Competing endogenous RNAs in human astrocytes: crosstalk and interacting networks in response to lipotoxicity.

Neurodegenerative diseases (NDs) are characterized by a progressive deterioration of neuronal function, leading to motor and cognitive damage in patients. Astrocytes are essential for maintaining brain homeostasis, and their functional impairment is increasingly recognized as central to the etiology of various NDs. Such impairment can be induced by toxic insults with palmitic acid (PA), a common fatty acid, that disrupts autophagy, increases reactive oxygen species, and triggers inflammation. Although the effects of PA on astrocytes have been addressed, most aspects of the dynamics of this fatty acid remain unknown. Additionally, there is still no model that satisfactorily explains how astroglia goes from being neuroprotective to neurotoxic. Current incomplete knowledge needs to be improved by the growing field of non-coding RNAs (ncRNAs), which is proven to be related to NDs, where the complexity of the interactions among these molecules and how they control other RNA expressions need to be addressed. In the present study, we present an extensive competing endogenous RNA (ceRNA) network using transcriptomic data from normal human astrocyte (NHA) cells exposed to PA lipotoxic conditions and experimentally validated data on ncRNA interaction. The obtained network contains 7 lncRNA transcripts, 38 miRNAs, and 239 mRNAs that showed enrichment in ND-related processes, such as fatty acid metabolism and biosynthesis, FoxO and TGF-ß signaling pathways, prion diseases, apoptosis, and immune-related pathways. In addition, the transcriptomic profile was used to propose 22 potential key controllers lncRNA/miRNA/mRNA axes in ND mechanisms. The relevance of five of these axes was corroborated by the miRNA expression data obtained in other studies. MEG3 (ENST00000398461)/hsa-let-7d-5p/ATF6B axis showed importance in Parkinson's and late Alzheimer's diseases, while AC092687.3/hsa-let-7e-5p/[SREBF2, FNIP1, PMAIP1] and SDCBP2-AS1 (ENST00000446423)/hsa-miR-101-3p/MAPK6 axes are probably related to Alzheimer's disease development and pathology. The presented network and axes will help to understand the PA-induced mechanisms in astrocytes, leading to protection or injury in the CNS under lipotoxic conditions as part of the intricated cellular regulation influencing the pathology of different NDs. Furthermore, the five corroborated axes could be considered study targets for new pharmacologic treatments or as possible diagnostic molecules, contributing to improving the quality of life of millions worldwide.

Palmitic Acid Upregulates Type I Interferon-Mediated Antiviral Response and Cholesterol Biosynthesis in Human Astrocytes.

Chronic intake of a high-fat diet increases saturated fatty acids in the brain causing the progression of neurodegenerative diseases. Palmitic acid is a free fatty acid abundant in the diet that at high concentrations may penetrate the blood-brain barrier and stimulate the production of pro-inflammatory cytokines, leading to inflammation in astrocytes. The use of the synthetic neurosteroid tibolone in protection against fatty acid toxicity is emerging, but its transcriptional effects on palmitic acid-induced lipotoxicity remain unclear. Herein, we performed a transcriptome profiling of normal human astrocytes to investigate the molecular mechanisms by which palmitic acid causes cellular damage to astrocytes, and whether tibolone could reverse its detrimental effects. Astrocytes undergo a profound transcriptional change at 2 mM palmitic acid, affecting the expression of 739 genes, 366 upregulated and 373 downregulated. However, tibolone at 10 nM does not entirely reverse palmitic acid effects. Additionally, the protein-protein interaction reveals two novel gene clustering modules. The first module involves astrocyte defense responses by upregulation of pathways associated with antiviral innate immunity, and the second is linked to lipid metabolism. Our data suggest that activation of viral response signaling pathways might be so far, the initial molecular mechanism of astrocytes in response to a lipotoxic insult by palmitic acid, triggered particularly upon increased expression levels of IFIT2, IRF1, and XAF1. Therefore, this novel approach using a global gene expression analysis may shed light on the pleiotropic effects of palmitic acid on astrocytes, and provide a basis for future studies addressed to elucidate these responses in neurodegenerative conditions, which is highly valuable for the design of therapeutic strategies.

