Irisin Levels in Cerebrospinal Fluid Correlate with Biomarkers and Clinical Dementia Scores in Alzheimer Disease.

OBJECTIVE: Irisin, released by muscles during exercise, was recently identified as a neuroprotective factor in mouse models of Alzheimer disease (AD). In a cohort of AD patients, we studied cerebrospinal fluid (CSF) and plasma irisin levels, sex interactions, and correlations with disease biomarkers. METHODS: Correlations between CSF and plasma irisin levels and AD biomarkers (amyloid ß 1-42, hyperphosphorylated tau, and total tau [t-tau]) and Clinical Dementia Rating Scale Sum of Boxes (CDR-SOB) were analyzed in a cohort of patients with Alzheimer dementia (n = 82), mild cognitive impairment (n = 44), and subjective memory complaint (n = 20) biologically characterized according to the recent amyloid/tau/neurodegeneration classification. RESULTS: CSF irisin was reduced in Alzheimer dementia patients (p < 0.0001), with lower levels in female patients. Moreover, CSF irisin correlated positively with Aß42 in both female (r = 0.379, p < 0.001) and male (r = 0.262, p < 0.05) patients, and negatively with CDR-SOB (r = -0.234, p < 0.05) only in female patients. A negative trend was also observed between CSF irisin and t-tau levels in all patients (r = -0.144, p = 0.082) and in the female subgroup (r = -0.189, p = 0.084). INTERPRETATION: The results highlight the relationship between irisin and biomarkers of AD pathology, especially in females. Our findings also offer perspectives toward the use of irisin as a marker of the AD continuum. ANN NEUROL 2024;96:61-73.

Narcissistic Personality Disorder as Prodromal Feature of Early-Onset, GRN-Positive bvFTD: A Case Report.

Background: Behavioral variant frontotemporal dementia (bvFTD) typically involves subtle changes in personality that can delay a timely diagnosis. Objective: Here, we report the case of a patient diagnosed of GRN-positive bvFTD at the age of 52 presenting with a 7-year history of narcissistic personality disorder, accordingly to DSM-5 criteria. Methods: The patient was referred to neurological and neuropsychological examination. She underwent 3 Tesla magnetic resonance imaging (MRI) and genetic studies. Results: The neuropsychological examination revealed profound deficits in all cognitive domains and 3T brain MRI showed marked fronto-temporal atrophy. A mutation in the GRN gene further confirmed the diagnosis. Conclusions: The present case documents an unusual onset of bvFTD and highlights the problematic nature of the differential diagnosis between prodromal psychiatric features of the disease and primary psychiatric disorders. Early recognition and diagnosis of bvFTD can lead to appropriate management and support for patients and their families. This case highlights the importance of considering neurodegenerative diseases, such as bvFTD, in the differential diagnosis of psychiatric disorders, especially when exacerbations of behavioral traits manifest in adults.

Survival in Incident Cases with Frontotemporal Lobar Degeneration: A Registry-Based Study.

Population-based registries represent a unique sample to estimate survival. The aim of the present study was to assess survival rates and predictors of outcome in incidental frontotemporal lobar degeneration (FTLD). Incident cases with FTLD, included between January 1, 2017 to December 31, 2017, have been followed for five years. Median survival was 8.16 years from disease onset and 5.38 years from diagnosis. Survival rates did not differ between phenotypes. Shorter disease duration from onset to diagnosis was associated with poorer outcome (p = 0.01). FTLD is a relatively homogeneous disease in terms of survival. Future multinational population-based studies are needed to confirm these findings.

Short-Term Irisin Treatment Enhanced Neurotrophin Expression Differently in the Hippocampus and the Prefrontal Cortex of Young Mice.

As a result of physical exercise, muscle releases multiple exerkines, such as "irisin", which is thought to induce pro-cognitive and antidepressant effects. We recently demonstrated in young healthy mice the mitigation of depressive behaviors induced by consecutive 5 day irisin administration. To understand which molecular mechanisms might be involved in such effect, we here studied, in a group of mice previously submitted to a behavioral test of depression, the gene expression of neurotrophins and cytokines in the hippocampus and prefrontal cortex (PFC), two brain areas frequently investigated in the depression pathogenesis. We found significantly increased mRNA levels of nerve growth factor (NGF) and fibroblast growth factor 2 (FGF-2) in the hippocampus and brain-derived growth factor (BDNF) in the PFC. We did not detect a difference in the mRNA levels of interleukin 6 (IL-6) and IL-1ß in both brain regions. Except for BDNF in the PFC, two-way ANOVA analysis did not reveal sex differences in the expression of the tested genes. Overall, our data evidenced a site-specific cerebral modulation of neurotrophins induced by irisin treatment in the hippocampus and the PFC, contributing to the search for new antidepressant treatments targeted at single depressive events with short-term protocols.

