Sustained Depolarization Induces Gene Expression Pattern Changes Related to Synaptic Plasticity in a Human Cholinergic Cellular Model.

Neuronal gene expression in the brain dynamically responds to synaptic activity. The interplay among synaptic activity, gene expression, and synaptic plasticity has crucial implications for understanding the pathophysiology of diseases such as Alzheimer's disease and epilepsy. These diseases are marked by synaptic dysfunction that affects the expression patterns of neuroprotective genes that are incompletely understood. In our study, we developed a cellular model of synaptic activity using human cholinergic neurons derived from SH-SY5Y cell differentiation. Depolarization induction modulates the expression of neurotrophic genes and synaptic markers, indicating a potential role in synaptic plasticity regulation. This hypothesis is further supported by the induction kinetics of various long non-coding RNAs, including primate-specific ones. Our experimental model showcases the utility of SH-SY5Y cells in elucidating the molecular mechanisms underlying synaptic plasticity in human cellular systems.

Genome-wide analyses reveal a potential role for the MAPT, MOBP, and APOE loci in sporadic frontotemporal dementia.

Frontotemporal dementia (FTD) is the second most common cause of early-onset dementia after Alzheimer disease (AD). Efforts in the field mainly focus on familial forms of disease (fFTDs), while studies of the genetic etiology of sporadic FTD (sFTD) have been less common. In the current work, we analyzed 4,685 sFTD cases and 15,308 controls looking for common genetic determinants for sFTD. We found a cluster of variants at the MAPT (rs199443; p = 2.5 × 10-12, OR = 1.27) and APOE (rs6857; p = 1.31 × 10-12, OR = 1.27) loci and a candidate locus on chromosome 3 (rs1009966; p = 2.41 × 10-8, OR = 1.16) in the intergenic region between RPSA and MOBP, contributing to increased risk for sFTD through effects on expression and/or splicing in brain cortex of functionally relevant in-cis genes at the MAPT and RPSA-MOBP loci. The association with the MAPT (H1c clade) and RPSA-MOBP loci may suggest common genetic pleiotropy across FTD and progressive supranuclear palsy (PSP) (MAPT and RPSA-MOBP loci) and across FTD, AD, Parkinson disease (PD), and cortico-basal degeneration (CBD) (MAPT locus). Our data also suggest population specificity of the risk signals, with MAPT and APOE loci associations mainly driven by Central/Nordic and Mediterranean Europeans, respectively. This study lays the foundations for future work aimed at further characterizing population-specific features of potential FTD-discriminant APOE haplotype(s) and the functional involvement and contribution of the MAPT H1c haplotype and RPSA-MOBP loci to pathogenesis of sporadic forms of FTD in brain cortex.

Expression of Insl3 Protein in Adult Danio rerio.

Insulin-like peptide 3 (INSL3) is a biomarker for Leydig cells in the testes of vertebrates, and it is principally involved in spermatogenesis through specific binding with the RXFP2 receptor. This study reports the insl3 gene transcript and the Insl3 prepropeptide expression in both non-reproductive and reproductive tissues of Danio rerio. An immunohistochemistry analysis shows that the hormone is present at a low level in the Leydig cells and germ cells at all stages of Danio rerio testis differentiation. Considering that the insl3 gene is transcribed in Leydig cells, our results highlight an autocrine and paracrine function of this hormone in the Danio rerio testis, adding new information on the Insl3 mode of action in reproduction. We also show that Insl3 and Rxfp2 belonging to Danio rerio and other vertebrate species share most of the amino acid residues involved in the ligand-receptor interaction and activation, suggesting a conserved mechanism of action during vertebrate evolution.

The long non-coding RNA transcript, LOC100130460 (CAND1.11) gene, encodes a novel protein highly expressed in cancer cells and tumor human testis tissues.

BACKGROUND: Testis-specific genes encoding for long non-coding RNA (lncRNA) have been detected in several cancers; many produce proteins with restricted or aberrant expression patterns in normal or cancer tissues. OBJECTIVE: To characterize new lncRNA involved in normal and/or pathological differentiation of testicular cells. METHODS: Using bioinformatics analysis, we found that lncRNA LOC100130460 (CAND1.11) is expressed in normal and tumor testis; its expression was assessed in several human cell lines by qRT-PCR. CAND1.11 protein, produced by a single nucleotide mutation, was studied by western blot and immunofluorescence analysis on normal, classic seminoma, and Leydig cell tumor testicular tissues. RESULTS: CAND1.11 gene is primate-specific; its expression was low in SH-SY5Y cells and increased when differentiated with retinoic acid treatment. CAND1.11 expression in PC3 cells was higher than in PNT2 cells. CAND1.11 protein is present in the human testis and overexpressed in testicular cancer tissues. CONCLUSIONS: This report is one of the few providing evidence that a lncRNA produces a protein expressed in normal human tissues and overexpressed in several testicular cancers, suggesting its involvement in regulating cell proliferation and differentiation. Although further studies are needed to validate the results, our data indicate that CAND1.11 could be a potential new prognostic biomarker to use in proliferation and cancer.

