia spectrum has driven growing scientific attention toward early diagnosis and, even more critically, toward the identification of more effective therapeutic strategies.
International Alzheimer's and Related Dementias Research Portfolio (https://iadrp.nia.nih.gov/), — one of the largest global databases, encompassing data from approximately 4,000 research projects focused on frontotemporal dementia (FTD) — underscores both the importance and complexity of this research field. Within this context, a major research direction has been the development of fluid biomarkers from cerebrospinal fluid (CSF) and blood, intended to enable early and minimally invasive detection of disease.
While substantial efforts were invested for determining the changes of blood-based protein biomarker levels for FTD diagnosis, less was done regarding the involvement of glycolipids, particularly GGs, although their valuable role as biomarkers in various disorders was demonstrated. In this context, The FTDBioMark project directly addresses this gap by developing a reliable strategy for the determination of blood-based ganglioside biomarkers with potential diagnostic value for FTD.
Through its objectives and methodologies, the project will: i) advance knowledge in blood-based glycolipidomics and biological mass spectrometry; ii) open a new research direction in the field of blood research; iii) place blood gangliosidome of FTD in the spotlight; and iv) stimulate national and international collaborations between the project team, the host institution, various medical institutions, research institutes and top universities.
Collectively, these advances will strengthen the scientific reputation and research capacity of the host institution, enhancing its visibility and competitiveness in the fields of glycobiology and biomolecular mass spectrometry.
Socio-Economic Impact
The major long-term impact of the FTDBioMark project lies in the creation of a ganglioside structural database specific to FTD diagnosis. This resource will represent a valuable outcome from both a scientific and technological perspective, serving as a reference for future studies and clinical applications.
The database and associated methodologies could be implemented in specialized blood screening assays for the early diagnosis of FTD, particularly during its preclinical stages—before structural brain atrophy becomes detectable. This would be especially beneficial for high-risk populations or families with a known incidence of FTD.
By contributing to earlier diagnosis and improved patient stratification, FTDBioMark has the potential to support personalized medical approaches, reduce diagnostic uncertainty, and ultimately lower healthcare costs associated with late-stage neurodegenerative disease management.
Furthermore, the project will have a positive cognitive and professional impact on team members by enhancing their technical expertise, fostering interdisciplinary collaboration, and strengthening their position in the international research landscape.