Molecular Taxonomy: Use of Transcriptional Profiles to Identify Different ALS Subtypes

Advances in diagnostic techniques and high-throughput biotechnologies provide a compelling opportunity to improve the diagnosis and treatment of diseases by developing a “New Taxonomy” that defines diseases on the basis of their underlying molecular and environmental factors rather than on traditional physical signs and symptoms. Oncology represents the first interesting example of how genomic medicine has changed the understanding of diseases and their therapy. However, much work remains to be completed on the molecular characterization and classification of complex and multifactorial diseases, including neurodegenerative disorders. Our research group has recently shown the genomic heterogeneity of sporadic amyotrophic lateral sclerosis (SALS), identifying two divergent subtypes associated with differentially expressed genes and pathways and providing several potential biomarkers and therapeutic targets. This chapter reviews the results emerged from our work, highlighting how molecular characterization of SALS patients may provide a framework for developing a more precise and accurate classification of diseases that could revolutionize the diagnosis, therapy, and clinical decisions of diseases, leading to more individualized treatments and improved outcomes for patients

A Diagnostic Gene-Expression Signature in Fibroblasts of Amyotrophic Lateral Sclerosis

myotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease with limited treatment options. Diagnosis can be difficult due to the heterogeneity and non-specific nature of the initial symptoms, resulting in delays that compromise prompt access to effective therapeutic strategies. Transcriptome profiling of patient-derived peripheral cells represents a valuable benchmark in overcoming such challenges, providing the opportunity to identify molecular diagnostic signatures. In this study, we characterized transcriptome changes in skin fibroblasts of sporadic ALS patients (sALS) and controls and evaluated their utility as a molecular classifier for ALS diagnosis. Our analysis identified 277 differentially expressed transcripts predominantly involved in transcriptional regulation, synaptic transmission, and the inflammatory response. A support vector machine classifier based on this 277-gene signature was developed to discriminate patients with sALS from controls, showing significant predictive power in both the discovery dataset and in six independent publicly available gene expression datasets obtained from different sALS tissue/cell samples. Taken together, our findings support the utility of transcriptional signatures in peripheral cells as valuable biomarkers for the diagnosis of ALS

Taxonomy Meets Neurology, the Case of Amyotrophic Lateral Sclerosis

Recent landmark publications from our research group outline a transformative approach to defining, studying and treating amyotrophic lateral sclerosis (ALS). Rather than approaching ALS as a single entity, we advocate targeting therapies to distinct “clusters” of patients based on their specific genomic and molecular features. Our findings point to the existence of a molecular taxonomy for ALS, bringing us a step closer to the establishment of a precision medicine approach in neurology practice

Algerian Olive Germplasm and Its Relationships with the Central-Western Mediterranean Varieties Contributes to Clarify Cultivated Olive Diversification

Olive tree with its main final product, olive oil, is an important element of Mediterranean history, considered the emblematic fruit of a civilization. Despite its wide diffusion and economic and cultural importance, its evolutionary and phylogenetic history is still difficult to clarify. As part of the Mediterranean basin, Algeria was indicated as a secondary diversification center. However, genetic characterization studies from Maghreb area, are currently underrepresented. In this context, we characterized 119 endemic Algerian accessions by using 12 microsatellite markers with the main goal to evaluate the genetic diversity and population structure. In order to provide new insights about the history of olive diversification events in the Central-Western Mediterranean basin, we included and analyzed a sample of 103 Italian accessions from Sicily and, a set of molecular profiles of cultivars from the Central-Western Mediterranean area. The phylogenetic investigation let us to evaluate genetic relationships among Central-Mediterranean basin olive germplasm, highlight new synonymy cases to support the importance of vegetative propagation in the cultivated olive diffusion and consolidate the hypothesis of more recent admixture events occurrence. This work provided new information about Algerian germplasm biodiversity and contributed to clarify olive diversification process