The hsa-miR-516a-5p and hsa-miR-516b-5p microRNAs reduce the migration and invasion on T98G glioblastoma cell line.

microRNAs (miRNAs) are involved in numerous functions and processes in the brain and other organs through the regulation of gene and protein expression. miRNA dysregulation is associated with the development of several diseases, including the brain and Central Nervous System cancer (CNS). The hsa-miR-516a-5p and hsa-miR-516b-5p are involved in proliferation, migration, and invasion in different tumor models, but their antitumor effect has not been evaluated in cancer of CNS. Therefore, we aimed to assess the effect of the miRNAs hsa-miR-516a-5p and miRNA hsa-miR-516b-5p on the Glioblastoma cell line (T98G). We used synthetic miRNA mimics to induce the overexpression of both miRNAs in the cell line, which was corroborated by RT-qPCR. Next, we evaluated the effect on proliferation, migration, and invasion using the CyQuant direct kit, ThinCert ™ inserts and invasion BioCoat ™ Matrigel® Invasion Chambers. We found upregulation of these miRNAs induced significant changes on the migration and invasion processes of T98G cells, but not affected the proliferation rate. These results suggest that both microRNAs could be playing an important role in the control of tumor progression towards metastasis. The bioinformatics analysis showed that target genes for these miRNAs are involved in different biological processes such as in cell adhesion molecule binding and cell junction disassembly, which are important for cancer progression. Further studies and experimental validation are needed to identify the genes regulated by microRNAs.

TERT silencing alters the expression of ARG1, GLUL, VIM, NES genes and hsa-miR-29b-3p in the T98G cell line.

The central function of telomerase is maintaining the telomere length. However, several extra-telomeric roles have been identified for this protein complex. In this study, we evaluated the effect of the silencing of the catalytic subunit of telomerase (TERT) on the expression of candidate microRNAs, cell activation markers and glial-related genes in a glioblastoma cell line (T98G). The silencing was performed by a siRNA and the qPCR method was used to analyze the expression of TERT and downstream genes. Flow cytometry was used to quantify the TERT protein, and bioinformatics analysis was carried out to analyze the functions of microRNA target genes. Here, it was observed that after a 50% reduction of the TERT gene, the expression of ARG1 (Arginase 1) was upregulated, whereas NES (Nestin), GLUL (Glutamate-Ammonia Ligase), VIM (Vimentin) and the hsa-miR-29b-3p microRNA were downregulated (P-value <0.05). A bioinformatic analysis showed that target genes of hsa-miR-29b are associated with focal adhesion, the PI3K-Akt signaling pathway, among others. These results are important because they contribute to the knowledge of extratelomeric functions by providing relevant evidence about novel genes modulated by TERT.

Control Theory and Systems Biology: Potential Applications in Neurodegeneration and Search for Therapeutic Targets.

Control theory, a well-established discipline in engineering and mathematics, has found novel applications in systems biology. This interdisciplinary approach leverages the principles of feedback control and regulation to gain insights into the complex dynamics of cellular and molecular networks underlying chronic diseases, including neurodegeneration. By modeling and analyzing these intricate systems, control theory provides a framework to understand the pathophysiology and identify potential therapeutic targets. Therefore, this review examines the most widely used control methods in conjunction with genomic-scale metabolic models in the steady state of the multi-omics type. According to our research, this approach involves integrating experimental data, mathematical modeling, and computational analyses to simulate and control complex biological systems. In this review, we find that the most significant application of this methodology is associated with cancer, leaving a lack of knowledge in neurodegenerative models. However, this methodology, mainly associated with the Minimal Dominant Set (MDS), has provided a starting point for identifying therapeutic targets for drug development and personalized treatment strategies, paving the way for more effective therapies.

Modulation of Small RNA Signatures by Astrocytes on Early Neurodegeneration Stages; Implications for Biomarker Discovery.