Long Non-Coding RNAs, Extracellular Vesicles and Inflammation in Alzheimer's Disease.

Alzheimer's Disease (AD) has currently no effective treatment; however, preventive measures have the potential to reduce AD risk. Thus, accurate and early prediction of risk is an important strategy to alleviate the AD burden. Neuroinflammation is a major factor prompting the onset of the disease. Inflammation exerts its toxic effect via multiple mechanisms. Amongst others, it is affecting gene expression via modulation of non-coding RNAs (ncRNAs), such as miRNAs. Recent evidence supports that inflammation can also affect long non-coding RNA (lncRNA) expression. While the association between miRNAs and inflammation in AD has been studied, the role of lncRNAs in neurodegenerative diseases has been less explored. In this review, we focus on lncRNAs and inflammation in the context of AD. Furthermore, since plasma-isolated extracellular vesicles (EVs) are increasingly recognized as an effective monitoring strategy for brain pathologies, we have focused on the studies reporting dysregulated lncRNAs in EVs isolated from AD patients and controls. The revised literature shows a positive association between pro-inflammatory lncRNAs and AD. However, the reports evaluating lncRNA alterations in EVs isolated from the plasma of patients and controls, although still limited, confirm the value of specific lncRNAs associated with AD as reliable biomarkers. This is an emerging field that will open new avenues to improve risk prediction and patient stratification, and may lead to the discovery of potential novel therapeutic targets for AD.

Relaxin ligand/receptor systems in the developing teleost fish brain: Conserved features with mammals and a platform to address neuropeptide system functions.

The relaxins (RLNs) are a group of peptide hormone/neuromodulators that can regulate a wide range of physiological processes ranging from reproduction to brain function. All the family members have originated from a RLN3-like ancestor via different rounds of whole genome and gene specific duplications during vertebrate evolution. In mammals, including human, the divergence of the different family members and the emergence of new members led to the acquisition of specific functions for the various relaxin family peptide and associated receptor genes. In particular, in mammals, it was shown, that the role of RLN3 is correlated to the modulation of arousal, stress responses, emotion, social recognition, and other brain functions, positioning this gene/peptide as a potential therapeutic target for neuropsychiatric disorders. This review highlights the evolutionary conservation of relaxin family peptide and receptor gene expression and their associated brain neural circuits. In the zebrafish, the expression pattern of the different relaxin family members has specific features that are conserved in higher species, including a likely similar functional role for the ancestral RLN3-like gene. The use of different model organisms, particularly the zebrafish, to explore the diversification and conservation of relaxin family ligands and receptor systems, provides a relatively high-throughput platform to identify their specific conserved or differential neuromodulatory roles in higher species including human.

CD33 rs2455069 SNP: Correlation with Alzheimer's Disease and Hypothesis of Functional Role.

The CD33 gene encodes for a member of the sialic-acid-binding immunoglobulin-type lectin (Siglec) family, and is one of the top-ranked Alzheimer's disease (AD) risk genes identified by genome-wide association studies (GWAS). Many CD33 polymorphisms are associated with an increased risk of AD, but the function and potential mechanism of many CD33 single-nucleotide polymorphisms (SNPs) in promoting AD have yet to be elucidated. We recently identified the CD33 SNP rs2455069-A>G (R69G) in a familial form of dementia. Here, we demonstrate an association between the G allele of the rs2455069 gene variant and the presence of AD in a cohort of 195 patients from southern Italy. We carried out in silico analysis of the 3D structures of CD33 carrying the identified SNP to provide insights into its functional effect. Structural models of the CD33 variant carrying the R69G amino acid change were compared to the CD33 wild type, and used for the docking analysis using sialic acid as the ligand. Our analysis demonstrated that the CD33-R69G variant may bind sialic acid at additional binding sites compared to the wild type, thus potentially increasing its affinity/specificity for this molecule. Our results led to a new hypothesis of rs2455069-A>G SNP as a risk factor for AD, suggesting that a long-term cumulative effect of the CD33-R69G variant results from the binding of sialic acid, acting as an enhancer of the CD33 inhibitory effects on amyloid plaque degradation.

Apoptosis, oxidative stress and genotoxicity in developing zebrafish after aluminium exposure.

