Disease Biosignatures in ALS/FTD Spectrum: New Impactful Biological Perspectives Beyond Clinical Approaches

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are considered two manifestations of the same disease continuum, sharing common clinical, genetic and pathological features. Both diseases can even co-occur in the same patient, however, the underlying mechanisms triggering ALS, FTD or their mixed phenotype are not fully understood. Unfortunately, reliable biomarkers for ALS/FTD clinical diagnosis are still lacking. The aim of this project is to employ a cutting-edge, multidisciplinary approach to investigate not only traditional cerebrospinal fluid (CSF) samples, but also other easily accessible tissues such as skin, olfactory mucosa (OM), serum and tears. This project intends to uncover new biomarkers able to recognize ALS/FTD clinical phenotypes, strengthen the clinical diagnosis of specific phenotypes (e.g. bulbar vs spinal onset ALS), predict patients at risk of developing a mixed disease phenotype (ALS+FTD or vice versa), evaluate the efficacy of therapeutic treatments and capable to unveiling specific biological mechanisms involved in the onset and progression of each disease. The added value of the project is that it will explore ALS/FTD from a peripheral point of view by using tissues that have yet to be thoroughly examined in this field of research. These tissues will either be subjected to direct analysis for biomarker discovery or processed to perform cell and structural studies useful to deepen our understanding of the ALS/FTD neurodegenerative process and uncover new druggable molecules or mechanisms. To achieve our objectives, the first year of the project will be dedicated to enrolling patients, performing also a retrospective study using already available CSF, serum, tears, and OM samples collected from an extensively characterized cohort of patients with bALS, sALS, FTD as well as subjects with other non-neurodegenerative neurological conditions (NNC). These samples will be subjected to the following analyses (1) NGS, (2) Simoa (to quantify NfL, tau, phospho-tau, and beta-amyloid proteins), (3) Microfluidic (to determine miRNA and long non-coding RNA profiles), (4) Bioplex (to evaluate the innate-adaptive immunity pathway); (5) Seed amplification assay (to detect peripheral pathological TDP-43), and (6) MiSeq Illumina (to analyze microbiota composition). For microfluidic and Bioplex analyses the retrospective study will serve for biomarker discovery. Whereas, for Simoa, SAA and MiSeq analyses this study will allow the collection of pivotal preliminary data and fine-tuning of the analytical procedures. The applicants have already obtained promising data showing that the miRNA profile in serum and TDP-43 content in OM (SAA) could discriminate between bALS and Sals. These findings suggest that molecular and biological profiling is feasible and could tangibly impact the diagnosis of ALS/FTD. The second year of the study will focus on subjecting the newly collected samples to the same analyses as previously described, either for biomarker validation or for collecting all the necessary and essential data to determine whether and to what extent some peripheral alterations actually differ among pathologies. All the data will be processed by the Data Science Center group (expert bioinformaticians, statisticians, mathematicians and neurobiologists) to assess whether our findings allow for the identification of disease biosignatures in ALS/FTD spectrum. The research group is composed of four operative units (OUs): Fondazione IRCCS Istituto Neurologico Carlo Besta (UO1), Università degli Studi di Napoli "Federico II" (UO2), Consorzio Interuniversitario Risonanze Magnetiche Metallo Proteine (UO3) and Azienda Ospedaliero Universitaria di Sassari (UO4). It has been decided to include a group from Sardinia (UO4) because it is one of the Italian regions with the highest incidence of ALS and will contribute to the enrollment of patients with more heterogeneous clinical presentations. OU1 and UO4 will be responsible for selecting retrospective biological samples and recruiting ALS/FTD and NNC patients in the first year of the study. CSF, blood, tears, skin, and OM samples have been or will be collected using standardized and shared procedures by OU1 and UO4 from a total of sALS (n=105), bALS (n=32), FTD (n=66) and NNC (n=27) patients. A subset of samples collected from sALS (n=45), bALS (n=12), FTD (n=30) and NNC (n=12) patients is already available at UO1 and UO4 and will be analyzed during the first year of new patient enrollment. All recruited subjects will undergo extensive clinical and neuropsychological assessments and will be subjected to the Burghart Sniffin' Sticks test before CSF, blood, OM, skin and tears sampling. Samples will be distributed and analyzed by each UO as follow: CSF will be prepared either for Simoa analysis at UO4 or for (1) miRNA and long non-coding RNA, and (2) SAA analyses at UO1. Blood will be immediately processed at UO1 and UO4 to isolate the serum fraction that will be analyzed at UO4 (Simoa measurement of NfL, amyloid-beta, tau and phosphorylated at threonine 181 tau (p-tau) levels) and UO1 (evaluation of miRNA and long non-coding RNA profiles and evaluation of the innate-adaptive immunity pathway). Tears will be prepared for miRNA and long non-coding RNA or for SAA analyses at UO1. OM will be prepared for (1) miRNA and long non-coding RNA, (2) SAA and (3) microbiota analyses at UO1. Finally, the skin will be prepared for (1) miRNA and long noncoding RNA or (2) SAA analyses at UO1 and will be processed to obtain primary cell fibroblasts that will be sent to UO2 for (3) cell biology studies including super-resolution microscopy analysis of intracellular aggregates and molecular characterization of their internalization/degradation pathways. Finally, UO3 will (1) produce recombinant TDP-43 (for SAA analyses performed at UO1) possessing special features useful to produce NMR spectra of good quality for detailed structural characterization and (2) analyze selected SAA products by protein-NMR. All data generated in this project, including all relevant clinical and psychological information, will be analyzed by members of the Data Science Center (DSC) group that has been recently created at UO1. DSC combines the expertise of bioinformaticians, statisticians, mathematicians and neurobiologists with the aim of applying sophisticated analytical methodologies to clinical, omics, and imaging data. Finally, other collaborators (see appropriate section) working at UO3 will assist with protein-NMR study. Thus, synergistic collaboration among all UOs is vital to achieving the aims of the project.