Diagnosis of neurodegenerative disease (NDD) is complex, therefore simpler, less invasive, more accurate biomarkers are needed. small non-coding RNA (sncRNA) dysregulates in NDDs and sncRNA signatures have been explored for the diagnosis of NDDs, however, the performance of previous biomarkers is still better. Astrocyte dysfunction promotes neurodegeneration and thus derived scnRNA signatures could provide a more precise way to identify of changes related to NDD course and pathogenesis, and it could be useful for the dissection of mechanistic insights operating in NDD. Often sncRNA are transported outside the cell by the action of secreted particles such as extracellular vesicles (EV), which protect sncRNA from degradation. Furthermore, EV associated sncRNA can cross the BBB to be found in easier to obtain peripheral samples, EVs also inherit cell-specific surface markers that can be used for the identification of Astrocyte Derived Extracellular Vesicles (ADEVs) in a peripheral sample. By the study of the sncRNA transported in ADEVs it is possible to identify astrocyte specific sncRNA signatures that could show astrocyte dysfunction in a more simpler manner than previous methods. However, sncRNA signatures in ADEV are not a copy of intracellular transcriptome and methodological aspects such as the yield of sncRNA produced in ADEV or the variable amount of ADEV captured after separation protocols must be considered. Here we review the role as signaling molecules of ADEV derived sncRNA dysregulated in conditions associated with risk of neurodegeneration, providing an explanation of why to choose ADEV for the identification of astrocyte-specific transcriptome. Finally, we discuss possible limitations of this approach and the need to improve the detection limits of sncRNA for the use of ADEV derived sncRNA signatures.

MicroRNA: A Linking between Astrocyte Dysfunction, Mild Cognitive Impairment, and Neurodegenerative Diseases.

The importance of miRNAs in cellular processes and their dysregulation has taken significant importance in understanding different pathologies. Due to the constant increase in the prevalence of neurodegenerative diseases (ND) worldwide and their economic impact, mild cognitive impairment (MCI), considered a prodromal phase, is a logical starting point to study this public health problem. Multiple studies have established the importance of miRNAs in MCI, including astrocyte regulation during stressful conditions. Additionally, the protection mechanisms exerted by astrocytes against some damage in the central nervous system (CNS) lead to astrocytic reactivation, in which a differential expression of miRNAs has been shown. Nevertheless, excessive reactivation can cause neurodegeneration, and a clear pattern defining the equilibrium point between a neuroprotective or detrimental astrocytic phenotype is unknown. Therefore, the miRNA expression has gained significant attention to understand the maintenance of brain balance and improve the diagnosis and treatment at earlier stages in the ND. Here, we provide a comprehensive review of the emerging role of miRNAs in cellular processes that contribute to the loss of cognitive function, including lipotoxicity, which can induce chronic inflammation, also considering the fundamental role of astrocytes in brain homeostasis.

Association between VDR Gene Polymorphisms and Melanoma Susceptibility in a Colombian Population.

BACKGROUND: The vitamin D receptor (VDR) is responsible for mediating the effects of vitamin D through regulation of other gene transcriptions. There are several polymorphisms that alter the gene expression or the function of this protein. We aimed to analyze the association between two SNPs  of VDR gene and melanoma cancer in Colombian patients. METHODS: We included 120 healthy individual as controls and 120 melanoma cancer patients as cases . Patients in both groups were matched in terms of gender and age. The genotyping of rs731236 and rs2228570 polymorphisms was performed using PCR-RFLP. The SNPStats program was used to carry out the statistical analysis through a logistic regression model. RESULTS: Under dominant model, we found that rs2228570 polymorphism was associated with melanoma cancer risk (C/C vs C/T-T/T, OR: 5.10, 95% CI: 2.85-9.14), whereas rs731236 polymorphism was associated with a protective effect against this cancer (T/T vs T/C, OR: 0.27, 95% CI: 0.14-0.53). CONCLUSION: Our results suggested that both polymorphisms were involved in the development of melanoma cancer, increasing or decreasing this risk.

Molecular genetics of substance use disorders: An umbrella review.