Cachrys libanotis L. Extracts: Photocytotoxic Effects on UVA-Irradiated Human Melanoma Cells

Melanoma is the most aggressive form of skin cancer. Photochemotherapy, combining the action of a light source and a chemical photosensitizer, is one of the most interesting current therapeutic approaches. Plants represent a rich source of photoactive compounds, and furanocoumarins are some of the most important naturally occurring phytoconstituents. The aim of this study was to evaluate the photocytotoxic potential of Cachrys libanotis L. (Apiaceae) from Southern Italy. This species belongs to a genus rich in furanocoumarins and widely distributed in Europe. The aerial parts were extracted through both traditional maceration and pressurized cyclic solid-liquid (PCSL) extraction using Naviglio extractor®. Qualitative and quantitative analyses were performed to detect the coumarins content using GC-MS, and the photocytotoxic effects of the extracts were assessed on UVA-irradiated C32 melanoma cells. The apoptotic responses were also evaluated. Furthermore, phenolic content and the in vitro antioxidant potential were also estimated. Xanthotoxin, bergapten and isopimpinellin were identified and quantified. Both extracts affected cell viability in a concentration-dependent manner after irradiation for 1 hour at a dose of 1.08 J/cm2. Sample obtained through PCSL extraction was the most effective, with an IC50 equal to 3.16 μg/mL, a very interesting value if compared with the positive control bergapten. This extract induced up-regulation of apoptotic signals such as BAX and PARP cleavage and, in the presence of UVA radiation, it caused a greater upregulation of p21 protein. Obtained results suggest that investigated species could be a good candidate for further studies aimed to find new drugs with photocytotoxic potential

FoxO3a Drives the Metabolic Reprogramming in Tamoxifen-Resistant Breast Cancer Cells Restoring Tamoxifen Sensitivity

Tamoxifen-resistant breast cancer cells (TamR-BCCs) are characterized by an enhanced metabolic phenotype compared to tamoxifen-sensitive cells. FoxO3a is an important modulator of cell metabolism, and its deregulation has been involved in the acquisition of tamoxifen resistance. Therefore, tetracycline-inducible FoxO3a was overexpressed in TamR-BCCs (TamR/TetOn-AAA), which, together with their control cell line (TamR/TetOn-V), were subjected to seahorse metabolic assays and proteomic analysis. FoxO3a was able to counteract the increased oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) observed in TamR by reducing their energetic activity and glycolytic rate. FoxO3a caused glucose accumulation, very likely by reducing LDH activity and mitigated TamR biosynthetic needs by reducing G6PDH activity and hindering NADPH production via the pentose phosphate pathway (PPP). Proteomic analysis revealed a FoxO3a-dependent marked decrease in the expression of LDH as well as of several enzymes involved in carbohydrate metabolism (e.g., Aldolase A, LDHA and phosphofructokinase) and the analysis of cBioPortal datasets of BC patients evidenced a significant inverse correlation of these proteins and FoxO3a. Interestingly, FoxO3a also increased mitochondrial biogenesis despite reducing mitochondrial functionality by triggering ROS production. Based on these findings, FoxO3a inducing/activating drugs could represent promising tools to be exploited in the management of patients who are refractory to antiestrogen therapy

The HFE p.H63D (p.His63Asp) Polymorphism Is a Modifier of ALS Outcome in Italian and French Patients with SOD1 Mutations

