Extracellular Vesicles in Multiple Sclerosis: Their Significance in the Development and Possible Applications as Therapeutic Agents and Biomarkers.

Extracellular vesicles (EVs) are "micro-shuttles" that play a role as mediators of intercellular communication. Cells release EVs into the extracellular environment in both physiological and pathological conditions and are involved in intercellular communication, due to their ability to transfer proteins, lipids, and nucleic acids, and in the modulation of the immune system and neuroinflammation. Because EVs can penetrate the blood-brain barrier and move from the central nervous system to the peripheral circulation, and vice versa, recent studies have shown a substantial role for EVs in several neurological diseases, including multiple sclerosis (MS). MS is a demyelinating disease where the main event is caused by T and B cells triggering an autoimmune reaction against myelin constituents. Recent research has elucidate the potential involvement of extracellular vesicles (EVs) in the pathophysiology of MS, although, to date, their potential role both as agents and therapeutic targets in MS is not fully defined. We present in this review a summary and comprehensive examination of EVs' involvement in the pathophysiology of multiple sclerosis, exploring their potential applications as biomarkers and indicators of therapy response.

The Role of Mitochondrial Copy Number in Neurodegenerative Diseases: Present Insights and Future Directions.

Neurodegenerative diseases are progressive disorders that affect the central nervous system (CNS) and represent the major cause of premature death in the elderly. One of the possible determinants of neurodegeneration is the change in mitochondrial function and content. Altered levels of mitochondrial DNA copy number (mtDNA-CN) in biological fluids have been reported during both the early stages and progression of the diseases. In patients affected by neurodegenerative diseases, changes in mtDNA-CN levels appear to correlate with mitochondrial dysfunction, cognitive decline, disease progression, and ultimately therapeutic interventions. In this review, we report the main results published up to April 2024, regarding the evaluation of mtDNA-CN levels in blood samples from patients affected by Alzheimer's (AD), Parkinson's (PD), and Huntington's diseases (HD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). The aim is to show a probable link between mtDNA-CN changes and neurodegenerative disorders. Understanding the causes underlying this association could provide useful information on the molecular mechanisms involved in neurodegeneration and offer the development of new diagnostic approaches and therapeutic interventions.

Genomics landscape of mitochondrial DNA variations in patients from South Italy affected by mitochondriopathies.

Mitochondrial DNA (mtDNA) is a 16,569 base pairs, double-stranded, circular molecule that contains 37 genes coding for 13 subunits of the respiratory chain plus 2 rRNAs and 22 tRNAs. Mutations in these genes have been identified in patients with a variety of disorders affecting every system in the body. The advent of next generation sequencing technologies has provided the possibility to perform the whole mitochondrial DNA sequencing, allowing the identification of disease-causing pathogenic variants in a single platform. In this study, the whole mtDNA of 100 patients from South Italy affected by mitochondrial diseases was analyzed by using an amplicon-based approach and then the enriched libraries were deeply sequenced on the ION Torrent platform (Thermofisher Scientific Waltham, MA, USA). After bioinformatics analysis and filtering, we were able to find 26 nonsynonymous variants with a MAF <1% that were associated with different pathological phenotypes, expanding the mutational spectrum of these diseases. Moreover, among the new mutations found, we have also analyzed the 3D structure of the MT-ATP6 A200T gene variation in order to confirm suspected functional alterations. This work brings light on new variants possibly associated with several mitochondriopathies in patients from South Italy and confirms that deep sequencing approach, compared to the standard methods, is a reliable and time-cost reducing strategy to detect all the variants present in the mitogenome, making the possibility to create a genomics landscape of mitochondrial DNA variations in human diseases.

A Novel Homozygous Variant in DYSF Gene Is Associated with Autosomal Recessive Limb Girdle Muscular Dystrophy R2/2B.

Mutations in the DYSF gene, encoding dysferlin, are responsible for Limb Girdle Muscular Dystrophy type R2/2B (LGMDR2/2B), Miyoshi myopathy (MM), and Distal Myopathy with Anterior Tibialis onset (MDAT). The size of the gene and the reported inter and intra familial phenotypic variability make early diagnosis difficult. Genetic analysis was conducted using Next Gene Sequencing (NGS), with a panel of 40 Muscular Dystrophies associated genes we designed. In the present study, we report a new missense variant c.5033G>A, p.Cys1678Tyr (NM_003494) in the exon 45 of DYSF gene related to Limb Girdle Muscular Dystrophy type R2/2B in a 57-year-old patient affected with LGMD from a consanguineous family of south Italy. Both healthy parents carried this variant in heterozygosity. Genetic analysis extended to two moderately affected sisters of the proband, showed the presence of the variant c.5033G>A in both in homozygosity. These data indicate a probable pathological role of the variant c.5033G>A never reported before in the onset of LGMDR2/2B, pointing at the NGS as powerful tool for identifying LGMD subtypes. Moreover, the collection and the networking of genetic data will increase power of genetic-molecular investigation, the management of at-risk individuals, the development of new therapeutic targets and a personalized medicine.