BACKGROUND: Substance use disorders (SUD) are a category of psychiatric disorders with a large epidemiological and societal impact around the world. In the last decades, a large number of genetic studies have been published for SUDs. METHODS: With the objective of having an overview and summarizing the evidence published up to date, we carried out an umbrella review of all the meta-analyses of genetic studies for the following substances: alcohol, tobacco, cannabis, cocaine, opioids, heroin and methamphetamines. Meta-analyses for candidate gene studies and genome-wide association studies (GWAS) were included. RESULTS: Alcohol and tobacco were the substances with the largest number of meta-analyses, and cannabis, opioids and cocaine the least studied. The following genes were associated with two or more SUDs: OPRM1, DRD2, DRD4, BDNF and SL6A4. The only genes that had an OR higher than two were the SLC6A4 for all addictions, the ADH1B for alcohol dependence, and BDNF for methamphetamine dependence. GWAS confirmed the possible role of CHRNA5 gene in nicotine dependence and identified novel candidate genes in other SUDs, such as FOXP2, PEX and, AUTS2, which need further functional analyses. CONCLUSIONS: This umbrella review summarizes the evidence of 16 years of research on the genetics of SUDs and provides a broad and detailed overview of results from more than 150 meta-analyses for SUD. The results of this umbrella review will guide the need for future genetic studies geared toward understanding, preventing and treating SUDs.

Common genes and pathways involved in the response to stressful stimuli by astrocytes: A meta-analysis of genome-wide expression studies.

Astrocytes play pivotal roles in the brain and they become reactive under stress conditions. Here, we carried out, for the first time, an integrative meta-analysis of genome-wide expression profiling of astrocytes from human and mouse exposed to different stressful stimuli (hypoxia, infections by virus and bacteria, cytokines, ethanol, among others). We identified common differentially expressed genes and pathways in human and murine astrocytes. Our results showed that astrocytes induce expression of genes associated with stress response and immune system regulation when they are exposed to stressful stimuli, whereas genes related to neurogenesis are found as downregulated. Several of the identified genes showed to be important hubs in the protein-protein interaction analysis (TRAF2, CDC37 and PAX6). This work demonstrates that despite astrocytes are highly heterogeneous and complex, there are common gene expression signatures that can be triggered under distinct detrimental stimuli, which opens an opportunity for exploring other possible markers of reactivity.

Association between resilience and a functional polymorphism in the serotonin transporter (SLC6A4) gene: A meta-analysis.

Resilience is a mechanism used by humans to adapt to adverse situations. It is a protective factor against mental health problems. This process can be influenced by environmental and genetic factors. Several genes have been associated with interindividual differences in resilience levels, but the results are inconclusive. Therefore, the aim of this meta-analysis was to evaluate the effect of a functional polymorphism (5-HTTLPR) in the SLC6A4 gene on resilience levels. A search in PubMed, HugeNavigator and Google Scholar databases was carried out and 16 studies about the association of 5-HTTLPR polymorphism and resilience in humans were identified. The OpenMeta[Analyst] program was employed to perform statistical analysis using a random-effects model. The final analysis included 9 studies, for a total of 4,080 subjects. Significant results were found when the standardized mean differences (SMD) of LL and SL carriers were compared, (SMD: -0.087 (confidence interval: -0.166 to -0.008; I 2 : 0 %); P value: 0.031). A significant result was also found in an analysis comparing SS/SL versus LL genotypes (SMD: -0.231; confidence interval: -0.400 to -0.061, P value: 0.008; I 2 : 0 %). This is the first meta-analysis performed to identify the pooled association of a functional polymorphism in the serotonin transporter gene and resilience. The current results suggest that the L/L genotype is associated with resilience. Further studies are necessary to elucidate the role of genetics on the resilience mechanisms.

Integrative In Silico Analysis of Genome-Wide DNA Methylation Profiles in Schizophrenia.

Schizophrenia (SZ) is a complex and severe psychiatric disorder, which has a global lifetime prevalence of 0.4% and a heritability of around 0.81. A number of epigenome-wide association studies (EWAS) have been carried out for SZ, with discordant results. The main aim of this study was to carry out an integrative in silico analysis of available genome-wide DNA methylation profiles in schizophrenia. In this work, an integration of multiple lines of evidence (top candidate genes from several EWAS and genome-wide expression and association data) was carried out, in order to identify top differentially methylated (DM) genes for SZ. In addition, functional enrichment and protein-protein interaction analyses were carried out. Several top differentially methylated genes, such as APC, CACNB2, and PRKN, were found, and an enrichment of binding sites for brain-expressed transcription factors, such as FOXO1, MYB, and ZIC3, was also observed. Moreover, a protein-protein interaction network showed a central role for DISC1 and ZNF688 genes, and experimentally validated targets of MIR-137, such as and KCNB2, NRXN1, and SYN2, were identified among DM genes. This is the first integrative in silico analysis of available genome-wide DNA methylation profiles in schizophrenia. This work identified novel candidate genes and pathways for SZ and provides the basis to explore their role in the pathogenesis of SZ in future studies.