Once-Daily Subcutaneous Irisin Administration Mitigates Depression- and Anxiety-like Behavior in Young Mice.

Major depression is one of the most common psychiatric disorders worldwide, usually associated with anxiety. The multi-etiological nature of depression has increased the search for new antidepressant molecules, including irisin, for which, in a previous study, we tested its effect in young mice when administered intraperitoneally in a long-term intermittent manner. Here, we evaluated the effect of subcutaneous short-term irisin administration (100 µg/Kg/day/5 days) in male and female mice subjected to behavioral paradigms: Tail Suspension Test (TST), Forced Swim Test (FST), Elevated Plus Maze (EPM), and Y Maze (YM). Moreover, a qRT-PCR assay was performed to analyze the impact of irisin treatment on Pgc-1α/FNDC5 expression in the brain. A significant reduction in immobility time in TST and FST was observed in irisin-treated mice. Furthermore, irisin treatment significantly increased the number of entries and time spent in open arms, demonstrating its anxiolytic effect. Memory-enhancing effects were not reported in YM. Interestingly, no gender differences were observed in all behavioral tests. Overall, these results suggest that short-term subcutaneous irisin administration can exert an antidepressant and anxiolytic role, probably due to the activation of the Pgc-1α/FNDC5 system in the brain. Further investigation could lead to the identification of irisin as a new agent for the treatment of psychiatric disorders.

Incidence of Syndromes Associated With Frontotemporal Lobar Degeneration in 9 European Countries.

Importance: Diagnostic incidence data for syndromes associated with frontotemporal lobar degeneration (FTLD) in multinational studies are urgent in light of upcoming therapeutic approaches. Objective: To assess the incidence of FTLD across Europe. Design, Setting, and Participants: The Frontotemporal Dementia Incidence European Research Study (FRONTIERS) was a retrospective cohort study conducted from June 1, 2018, to May 31, 2019, using a population-based registry from 13 tertiary FTLD research clinics from the UK, the Netherlands, Finland, Sweden, Spain, Bulgaria, Serbia, Germany, and Italy and including all new FTLD-associated cases during the study period, with a combined catchment population of 11 023 643 person-years. Included patients fulfilled criteria for the behavioral variant of frontotemporal dementia (BVFTD), the nonfluent variant or semantic variant of primary progressive aphasia (PPA), unspecified PPA, progressive supranuclear palsy, corticobasal syndrome, or frontotemporal dementia with amyotrophic lateral sclerosis (FTD-ALS). Data were analyzed from July 19 to December 7, 2021. Main Outcomes and Measures: Random-intercept Poisson models were used to obtain estimates of the European FTLD incidence rate accounting for geographic heterogeneity. Results: Based on 267 identified cases (mean [SD] patient age, 66.70 [9.02] years; 156 males [58.43%]), the estimated annual incidence rate for FTLD in Europe was 2.36 cases per 100 000 person-years (95% CI, 1.59-3.51 cases per 100 000 person-years). There was a progressive increase in FTLD incidence across age, reaching its peak at the age of 71 years, with 13.09 cases per 100 000 person-years (95% CI, 8.46-18.93 cases per 100 000 person-years) among men and 7.88 cases per 100 000 person-years (95% CI, 5.39-11.60 cases per 100 000 person-years) among women. Overall, the incidence was higher among men (2.84 cases per 100 000 person-years; 95% CI, 1.88-4.27 cases per 100 000 person-years) than among women (1.91 cases per 100 000 person-years; 95% CI, 1.26-2.91 cases per 100 000 person-years). BVFTD was the most common phenotype (107 cases [40.07%]), followed by PPA (76 [28.46%]) and extrapyramidal phenotypes (69 [25.84%]). FTD-ALS was the rarest phenotype (15 cases [5.62%]). A total of 95 patients with FTLD (35.58%) had a family history of dementia. The estimated number of new FTLD cases per year in Europe was 12 057. Conclusions and Relevance: The findings suggest that FTLD-associated syndromes are more common than previously recognized, and diagnosis should be considered at any age. Improved knowledge of FTLD incidence may contribute to appropriate health and social care planning and in the design of future clinical trials.

