BACKGROUND: Neurodegenerative diseases (NDDs) are a miscellaneous group of disorders that
variably affect individuals, with many subtle distinctions and different speeds across
individuals and syndromes. The evolution of NDDs involves cognition, behavior and motor
domains of clinical assessment that result in lifelong functional and social impairments
with high economic and social costs. The biochemical pathophysiological drivers occur far
earlier than symptoms appearance making the identification of subjects with preclinical
disease the basis for early diagnosis, needed for an effective therapy.
The clinical definition of NDDs is basically insufficient, but the molecular signals from
the brain can lead to the identification of a biomarker that can be measured periodically
in a non-invasive way. Therefore, a disease-specific biomarker is needed.
The possibility of identifying specific markers for NDDs within saliva has recently
emerged. Saliva and salivary Extracelluler Vesicles (sEVs) are vehicles for molecules
associated with neuronal damage and neuroinflammation. Their isolation allows an
enrichment of the molecules involved in the pathogenetic mechanisms of NDDs, improving
their quantification.
Raman spectroscopy (RS) is a method useful for the exhaustive biochemical
characterization of saliva and its vesicular component, without staining and labeling
procedures, highly informative, rapid and sustainable. In a rapid, sensitive and
non-destructive way, RS provides with a spectrum that can be used as a highly specific
"fingerprint" for the selected sample (e.g. saliva, blood, EV) representing the
diagnostic biomarker itself.
The RS study of saliva has already demonstrated the possibility of profiling patients
with progressive pathologies with good accuracy and, specifically, of distinguishing
subjects suffering from NDDs, with no further investigation of the ability to distinguish
the NDDs at an early stage, the verification of the possibility to monitor its
progression, nor the investigation of the biomolecular moieties involved in the observed
differences.
Raman spectroscopy is proposed as a reliable method for the rapid and exhaustive
biochemical characterization of salivary and vesicular component present in the sample,
without the need for staining or labeling procedures.
OBJECTIVES: The objective of this project is the validation of a Raman molecular
fingerprint for the considered exerimental groups, leading to the identification of a
complex biomarker useful for 1) the early identification, 2) phenotyping and 3) molecular
profiling of subjects with NDDs, leading to the prompt identification of tailored
therapeutic strategies, including optimal pharmacological and rehabilitation therapies
for each subject, with a significant impact on patients' quality of life and, in the
future, on the increased probability of slowing down the progression of NDDs with optimal
effective therapies. At a national level, early personalized intervention can reduce
patient management times and costs.
SAMPLE SIZE: Sample size was calculated with G-Power (medium effect size f=0.25,
statistical power 85%, a=0.05, for ANOVA omnibus statistical test with 5 experimental
groups AD, PD, AtP, pPD, MCI, drop-out rate of about 10%). The minimum number of subjects
to be involved is 242. Considering the different incidence of the considered NDDs,
distribution is not equal among groups.
DATA COLLECTION: Demographic, clinical and research data will be pseudonymized and stored
in a custom made REDCap database. NDDs diagnosis (AD, PD, AtPD, pPD or MCI), demographic
(age, sex), clinical history, and comorbidities (Cumulative Illness Rating Scale) data
will be stored. Saliva will be collected using an optimized protocol.
SAMPLE COLLECTION: At recruitment (T0), at least 60min after the intake of food and/or
drinks, saliva will be collected using Salivette tubes(Starstedt®). After 12 months(T12),
subjects will be asked for a second saliva sample. Pre-analytical parameters, dietary and
smoking habit will be recorded. Samples will be frozen until used.
SAMPLE BIOMOLECULAR EVALUATION: SiMoA technology will be used to quantify NDDs related
markers: asyn for PD, AtP and pPD, Aβ1-42 for AD and MCI, NfL as general biomarkers of
neurological damage in all groups.
EV ISOLATION AND CHARACTERIZATION: Saliva will be used for the isolation of EVs by Size
Exclusion Chromatography (SEC) and by ultracentrifugation.
Effective isolation will be verified with dot blot for protein markers, Nanoparticle
Tracking Analysis (NTA) for size distribution and Transmission Electron Microscopy for
morphology.
RAMAN ANALYSIS: Salivary and saliva derived EV spectra will be acquired using an Aramis
Raman microscope (Horiba Jobin-Yvon, France) equipped with a laser light source operating
at 785 nm and 532 nm (Carlomagno et al., Frontiers, 2021; Mangolini et al., Biology,
2023).
DATA PROCESSING: Acquired spectra will be baseline corrected and normalized by unit
vector, to homogenize the dataset using the LabSpec6(i.e. baseline, normalization).
Multivariate analysis will be used to create a classification model for AD, PD, AtPD, pPD
and MCI at T0, obtaining the dispersion of the Canonical Variables. The accuracy,
specificity and sensitivity of saliva and sEV RS will be calculated after the Leave-One
Out Cross-Validation (LOOCV). ROC curve will be calculated. Data obtained from the
molecular profiling of NDDs patients and the Raman databases will be used to interpret
the spectral variation in the different experimental groups.
The correlation between Raman biomolecular and clinical data will be performed to
evaluate the ability of the Raman platform to stratify patients at different disease
stages.
The changes in the RS fingerprint between T0 and T12 will be investigated (longitudinal
study) and correlated with the changes in the clinical scale scores.