A functional SNP in the synaptic SNAP25 gene is associated with impulsivity in a Colombian sample.

The aim of the current study was to test the hypothesis that a functional polymorphism in the synaptosome associated protein 25 (SNAP25) gene could be associated with impulsivity scores in a sample of young Colombian subjects. Impulsivity has been postulated as an endophenotype for several psychiatric disorders of high epidemiological relevance. There is a need for the study of additional candidate genes for impulsivity. One hundred seventy-five young Colombian subjects completed the Spanish version of the short form of the Barratt Impulsiveness Scale (BIS-15S). A TaqMan assay was used to genotype a functional polymorphism (rs3746544) in the SNAP25 gene. A significant association was found between the functional polymorphism in the SNAP25 gene and impulsivity in the Colombian sample, with subjects carrying T/T and G/G genotypes showing lower mean scores in the non-planning subfactor (p = 0.02). This is the first report of an association of a functional polymorphism in the SNAP25 gene and a subfactor of the BIS-15S scale of impulsivity. In addition, this the first genetic study of impulsivity scores in a Latin American sample. Future studies should explore additional variants in brain-expressed miRNAs and in their binding sites as candidates for impulsivity in different populations.

Convergent functional genomics of cocaine misuse in humans and animal models.

Background: Data from the Global Burden of Disease Study 2016 recently estimated that after opioid and cannabis use disorders, cocaine use disorders were among the most common, with around 5.8 million cases around the world. Several genome-wide expression studies (GWES) for cocaine misuse have been carried out in brain tissues from patients and controls and in mouse and rat models.Objectives: In the current work, we used a convergent functional genomics approach to identify novel candidate genes and pathways for cocaine misuse.Methods: We carried out meta-analyses for available GWES for cocaine misuse in humans and mouse and rat models (three, four, and two GWES, respectively). Multiple lines of evidence (GWES, genome-wide association and epigenomic data) were integrated to prioritize top candidate genes, and a functional enrichment analysis was carried out.Results: Several top candidate genes supported by multiple lines of genomic evidence, and with known roles in brain plasticity, were identified: APP, GRIN2A, GRIN2B, KCNA2, MAP4, PCDH10, PPP3CA, SNCB, and SV2C. An enrichment of genes regulated by the AP1 transcription factor was found.Conclusion: This is the first meta-analysis of GWES for cocaine misuse in humans and mouse and rat models. The analysis of convergence of multiple lines of genome-wide evidence identified novel candidate genes and pathways for cocaine misuse, which are of basic and clinical importance.

Scientific productivity in neurosciences in Latin America: a scientometrics perspective.

Background: Neurosciences research has increased significantly in recent years around the world. It has led to the development of interdisciplinary work, moving from activities from isolated fields (such as biology, psychology or neurology) to research that involves different scientific perspectives. In developing regions, such as Latin America, it has additional challenges, related to available funding and infrastructure.Aim: To analyze key factors in scientific productivity in neurosciences in Latin America.Methods: A bibliometric analysis of the scientific productivity in neurosciences in main five Latin American countries (Argentina, Brazil, Chile, Colombia and Mexico) was carried out.Results: Brazil was the largest producer of scientific articles, and receptor of citations, in neurosciences in 1998-2017, followed by Mexico. We identified highly cited papers, top institutions, networks of authors, main journals and key areas in neurosciences for this period in the 5 countries.Conclusions: Scientific productivity in neurosciences in Latin America would benefit from the consolidation of more regional, interdisciplinary and international research networks. In this work, we discuss key elements for the consolidation of neurosciences research in Latin America.

qPCR-based methods for expression analysis of miRNAs.

Several approaches for miRNA expression analysis have been developed in recent years. In this article, we provide an updated and comprehensive review of available qPCR-based methods for miRNA expression analysis and discuss their advantages and disadvantages. Existing techniques involve the use of stem-loop reverse transcriptase-PCR, polyadenylation of RNAs, ligation of adapters or RT with complex primers, using universal or miRNA-specific qPCR primers and/or probes. Many of these methods are oriented towards the expression analysis of mature miRNAs and few are designed for the study of pre-miRNAs and pri-miRNAs. We also discuss findings from articles that compare results from existing methods. Finally, we suggest key points for the improvement of available techniques and for the future development of additional methods.