Genotype-phenotype correlation in the spectrum of frontotemporal dementia-parkinsonian syndromes and advanced diagnostic approaches.

The term frontotemporal dementia (FTD) refers to a group of progressive neurodegenerative disorders characterized mainly by atrophy of the frontal and anterior temporal lobes. Based on clinical presentation, three main clinical syndromes have traditionally been described: behavioral variant frontotemporal dementia (bvFTD), non-fluent/agrammatic primary progressive aphasia (nfPPA), and semantic variant PPA (svPPA). However, over the last 20 years, it has been recognized that cognitive phenotypes often overlap with motor phenotypes, either motor neuron diseases or parkinsonian signs and/or syndromes like progressive supranuclear palsy (PSP) and cortico-basal syndrome (CBS). Furthermore, FTD-related genes are characterized by genetic pleiotropy and can cause, even in the same family, pure motor phenotypes, findings that underlie the clinical continuum of the spectrum, which has pure cognitive and pure motor phenotypes as the extremes. The genotype-phenotype correlation of the spectrum, FTD-motor neuron disease, has been well defined and extensively investigated, while the continuum, FTD-parkinsonism, lacks a comprehensive review. In this narrative review, we describe the current knowledge about the genotype-phenotype correlation of the spectrum, FTD-parkinsonism, focusing on the phenotypes that are less frequent than bvFTD, namely nfPPA, svPPA, PSP, CBS, and cognitive-motor overlapping phenotypes (i.e. PPA + PSP). From a pathological point of view, they are characterized mainly by the presence of phosphorylated-tau inclusions, either 4 R or 3 R. The genetic correlate of the spectrum can be heterogeneous, although some variants seem to lead preferentially to specific clinical syndromes. Furthermore, we critically review the contribution of genome-wide association studies (GWAS) and next-generation sequencing (NGS) in disentangling the complex heritability of the FTD-parkinsonism spectrum and in defining the genotype-phenotype correlation of the entire clinical scenario, owing to the ability of these techniques to test multiple genes, and so to allow detailed investigations of the overlapping phenotypes. Finally, we conclude with the importance of a detailed genetic characterization and we offer to patients and families the chance to be included in future randomized clinical trials focused on autosomal dominant forms of FTLD.

Cerebrospinal fluid level of proNGF as potential diagnostic biomarker in patients with frontotemporal dementia.

Introduction: Frontotemporal dementia (FTD) is an extremely heterogeneous and complex neurodegenerative disease, exhibiting different phenotypes, genetic backgrounds, and pathological states. Due to these characteristics, and to the fact that clinical symptoms overlap with those of other neurodegenerative diseases or psychiatric disorders, the diagnosis based only on the clinical evaluation is very difficult. The currently used biomarkers help in the clinical diagnosis, but are insufficient and do not cover all the clinical needs. Methods: By the means of a new immunoassay, we have measured and analyzed the proNGF levels in 43 cerebrospinal fluids (CSF) from FTD patients, and compared the results to those obtained in CSF from 84 Alzheimer's disease (AD), 15 subjective memory complaints (SMC) and 13 control subjects. Results: A statistically significant difference between proNGF levels in FTD compared to AD, SMC and controls subjects was found. The statistical models reveal that proNGF determination increases the accuracy of FTD diagnosis, if added to the clinically validated CSF biomarkers. Discussion: These results suggest that proNGF could be included in a panel of biomarkers to improve the FTD diagnosis.

Role of plasma phosphorylated neurofilament heavy chain (pNfH) in amyotrophic lateral sclerosis.

The phosphorylated neurofilament heavy chain (pNfH) is a promising biomarker in amyotrophic lateral sclerosis (ALS). We examined plasma pNfH concentrations in order to corroborate its role as a diagnostic and prognostic biomarker in ALS. Incident ALS cases enrolled in a population-based registry were retrospectively selected and matched by sex and age with a cohort of healthy volunteers. Plasma pNfH levels were measured by an ELISA kit and correlated with clinical parameters. Discrimination ability of pNfH was tested using receiving operating characteristic (ROC) curves. Kaplan-Meier (KM) analysis and Cox proportional hazard models were used for survival analysis. Plasma pNfH was significantly higher in patients compared to controls. An optimal cut-off of 39.74 pg/ml discriminated cases from controls with an elevated sensitivity and specificity. Bulbar-onset cases had higher plasma pNfH compared to spinal onset (p = 0.0033). Furthermore, plasma pNfH positively correlated with disease progression rate (r = 0.19, p = 0.031). Baseline plasma pNfH did not influence survival in our cohort. Our findings confirmed the potential utility of plasma pNfH as a diagnostic biomarker in ALS. However, further studies with longitudinal data are needed to corroborate its prognostic value.