Circulating miRNAs in progressive supranuclear palsy: Recent evidence and future challenges.

Numerous studies have highlighted the importance of microRNA (miRNAs) in neurodegenerative diseases. However, the miRNA profiles in progressive supranuclear palsy (PSP) patients have been rarely reported. Recent evidence suggested a possible role of some dysregulated miRNAs in the cerebrospinal fluid (CSF) of PSP patients in the pathogenesis of the disease.

Non-Coding RNAs: New Biomarkers and Therapeutic Targets for Temporal Lobe Epilepsy.

Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy; it is considered a network disorder associated with structural changes. Incomplete knowledge of the pathological changes in TLE complicates a therapeutic approach; indeed, 30 to 50% of patients with TLE are refractory to drug treatment. Non-coding RNAs (ncRNAs), acting as epigenetic factors, participate in the regulation of the pathophysiological processes of epilepsy and are dysregulated during epileptogenesis. Abnormal expression of ncRNA is observed in patients with epilepsy and in animal models of epilepsy. Furthermore, ncRNAs could also be used as biomarkers for the diagnosis and prognosis of treatment response in epilepsy. In summary, ncRNAs can represent important mechanisms and targets for the modulation of brain excitability and can provide information on pathomechanisms, biomarkers and novel therapies for epilepsy. In this review, we summarize the latest research advances concerning mainly molecular mechanisms, regulated by ncRNA, such as synaptic plasticity, inflammation and apoptosis, already associated with the pathogenesis of TLE. Moreover, we discuss the role of ncRNAs, such as microRNAs, long non-coding RNAs and circular RNAs, in the pathophysiology of epilepsy, highlighting their use as potential biomarkers for future therapeutic approaches.

Exosomal miRNA as peripheral biomarkers in Parkinson's disease and progressive supranuclear palsy: A pilot study.

INTRODUCTION: Parkinson's disease (PD), a progressive neurodegenerative disease, can be misdiagnosed with atypical conditions such as Progressive Supranuclear Paralysis (PSP) due to overlapping clinical features. MicroRNAs (miRNAs) are small non-coding RNAs with a key role in post-transcriptional gene regulation. The aim was to identify a set of differential exosomal miRNAs biomarkers, which may aid in diagnosis. METHODS: We analyzed the serum level of 188 miRNAs in a discovery set, by using RTqPCR based TaqMan assay, in a small cohort of healthy controls, PD and PSP patients. Subsequently, the differentially expressed miRNAs, between PSP and PD patients, were further tested in a larger and independent cohort of 33 healthy controls, 40 PD and 20 PSP patients. The most accurate diagnostic exosomal miRNAs classifiers were identified in a logistic regression model. RESULTS: A statistically significant set of three exosomal miRNAs: miR-21-3p, miR-22-3p and miR-223-5p, discriminated PD from HC (area under the curve of 0.75), and a set of three exosomal miRNAs, miR-425-5p, miR-21-3p, and miR-199a-5p, discriminated PSP from PD with good diagnostic accuracy (area under the curve of 0.86). Finally, the classifier that best discriminated PSP from PD consisted of six exosomal miRNAs (area under the curve = 0.91), with diagnostic sensitivity and specificity of 0.89 and 0.90, respectively. CONCLUSIONS: Based on our analysis, these data showed that exosomal miRNAs could act as biomarkers to differentiate between PSP and PD.

Roles of Non-Coding RNAs as Novel Diagnostic Biomarkers in Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, affecting 5%of the elderly population. Currently, the diagnosis of PD is mainly based on clinical features and no definitive diagnostic biomarkers have been identified. The discovery of biomarkers at the earliest stages of PD is of extreme interest. This review focuses on the current findings in the field of circulating non-coding RNAs in PD. We briefly describe the more established circulating biomarkers in PD and provide a more thorough review of non-coding RNAs, in particular microRNAs, long non-coding RNAs and circular RNAs, differentially expressed in PD, highlighting their potential for being considered as biomarkers for diagnosis. Together, these studies hold promise for the use of peripheral biomarkers for the diagnosis of PD.

LC-MALDI-TOF ISD MS analysis is an effective, simple and rapid method of investigation for histones characterization: Application to EBV lymphoblastoid cell lines.

This contribution is the result of our progressive engagement to develop and to apply a top-down liquid chromatography (LC) matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) (LC-MALDI-TOF) analysis for the histone post-translational modifications (PTMs) and variants characterization, mainly in order to provide comprehensive and fast results. The histone post-translational modifications and the differential expression of the histone variants play an essential role both in the DNA packaging mechanism in chromosomes and in the regulation of gene expression in different cellular processes, also in response to molecular agents of environmental origin. This epigenetic mechanism is widely studied in different field such as cellular differentiation, development and in the understanding of mechanisms underlying diseases. The characterization of histone PTMs has traditionally performed by antibodies-based assay, but immunological methods have significant limits, and today systems that use mass spectrometry are increasingly employed. We evaluated an in-source decay (ISD) analysis for the histone investigation on human lymphoblastoid cells, and by this approach, we were able to identify and quantify several PTMs such as the di-methylation in the lysine 20 and the acetylation in the lysine 16 in H4 and the mono-methylation, di-methylation and trimethylations at K9 of the histone H3.1. Moreover, we detected and quantified in the same H2B spectrum the prevalent H2B 1C/2E type but also the minor H2B 1D, 1M and 1B/1L/1N, 1O/2F, 1J/1K variants. In this work, we show that MALDI-ISD represents an excellent methodology to obtain global information on histone PTMs and variants from cells in culture, with rapidity and simplicity of execution. Finally, this is a useful approach to get label-free relative quantitative data of histone variants and PTMs.