Background: Data from published studies about the effect of HFE polymorphisms on ALS risk, phenotype, and survival are still inconclusive. We aimed at evaluating whether the p.H63D polymorphism is a modifier of phenotype and survival in SOD1-mutated patients. Methods: We included 183 SOD1-mutated ALS patients. Mutations were classified as severe or mild according to the median survival of the study population. Patients were screened for the HFE p.H63D polymorphism. Survival was calculated using the Kaplan-Meier modeling, and differences were measured by the log-rank test. Multivariable analysis was performed with the Cox proportional hazards model (stepwise backward). Results: SOD1 severe mutation carriers show more frequent familial history for ALS and shorter survival compared to mild mutation carriers. Carriers and non-carriers of the p.H63D polymorphism did not differ in terms of sex ratio, frequency of positive familial history, age at onset, and bulbar/spinal ratio. In univariate and in Cox multivariable analysis using sex, age at onset, site of onset, family history, country of origin, and mutation severity as covariates, p.H63D carriers had a longer survival (p = 0.034 and p = 0.004). Conclusions: We found that SOD1-mutated ALS patients carrying the p.H63D HFE polymorphism have a longer survival compared to non-carriers, independently of sex, age and site of onset, family history, nation of origin, and severity of mutations, suggesting a possible role as disease progression modifier for the p.H63D HFE polymorphism in SOD1-ALS

Thymic Epithelium Abnormalities in DiGeorge and Down Syndrome Patients Contribute to Dysregulation in T Cell Development

The thymus plays a fundamental role in establishing and maintaining central and peripheral tolerance and defects in thymic architecture or AIRE expression result in the development of autoreactive lymphocytes. Patients with partial DiGeorge Syndrome (pDGS) and Down Syndrome (DS) present alterations in size and architecture of the thymus and higher risk to develop autoimmunity. We sought to evaluate thymic architecture and thymocyte development in DGS and DS patients and to determine the extent to which thymic defects result in immune dysregulation and T cell homeostasis perturbation in these patients. Thymi from pediatric patients and age-matched controls were obtained to evaluate cortex and medullary compartments, AIRE expression and thymocyte development. In the same patients we also characterized immunophenotype of peripheral T cells. Phenotypic and functional characterization of thymic and peripheral regulatory T (Treg) cells was finally assessed. Histologic analysis revealed peculiar alterations in thymic medulla size and maturation in DGS and DS patients. Perturbed distribution of thymocytes and altered thymic output was also observed. DGS patients showed lower mature CD4+ and CD8+ T cell frequency, associated with reduced proportion and function of Tregs both in thymus and peripheral blood. DS patients showed increased frequency of single positive (SP) thymocytes and thymic Treg cells. However, Tregs isolated both from thymus and peripheral blood of DS patients showed reduced suppressive ability. Our results provide novel insights on thymic defects associated with DGS and DS and their impact on peripheral immune dysregulation. Indeed, thymic abnormalities and defect in thymocyte development, in particular in Treg cell number and function could contribute in the pathogenesis of the immunodysregulation present in pDGS and in DS patients

Sporadic ALS is not associated with VAPB gene mutations in Southern Italy

Mutations in the Cu/Zn superoxide dismutase (Sod1) gene have been reported to cause adult-onset autosomal dominant Amyotrophic Lateral Sclerosis (FALS). In sporadic cases (SALS) de novo mutations in the Sod1 gene have occasionally been observed. The recent finding of a mutation in the VAMP/synaptobrevin-associated membrane protein B (VAPB) gene as the cause of amyotrophic lateral sclerosis (ALS8), prompted us to investigate the entire coding region of this gene in SALS patients. One hundred twenty-five unrelated patients with adult-onset ALS and 150 healthy sex-age-matched subjects with the same genetic background were analyzed. Genetic analysis for all exons of the VAPB gene by DHPLC revealed 5 variant profiles in 83 out of 125 SALS patients. Direct sequencing of these PCR products revealed 3 nucleotide substitutions. Two of these were found within intron 3 of the gene, harbouring 4 variant DHPLC profiles. The third nucleotide variation (Asp130Glu) was the only substitution present in the coding region of the VAPB gene, and it occurred within exon 4. It was found in three patients out of 125. The frequency of the detected exon variation in the VAPB gene was not significantly different between patients and controls. In conclusion, our study suggests that VAPB mutations are not a common cause of adult-onset SALS