Neuroimaging Findings in a Patient with Anti-IgLON5 Disease: Cerebrospinal Fluid Dynamics Abnormalities.

Anti-IgLON5 disease is a recently described autoimmune neurodegenerative disorder characterized by insidious onset, slow progression and a variety of neurological features. Neuroimaging in most patients with anti-IgLON5 disease is normal or shows nonspecific findings. Here, we report a case of anti-IgLON5 disease presenting with parkinsonism, falls, sleep problems with severe nocturnal dyspnea attacks, dysphagia, and dysautonomia. Imaging findings were initially suggestive of progressive supranuclear palsy. An altered cerebrospinal fluid dynamic was found on an MRI as well as high-convexity hyperperfusion on a brain SPECT. Further case descriptions with neuroimaging are required to characterize cerebral and cerebrospinal fluid dynamics abnormalities in this rare condition.

proNGF Measurement in Cerebrospinal Fluid Samples of a Large Cohort of Living Patients With Alzheimer's Disease by a New Automated Immunoassay.

The discovery of new biomarkers for Alzheimer's disease (AD) is essential for an accurate diagnosis, to conceive new strategies of treatments, and for monitoring the efficacy of potential disease-modifying therapies in clinical trials. proNGF levels in the cerebrospinal fluid (CSF) represent a promising diagnostic biomarker for AD, but its validation was hampered by the absence of a reliable immunoassay. In the literature, proNGF is currently measured in postmortem brain tissue by semiquantitative immunoblot. Here we describe the development and validation of a new method to measure proNGF in the CSF of living patients. This method, based on molecular size separation by capillary electrophoresis, is automated and shows a 40-fold increase in sensitivity with respect to the proNGF immunoblot, largely used in literature, and is robust, specific, and scalable to high-throughput. We have measured proNGF in the cerebrospinal fluid of 84 living patients with AD, 13 controls, and 15 subjective memory complaints (SMC) subjects. By comparing the proNGF levels in the three groups, we found a very significant difference between proNGF levels in AD samples compared with both controls and SMC subjects, while no significant difference was found between SMC and controls. Because of the development of this new immunoassay, we are ready to explore the potentiality of proNGF as a new biomarker for AD or subgroups thereof, as well as for other neurodegenerative diseases.

The Role of Age on Beta-Amyloid1-42 Plasma Levels in Healthy Subjects.

Beta-amyloid (Aß) plaques have been observed in the brain of healthy elderlies with frequencies strongly influenced by age. The aim of the study is to evaluate the role of age and other biochemical and hematological parameters on Aß1-42 plasma levels in cognitively and neurologically normal individuals. Two-hundred and seventy-five normal subjects stratified by age groups (<35 years, 35-65 years, and >65 years) were included in the study. Aß1-42 plasma levels significantly correlated with age (rs = 0.27; p < 0.0001) in the whole sample, inversely correlated with age in the first age group (rs = -0.25, p = 0.01), positively correlated in the second group (rs = 0.22, p = 0.03), while there was no significant correlation in the older group (rs = 0.02, p = 0.86). Both age (ß-estimate = 0.08; p < 0.001) and cholesterol (ß-estimate = 0.03; p = 0.009) were significantly associated with Aß1-42 plasma level in multivariable analysis. However, only the association with age survived post hoc adjustment for multiple comparisons. The different effects of age on the Aß level across age groups should be explored in further studies to better understand the age-dependent variability. This could better define the value of plasma Aß as a biomarker of the Alzheimer neuropathology.

A New Presenilin 1 (Psen1) Mutation (p.Cys263Trp) as a Cause of Both Early and Late-Onset Alzheimer's Disease in a Large Italian Family.

