Diagnosis of ALS/FTD disease spectrum is challenging because it largely relies on clinical symptoms. Identifying novel biomarkers is essential for a paradigm shift towards a more precise biological-based diagnosis. To achieve this aim, having access to proper specimens and analytical methods is crucial. Our team of experts in neurology, biology, chemistry, physics, and AI will explore ALS/FTD from novel perspectives using transcriptomics, proteomics, genomics and other innovative approaches to analyzing easily accessible tissues. The seed amplification assay (SAA) will be also exploited to detect pathological TDP-43. This project aims to create disease fingerprints useful for patient stratification and monitoring of disease progression, and to evaluate the therapeutic efficacy in clinical trials, thus overcoming the limits of clinical interpretation. Discovering new biomarkers and cellular pathways will improve the diagnosis and treatment of these devastating diseases.
Clinical criteria for ALS (Brooks et al., 2000; de Carvalho M., 2008), FTD (GornoTempini et al., 2011; Rascovsky et al., 2011)
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Spinal-ALS (sALS, n=60), Bulbar-ALS (Bals,n=20), FTD (n=36), and NNC (n=10)
Cohort
Prospective
Not listed
Evaluation of SAA accuracy in detecting misfolded TDP-43 in CSF, skin, OM, and tears of ALS and FTD patients.
the primary outcome of the study will involve investigating the distribution of misfolded TDP-43, a protein associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), using ultrasensitive techniques namely Seed Amplification Assays (SAA). These assays will be employed to detect minute amounts of misfolded TDP-43 in cerebrospinal fluid (CSF), olfactory mucosa (OM), serum, skin, and tears from patients, with a clinical diagnosis of bulbar ALS (bALS; n=32), spinal ALS (sALS; n=105), FTD (n=66), and other non-neurodegenerative neurological conditions (NNC; n=27). By analyzing multiple peripheral tissues from the same patients, the generation of a disease biological fingerprint, obtained by integrating SAA analysis, other biochemical tests together with clinical and instrumental findings will be attempted. We aim to explore the reliability of this approach to improve the clinical diagnosis of ALS and FTD by allowing patient stratification.
Diagnostic and prognostic peripheral biomarkers identification through Simoa
To analyze if the levels of total-tau (pg/mL), phosphorylated tau (pg/mL), neurofilament light chains (NfL, pg/mL) and beta-amyloid proteins (pg/mL) are altered in the biological samples analysed (serum and CSF) compared to NNC and if these values differ in such a way that might help us to discriminate the pathologies (sALS, bALS, FTD). The assessment of these components will be pursued using Simoa.
Diagnostic and prognostic peripheral biomarkers identification through Microfluidic analysis
To determine the miRNA and long non-coding RNA profiles of the biological samples analysed (serum, CSF, OM, skin and tears), the TaqManTM Array Human MicroRNA-Card will be used. Bioinformatic analysis will be performed using tools and customized pipelines to identify differentially expressed (DE) miRNAs/lncRNAs in ALS and FTD samples compared to NNC. Validation of the top-ranked DE miRNA/lncRNA will be performed. Receiver operating characteristic (ROC) curves and the area under the curve (AUC) will be calculated to assess the ability of each miRNA/lncRNA to differentiate among groups.
Diagnostic and prognostic peripheral biomarkers identification through multiplex immunoassays
To evaluate the innate-adaptive immunity pathway, serum samples will undergo multiplex immunoassays analysis with the Bio-Plex Pro Human Cytokine multiplex kits on the Bioplex 200 system to analyze the levels of molecules, including interleukin (IL)-1ß, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-31, IL-33 (pg/mL), tumor necrosis factor-alpha (TNF-a; pg/mL), and interferon-gamma (IFN-y; pg/mL)
Metagenomic analysis to evaluate the microbiota composition
CSF and OM will be subjected to metagenomic analysis to evaluate the microbiota composition (eubiosis/dysbiosis), by MiSeq Illumina analysis. Total bacterial DNA will be extracted from CSF and OM samples using the QIAamp DNA Microbiome Kit. The V3-V4 region of the 16S rRNA gene will be amplified using gene-specific primers. 16S libraries will be prepared and completed using the KAPA HiFi HT ReadyMix, following Illumina Nextera DNA Flex Library Prep Kit. DNA yield will be determined fluorometrically using the High Sensitivity dsDNA kit on the Qubit® Fluorometer.
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