High adenosine extracellular levels induce glioblastoma aggressive traits modulating the mesenchymal stromal cell secretome

Glioblastoma is an aggressive, fast-growing brain tumor influenced by the composition of the tumor microenvironment (TME) in which mesenchymal stromal cell (MSCs) play a pivotal role. Adenosine (ADO), a purinergic signal molecule, can reach up to high micromolar concentrations in TME. The activity of specific adenosine receptor subtypes on glioma cells has been widely explored, as have the effects of MSCs on tumor progression. However, the effects of high levels of ADO on glioma aggressive traits are still unclear as is its role in cancer cells-MSC cross-talk. Herein, we first studied the role of extracellular Adenosine (ADO) on isolated human U343MG cells as a glioblastoma cellular model, finding that at high concentrations it was able to prompt the gene expression of Snail and ZEB1, which regulate the epithelial–mesenchymal transition (EMT) process, even if a complete transition was not reached. These effects were mediated by the induction of ERK1/2 phosphorylation. Additionally, ADO affected isolated bone marrow derived MSCs (BM-MSCs) by modifying the pattern of secreted inflammatory cytokines. Then, the conditioned medium (CM) of BM-MSCs stimulated with ADO and a co-culture system were used to investigate the role of extracellular ADO in GBM–MSC cross-talk. The CM promoted the increase of glioma motility and induced a partial phenotypic change of glioblastoma cells. These effects were maintained when U343MG cells and BM-MSCs were co-cultured. In conclusion, ADO may affect glioma biology directly and through the modulation of the paracrine factors released by MSCs overall promoting a more aggressive phenotype. These results point out the importance to deeply investigate the role of extracellular soluble factors in the glioma cross-talk with other cell types of the TME to better understand its pathological mechanisms

Memantine prodrug as a new agent for alzheimer’s disease

Hydrogen sulphide has recently drawn much attention due to its potent anti-inflammatory and neuroprotective roles in brain functions. The purpose of the current study was to exploit these beneficial properties of H 2 S to design a new agent for the treatment of Alzheimer’s disease (AD). To pursue our aims, we replaced the free amine group of memantine with an isothiocyanate functionality as a putative H 2 S-donor moiety. The new chemical entity, named memit, was then tested in vitro to determine whether it retains the pharmacological profile of the “native drug”, while also providing a source of H 2 S in the CNS. Indeed, Memit showed the ability to release H 2 S through a cysteine-mediated mechanism, thus generating memantine. Moreover, the new hybrid molecule exerts protective effects against neuronal inflammation and induces a drastic fall in ROS production. In addition, memit was also able to reduce the Aβ(1-42) self-induced aggregation and exerted cytoprotective effect against Aβ oligomers-induced damage in both human neurons and rat microglia cells. Finally, similarly to memantine, the new compound promotes autophagy, a complex process required for cellular homeostasis in cell survival that results to be altered in neurodegenerative diseases. In conclusion, our study revealed that memit is a prodrug of memantine. Further in vivo studies will be necessary to fully investigate the synergic or cumulative effects due to the H 2 S-releasing moiety and the native drug

Epigenetic Modifications of the α-Synuclein Gene and Relative Protein Content Are Affected by Ageing and Physical Exercise in Blood from Healthy Subjects

Epigenetic regulation may contribute to the beneficial effects of physical activity against age-related neurodegeneration. For example, epigenetic alterations of the gene encoding for α-synuclein (SNCA) have been widely explored in both brain and peripheral tissues of Parkinson’s disease samples. However, no data are currently available about the effects of physical exercise on SNCA epigenetic regulation in ageing healthy subjects. The present paper explored whether, in healthy individuals, age and physical activity are related to blood intron1-SNCA (SNCAI1) methylation, as well as further parameters linked to such epigenetic modification (total, oligomeric α-synuclein and DNA methyltransferase concentrations in the blood). Here, the SNCAI1 methylation status increased with ageing, and consistent with this result, low α-synuclein levels were found in the blood. The direct relationship between SNCAI1 methylation and α-synuclein levels was observed in samples characterized by blood α-synuclein concentrations of 76.3 ng/mg protein or lower (confidence interval (CI) = 95%). In this selected population, higher physical activity reduced the total and oligomeric α-synuclein levels. Taken together, our data shed light on ageing- and physical exercise-induced changes on the SNCA methylation status and protein levels of α-synuclein