Mutations in the PSEN1 gene are the most common cause of autosomal dominant Alzheimer's disease, and are characterized by a high phenotype variability. This study describes a five-generation family, with a prevalent late-onset of the disease and a high frequency of depression, in which a new missense mutation (c.789T > G, p.Cys263Trp) in exon 8 of the PSEN1 gene was found. Only the proband presented an early onset at the age of 45 with attention deficit, followed by spatial disorientation, psychiatric symptoms and parkinsonian signs. The other two cases had a late onset of the disease and a typical presentation with memory loss. Both were characterized by a high level of anxiety and depression. The disease course was different with signs of Lewy body dementia for the proband's mother, and pyramidal involvement and a shorter disease duration for the proband's maternal aunt. The other eight cases with late-onset dementia and three cases with a long history of depression have been reported in the family pedigree, underlying the high phenotype variability of PSEN1 mutations.

FNDC5/Irisin System in Neuroinflammation and Neurodegenerative Diseases: Update and Novel Perspective.

Irisin, the circulating peptide originating from fibronectin type III domain-containing protein 5 (FNDC5), is mainly expressed by muscle fibers under peroxisome proliferator-activated receptor gamma coactivator 1-alpha PGC1α control during exercise. In addition to several beneficial effects on health, physical activity positively affects nervous system functioning, particularly the hippocampus, resulting in amelioration of cognition impairments. Recently, FNDC5/irisin detection in hippocampal neurons and the presence of irisin in the cerebrospinal fluid opened a new intriguing chapter in irisin history. Interestingly, in the hippocampus of mice, exercise increases FNDC5 levels and upregulates brain-derived neurotrophic factor (BDNF) expression. BDNF, displaying neuroprotection and anti-inflammatory effects, is mainly produced by microglia and astrocytes. In this review, we discuss how these glial cells can morphologically and functionally switch during neuroinflammation by modulating the expression of a plethora of neuroprotective or neurotoxic factors. We also focus on studies investigating the irisin role in neurodegenerative diseases (ND). The emerging involvement of irisin as a mediator of the multiple positive effects of exercise on the brain needs further studies to better deepen this issue and the potential use in therapeutic approaches for neuroinflammation and ND.

Plasma Inflammatory Cytokines Are Elevated in ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease which leads to death in a median time of 2-3 years. Inflammation has been claimed important to the ALS pathogenesis, but its role is still not well-characterized. In the present study, a panel of five cytokines (IL-2, IL-6, IL-10, IFN-gamma, and TNF-alpha) measured in plasma has been investigated in ALS. These biomarkers of inflammation were measured in a population-based cohort of 79 patients with ALS and 79 age- and sex-matched healthy controls using the Bio-Plex technology (Bio-Rad). All the five cytokines were significantly increased in plasma samples of patients compared with controls (p < 0.0001), with IL-6 having the highest median concentration (10.11 pg/ml) in the ALS group. Furthermore, IL-6 was the plasma cytokine with the highest discrimination ability between patients and controls according to the receiver operating characteristic analysis (area under the curve = 0.93). At a cut-off point of 5.71 pg/ml, it was able to classify patients and controls with 91% of sensitivity and 87% of specificity. In the ALS group, plasma IL-6 concentration correlated with demographic (age: rs = 0.25, p = 0.025) and clinical (revised ALS Functional Rating Scale at evaluation: rs = -0.32, p = 0.007; Manual Muscle Testing: rs = -0.33, p = 0.004; progression: rs = 0.29, p = 0.0395) parameters. In line with previous studies, our results confirm that inflammatory cytokines are elevated in ALS, supporting a possible role of inflammation in disease mechanism and progression. However, the precise role of inflammation in ALS needs to be further investigated on larger samples and with more mechanistic studies.

Telemedicine for Delivery of Care in Frontotemporal Lobar Degeneration During COVID-19 Pandemic: Results from Southern Italy.