Circulating microRNA: The Potential Novel Diagnostic Biomarkers to Predict Drug Resistance in Temporal Lobe Epilepsy, a Pilot Study.

MicroRNAs (miRNAs) are small noncoding RNAs that have emerged as new potential epigenetic biomarkers. Here, we evaluate the efficacy of six circulating miRNA previously described in the literature as biomarkers for the diagnosis of temporal lobe epilepsy (TLE) and/or as predictive biomarkers to antiepileptic drug response. We measured the differences in serum miRNA levels by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assays in a cohort of 27 patients (14 women and 13 men; mean ± SD age: 43.65 ± 17.07) with TLE compared to 20 healthy controls (HC) matched for sex, age and ethnicity (11 women and 9 men; mean ± SD age: 47.5 ± 9.1). Additionally, patients were classified according to whether they had drug-responsive (n = 17) or drug-resistant (n = 10) TLE. We have investigated any correlations between miRNAs and several electroclinical parameters. Three miRNAs (miR-142, miR-146a, miR-223) were significantly upregulated in patients (expressed as average expression ± SD). In detail, miR-142 expression was 0.40 ± 0.29 versus 0.16 ± 0.10 in TLE patients compared to HC (t-test, p < 0.01), miR-146a expression was 0.15 ± 0.11 versus 0.07 ± 0.04 (t-test, p < 0.05), and miR-223 expression was 6.21 ± 3.65 versus 1.23 ± 0.84 (t-test, p < 0.001). Moreover, results obtained from a logistic regression model showed the good performance of miR-142 and miR-223 in distinguishing drug-sensitive vs. drug-resistant TLE. The results of this pilot study give evidence that miRNAs are suitable targets in TLE and offer the rationale for further confirmation studies in larger epilepsy cohorts.

Exosomal miRNAs as Potential Diagnostic Biomarkers in Alzheimer's Disease.

Alzheimer's disease (AD), a neurodegenerative disease, is linked to a variety of internal and external factors present from the early stages of the disease. There are several risk factors related to the pathogenesis of AD, among these exosomes and microRNAs (miRNAs) are of particular importance. Exosomes are nanocarriers released from many different cell types, including neuronal cells. Through the transfer of bioactive molecules, they play an important role both in the maintenance of physiological and in pathological conditions. Exosomes could be carriers of potential biomarkers useful for the assessment of disease progression and for therapeutic applications. miRNAs are small noncoding endogenous RNA sequences active in the regulation of protein expression, and alteration of miRNA expression can result in a dysregulation of key genes and pathways that contribute to disease development. Indeed, the involvement of exosomal miRNAs has been highlighted in various neurodegenerative diseases, and this opens the possibility that dysregulated exosomal miRNA profiles may influence AD disease. The advances in exosome-related biomarker detection in AD are summarized. Finally, in this review, we highlight the use of exosomal miRNAs as essential biomarkers in preclinical and clinical studies in Alzheimer's disease, also taking a look at their potential clinical value.

Actively Targeted and Redox Responsive Delivery of Anticancer Drug by Chitosan Nanoparticles.

The clinical efficacy of methotrexate (MTX) is limited by its poor water solubility, its low bioavailability, and the development of resistance in cancer cells. Herein, we developed novel folate redox-responsive chitosan (FTC) nanoparticles for intracellular MTX delivery. l-Cysteine and folic acid molecules were selected to be covalently linked to chitosan in order to confer it redox responsiveness and active targeting of folate receptors (FRs). NPs based on these novel polymers could possess tumor specificity and a controlled drug release due to the overexpression of FRs and high concentration of reductive agents in the microenvironment of cancer cells. Nanoparticles (NPs) were prepared using an ionotropic gelation technique and characterized in terms of size, morphology, and loading capacity. In vitro drug release profiles exhibited a glutathione (GSH) dependence. In the normal physiological environment, NPs maintained good stability, whereas, in a reducing environment similar to tumor cells, the encapsulated MTX was promptly released. The anticancer activity of MTX-loaded FTC-NPs was also studied by incubating HeLa cells with formulations for various time and concentration intervals. A significant reduction in viability was observed in a dose- and time-dependent manner. In particular, FTC-NPs showed a better inhibition effect on HeLa cancer cell proliferation compared to non-target chitosan-based NPs used as control. The selective cellular uptake of FTC-NPs via FRs was evaluated and confirmed by fluorescence microscopy. Overall, the designed NPs provide an attractive strategy and potential platform for efficient intracellular anticancer drug delivery.