High Levels of β-Amyloid, Tau, and Phospho-Tau in Red Blood Cells as Biomarkers of Neuropathology in Senescence-Accelerated Mouse

Alzheimer’s Disease (AD) is the most common Neurodegenerative Disease (ND), primarily characterised by neuroinflammation, neuronal plaques of β-amyloid (Aβ), and neurofibrillary tangles of hyperphosphorylated tau. α-Synuclein (α-syn) and its heteroaggregates with Aβ and tau have been recently included among the neuropathological elements of NDs. These pathological traits are not restricted to the brain, but they reach peripheral fluids as well. In this sense, Red Blood Cells (RBCs) are emerging as a good model to investigate the biochemical alterations of aging and NDs. Herein, the levels of homo- and heteroaggregates of ND-related proteins were analysed at different stages of disease progression. In particular, a validated animal model of AD, the SAMP8 (Senescence-Accelerated Mouse-Prone) and its control strain SAMR1 (Senescence-Accelerated Mouse-Resistant) were used in parallel experiments. The levels of the aforementioned proteins and of the inflammatory marker interleukin-1β (IL-1β) were examined in both brain and RBCs of SAMP8 and SAMR1 at 6 and 8 months. Brain Aβ, tau, and phospho-tau (p-tau) were higher in SAMP8 mice than in control mice and increased with AD progression. Similar accumulation kinetics were found in RBCs, even if slower. By contrast, α-syn and its heterocomplexes (α-syn-Aβ and α-syn-tau) displayed different accumulation kinetics between brain tissue and RBCs. Both brain and peripheral IL-1β levels were higher in SAMP8 mice, but increased sooner in RBCs, suggesting that inflammation might initiate at a peripheral level before affecting the brain. In conclusion, these results confirm RBCs as a valuable model for monitoring neurodegeneration, suggesting peripheral Aβ, tau, and p-tau as potential early biomarkers of AD

α-Synuclein Aggregated with Tau and β-Amyloid in Human Platelets from Healthy Subjects: Correlation with Physical Exercise

The loss of protein homeostasis that has been associated with aging leads to altered levels and conformational instability of proteins, which tend to form toxic aggregates. In particular, brain aging presents characteristic patterns of misfolded oligomers, primarily constituted of β-amyloid (Aβ), tau, and α-synuclein (α-syn), which can accumulate in neuronal membranes or extracellular compartments. Such aging-related proteins can also reach peripheral compartments, thus suggesting the possibility to monitor their accumulation in more accessible fluids. In this respect, we have demonstrated that α-syn forms detectable hetero-aggregates with Aβ or tau in red blood cells (RBCs) of healthy subjects. In particular, α-syn levels and its heteromeric interactions are modulated by plasma antioxidant capability (AOC), which increases in turn with physical activity. In order to understand if a specific distribution of misfolded proteins can occur in other blood cells, a cohort of human subjects was enrolled to establish a correlation among AOC, the level of physical exercise and the concentrations of aging-related proteins in platelets. The healthy subjects were divided depending on their level of physical exercise (i.e., athletes and sedentary subjects) and their age (young and older subjects). Herein, aging-related proteins (i.e., α-syn, tau and Aβ) were confirmed to be present in human platelets. Among such proteins, platelet tau concentration was demonstrated to decrease in athletes, while α-syn and Aβ did not correlate with physical exercise. For the first time, α-syn was shown to directly interact with Aβ and tau in platelets, forming detectable hetero-complexes. Interestingly, α-syn interaction with tau was inversely related to plasma AOC and to the level of physical activity. These results suggested that α-syn heterocomplexes, particularly with tau, could represent novel indicators to monitor aging-related proteins in platelets

Ovarian dysfunction and FMR1 alleles in a large Italian family with POF and FRAXA disorders: case report