BACKGROUND: The COVID-19 pandemic is changing clinical practice in neurology, after the governments decided the introduction of social distancing and interruption of medical non-emergency services in many countries. Teleneurology is an effective tool for the remote evaluation of patients but its adoption for frontotemporal lobar dementia (FTD) is in a preliminary stage. OBJECTIVE: We evaluated multidisciplinary assessment of patients with FTD using telehealth during the COVID-19 pandemic. METHODS: All patients received a diagnosis of FTD during 2018-2019 according to international criteria. A structured questionnaire and Clinical Dementia Rating Scale (CDR)-FTD were used by the neurologist with patients and/or caregivers. Index symptoms of COVID-19 infection were searched. RESULTS: Twenty-eight clinical interviews were completed with caregivers and four with both patients/caregivers. Most patients and caregivers were satisfied with the neurological interview and expressed their willingness to continue to be included in remote evaluation programs (90%). Fifty percent of patients experienced significant worsening of clinical picture and quality of life since the start of social distancing. The CDR-FTD scale revealed a significant worsening of behavior (p = 0.01) and language functions (p = 0.009), compared to the last in-person evaluation at the center. One patient presented index symptoms of COVID-19 infection and was confirmed to be positive for COVID-19 with pharyngeal swab. CONCLUSION: The study was conducted in Italy, one of the countries hit particularly hard by the COVID-19 pandemic, with interruption of all non-emergency medical services. Our study indicates that telemedicine is a valid tool to triage patients with FTD to increase practice outreach and efficiency.

Metabolites response to onion yellow dwarf virus (OYDV) infection in 'Rossa di Tropea' onion during storage: a 1 H HR-MAS NMR study.

BACKGROUND: Plant viral infections induce changes in metabolic components in the host plant, with potential effects on compositional, organoleptic and storability features of agricultural products. Identification of modulated metabolites may provide clues concerning pathways implementing responses in plant-pathogen interactions. A time course study of metabolic fingerprinting of onion yellow dwarf virus (OYDV)-infected versus healthy 'Rossa di Tropea' onion bulbs was performed using proton high-resolution magic angle spinning nuclear magnetic resonance (1 H HR-MAS NMR) and ultra-performance liquid chromatography (UPLC), providing an overview of the metabolic state of the bulb in response to OYDV infection during storage. RESULTS: Metabolites accumulated/depleted upon infection were identified, belonging to flavonoid, saccharide, amino acid and organic acid classes. A decrease in quercetin glucosides content and antioxidant activity was observed in infected bulbs; some amino acids (Arg, Asn, Phe, Val) accumulated, while others were depleted (Leu); for some metabolites, a bimodal time-course was observed during storage (Glc, Lys). Virus interference on metabolic pathways, and the effects of the metabolic shift on edible product storability, organoleptic and nutritional quality were discussed. CONCLUSIONS: OYDV infection induces a metabolic shift in 'Rossa di Tropea' onion during bulb storage, involving several pathways and affecting storability and organoleptic and nutritional quality of bulbs at marketable stage. © 2020 Society of Chemical Industry.

Combined magnetic resonance imaging and high resolution spectroscopy approaches to study the fertilization effects on metabolome, morphology and yeast community of wine grape berries, cultivar Nero di Troia.

Effects of fertilization practices, mineral (M) and organo-mineral (OM), on molecular composition of Nero di Troia cultivar grape berries was studied using conventional chemical analysis, Magnetic Resonance Imaging (MRI) and 1H NMR spectroscopy on intact berries and extracts, respectively, and through analysis of yeast species developed on grape skins. Plants vegetative status did not differ between the two fertilization practices, whereas some grape juice chemical characteristics differed in fertilized grapes. MRI provided information on grape berries morphology through weighted images depending on spin-spin (T2) and spin-lattice (T1) relaxation times. T1 values were the highest in OM grape berries. 1H NMR metabolic profile, combined with chemometric analysis, evidenced significant differences for some metabolites (valine, leucine, isoleucine, proline, and malic acid). Furthermore, higher frequency of yeasts genus Starmella sp., isolated from OM grape berries contributed to reinforcing the found results on the physiological response of wine grape Nero di Troia to fertilization.

A multifactorial approach in characterizing geographical origin of Sicilian cherry tomatoes using 1H-NMR profiling.

In this study, metabolomic analysis of chloroform extracts was performed to characterize cherry tomatoes (cv Naomi and Shiren) grown in different Sicilian areas, using 1H NMR spectroscopy coupled with multivariate statistical analysis. Principal components analysis showed clear discrimination between extracts of cherry tomatoes cultivated in two different seasons (winter and summer) and grown in three areas of Sicily (Gela, Licata, Pachino). In particular, carotenoids and phospholipids mainly were found to be more discriminating metabolites for both cultivars in summer and winter. In the present study, the simple separation only based on production area was found to be inadequate to distinguish the three groups of tomatoes. A clear separation among the different samples groups was obtained using a multifactorial approach not only based on the geographical origin classification, but considering also cultivar, year and seasonality.