TERT inhibition leads to reduction of IL-6 expression induced by palmitic acid and interferes with the protective effects of tibolone in an astrocytic cell model.

Although it has been shown that telomerase has neuroprotective effects, mainly as a result of its non-canonical functions in neuronal cells, its role with respect to glial cells remains unknown. There is growing evidence indicating that telomerase plays an important role with respect to inflammation, especially in the regulation of pro-inflammatory cytokine gene expression. The present study aimed to evaluate the role of telomerase in an astrocyte cell model treated with palmitic acid (PA) and tibolone. Cell death, reactive oxygen species production and interleukin-6 expression were evaluated under telomerase inhibition with the BIBR1532 compound in T98G cells treated with tibolone and PA, using fluorometry, flow cytometry, enzyme-linked immunosorbent assays and the quantitative polymerase chain reaction. The results obtained showed that telomerase protein was increased by PA after 36 hours, alone or in combination with tibolone, and that its activity was affected by PA. Telomerase inhibition reduced interleukin-6 expression and it interfered with the protective effects of tibolone on cell death. Moreover, tibolone increased Tyr707 phosphorylation in PA-treated cells. In the present study, we provide novel findings about the regulation of telomerase by PA and tibolone. Telomerase was involved in inflammation by PA and in protective effects of tibolone. Therefore, we conclude that telomerase could play a dual role in these cells.

Tibolone attenuates inflammatory response by palmitic acid and preserves mitochondrial membrane potential in astrocytic cells through estrogen receptor beta.

Palmitic acid (PA) induces several metabolic and molecular changes in astrocytes, and, it is involved in pathological conditions related to neurodegenerative diseases. Previously, we demonstrated that tibolone, a synthetic steroid with estrogenic, progestogenic and androgenic actions, protects cells from mitochondrial damage and morphological changes induced by PA. Here, we have evaluated which estrogen receptor is involved in protective actions of tibolone and analyzed whether tibolone reverses gene expression changes induced by PA. Tibolone actions on astrocytic cells were mimicked by agonists of estrogen receptor α (ERα) and ß (ERß), but the blockade of both ERs suggested a predominance of ERß on mitochondria membrane potential. Expression analysis showed a significant effect of tibolone on genes associated with inflammation such as IL6, IL1B and miR155-3p. It is noteworthy that tibolone attenuated the increased expression of TERT, TERC and DNMT3B genes induced by palmitic acid. Our results suggest that tibolone has anti-inflammatory effects and can modulate pathways associated with DNA methylation and telomeric complex. However, future studies are needed to elucidate the role of epigenetic mechanisms and telomere-associated proteins on tibolone actions.

Situation Awareness Performance in Healthy Young Adults Is Associated With a Serotonin Transporter Gene Polymorphism.

Background Situation awareness (SA) is defined in three levels: SA1 is the perception of the elements in a specific context, SA2 is the comprehension of their meaning, and SA3 is the projection of their status. Purpose To analyze the possible association of a genetic polymorphism in the serotonin transporter ( SLC6A4) gene and performance on the Situational Awareness test (SAtest). Methods SAtest was applied to a sample of 230 healthy Colombian subjects, using the Psychology Experiment Building Language platform and a functional polymorphism in the SLC6A4 gene was genotyped by polymerase chain reaction. Results In the SA1 level, s/s genotype carriers had worse accuracy, in comparison with s/l and l/l genotypes. At SA2 level, l/l genotype carriers had better accuracy than s/s and s/l individuals and that in the SA3 level, l/l carriers also had better accuracy. These associations were significant after correction for multiple testing. Conclusions It is possible that l/l carriers have a better ability to perceive and focus their attention on the elements of their environment and to have the capacity to understand and predict what will happen with those elements. This is the first genetic study of SA performance in healthy participants. Additional investigations of other genes could contribute to the understanding of the molecular correlates of SA in healthy subjects and in neuropsychiatric patients.