BACKGROUND: The association between premature ovarian failure (POF) and the FMR1 repeat number (41> CGG(n)< 200) has been widely investigated. Current findings suggest that the risk estimation for POF can be calculated in the offspring of women with pre-mutated FMR1 alleles. CASE PRESENTATION: We describe the coexistence in a large Italian kindred of Fragile X syndrome and familial POF in females with ovarian dysfunctions who carried normal or expanded FMR1 alleles. Genetic analysis of the FMR1 gene in over three generations of females revealed that six carried pre-mutated alleles (61–200), of which two were also affected by POF. However a young woman, who presented a severe ovarian failure with early onset, carried normal FMR1 alleles (<40). The coexistence within the same family of two dysfunctional ovarian conditions, one FMR1-related and one not FMR1-related, suggests that the complexity of familial POF conditions is larger than expected. CONCLUSION: Our case study represents a helpful observation and will provide familial cases with heterogeneous etiology that could be further studied when candidate genes in addition to the FMR1 premutation will be available

Long intronic GAA•TTC repeats induce epigenetic changes and reporter gene silencing in a molecular model of Friedreich ataxia

Friedreich ataxia (FRDA) is caused by hyperexpansion of GAA•TTC repeats located in the first intron of the FXN gene, which inhibits transcription leading to the deficiency of frataxin. The FXN gene is an excellent target for therapeutic intervention since (i) 98% of patients carry the same type of mutation, (ii) the mutation is intronic, thus leaving the FXN coding sequence unaffected and (iii) heterozygous GAA•TTC expansion carriers with ∼50% decrease of the frataxin are asymptomatic. The discovery of therapeutic strategies for FRDA is hampered by a lack of appropriate molecular models of the disease. Herein, we present the development of a new cell line as a molecular model of FRDA by inserting 560 GAA•TTC repeats into an intron of a GFP reporter minigene. The GFP_(GAA•TTC)560 minigene recapitulates the molecular hallmarks of the mutated FXN gene, i.e. inhibition of transcription of the reporter gene, decreased levels of the reporter protein and hypoacetylation and hypermethylation of histones in the vicinity of the repeats. Additionally, selected histone deacetylase inhibitors, known to stimulate the FXN gene expression, increase the expression of the GFP_(GAA•TTC)560 reporter. This FRDA model can be adapted to high-throughput analyses in a search for new therapeutics for the disease

Histone deacetylases suppress cgg repeat-induced neurodegeneration via transcriptional silencing in models of Fragile X Tremor Ataxia Syndrome

Fragile X Tremor Ataxia Syndrome (FXTAS) is a common inherited neurodegenerative disorder caused by expansion of a CGG trinucleotide repeat in the 59UTR of the fragile X syndrome (FXS) gene, FMR1. The expanded CGG repeat is thought to induce toxicity as RNA, and in FXTAS patients mRNA levels for FMR1 are markedly increased. Despite the critical role of FMR1 mRNA in disease pathogenesis, the basis for the increase in FMR1 mRNA expression is unknown. Here we show that overexpressing any of three histone deacetylases (HDACs 3, 6, or 11) suppresses CGG repeat-induced neurodegeneration in a Drosophila model of FXTAS. This suppression results from selective transcriptional repression of the CGG repeat-containing transgene. These findings led us to evaluate the acetylation state of histones at the human FMR1 locus. In patient-derived lymphoblasts and fibroblasts, we determined by chromatin immunoprecipitation that there is increased acetylation of histones at the FMR1 locus in pre-mutation carriers compared to control or FXS derived cell lines. These epigenetic changes correlate with elevated FMR1 mRNA expression in pre-mutation cell lines. Consistent with this finding, histone acetyltransferase (HAT) inhibitors repress FMR1 mRNA expression to control levels in pre-mutation carrier cell lines and extend lifespan in CGG repeat-expressing Drosophila. These findings support a disease model whereby the CGG repeat expansion in FXTAS promotes chromatin remodeling in cis, which in turn increases expression of the toxic FMR1 mRNA. Moreover, these results provide proof of principle that HAT inhibitors or HDAC activators might be used to selectively repress transcription at the FMR1 locus.open293