Aluminium is a non-essential metal and potentially toxic to organisms whose environmental concentration increases due to pollution. In our previous studies, the behavioral changes induced by aluminium were already shown on zebrafish, a model organism widely used for ecotoxicology screening. To examine in depth the knowledge about the toxicity mechanism induced by this metal, zebrafish embryos, at 6 hpf, have been exposed to 50, 100 and 200 µM of AlCl3 for 72 h. Phenotypic alterations, apoptosis and oxidative stress responses have been assessed by evaluations of antioxidant defence and changes in metabolism at the end of treatment. The mRNA expression level of c-fos, appa and appb as marker genes of neural development and function were analyzed by qPCR for the highest used concentration. The data showed that aluminium significantly affected the development of zebrafish inducing morphological alterations and cell death. The oxidative state of larvae was altered, although the formation of reactive oxygen species and the levels of metallothioneins, and the activity of some antioxidant enzymes, decreased at the maximum concentration tested. In addition, at this concentration, the expression of the evaluated genes increased. The comprehensive information obtained gives a realistic snapshot of the aluminium toxicity and provides new information on the mechanism of action of this metal.

LINC00473 as an Immediate Early Gene under the Control of the EGR1 Transcription Factor.

Immediate early genes play an essential role in cellular responses to different stimuli. Many of them are transcription factors that regulate the secondary response gene expression. Non-coding RNAs may also be involved in this regulatory cascade. In fact, they are emerging as key actors of gene expression regulation, and evidence suggests that their dysregulation may underly pathological states. We previously took a snapshot of both coding and long non-coding RNAs differentially expressed in neuronal cells after brain-derived neurotrophic factor stimulation. Among these, the transcription factor EGR1 (a well-known immediate early gene) and LINC00473 (a primate-specific long non-coding RNA) that has emerged as an interesting RNA candidate involved in neuronal function and in cancer. In this work, we demonstrated that LINC00473 gene expression kinetics resembled that of immediate early genes in SH-SY5Y and HEK293T cells under different cell stimulation conditions. Moreover, we showed that the expression of LINC00473 is under the control of the transcription factor EGR1, providing evidence for an interesting functional relationship in neuron function.

EH domain binding protein 1-like 1 (EHBP1L1), a protein with calponin homology domain, is expressed in the rat testis.

In this paper, with the aim to find new genes involved in mammalian spermatogenesis, we isolated, for the first time in the rat testis, a partial cDNA clone that encoded EH domain binding protein 1-like 1 (Ehbp1l1), a protein that has a single calponin homology domain (CH). Bioinformatic analysis showed that EHBP1l1 contains three domains: the N-terminal C2-like, the CH and the C-terminal bivalent Mical/EHBP Rab binding (bMERB) domains, which are evolutionarily conserved in vertebrates. We found that Ehbp1l1 mRNA was expressed in several rat tissues, including the liver, intestine, kidney and also in the testis during its development, with a higher level in testis from 12-month-old animals. Interestingly, in situ hybridization experiments revealed that Ehbp1l1 is specifically expressed by types I and II spermatocytes, this result was validated by RT-PCR performed on total RNA obtained from enriched fractions of different testicular cell types. As EHBP1l1 has been described as linked to vesicular transport to the actin cytoskeleton and as an effector of the small GTPase Rab8, we hypothesized that it could participate both in cytoskeletal remodelling and in the regulation of vesicle sorting from the trans-Golgi network to the apical plasma membrane. Our findings provide a better understand of the molecular mechanisms of the differentiation process of spermatogenesis; Ehbp1l1 may also be used as a new marker of testicular activity.

CD33 and SIGLECL1 Immunoglobulin Superfamily Involved in Dementia.

Sialic acid-binding immunoglobulin-type lectins, which are predominantly expressed in immune cells, represent a family of immunomodulatory receptors with inhibitory and activating signals, in both healthy and disease states. Genetic factors are important in all forms of dementia, especially in early onset dementia. CD33 was recently recognized as a genetic risk factor for Alzheimer disease (AD). Here, we present a 2-generation family with 4 members, the father and the 3 siblings, characterized by an early form of unusual dementia exhibiting a behavioral variant close to behavioral variant frontotemporal dementia phenotype and severe forms of memory loss suggestive of AD. We analyzed the CD33 gene in this family and identified 10 single nucleotide polymorphisms (SNPs) in a linkage disequilibrium block associated with the disease. We also identified a tag SNP, rs2455069-A>G, in CD33 exon 2 that could be involved with dementia risk. Additionally, we excluded the presence of C9orf72 expansion mutations and other mutations previously associated with sporadic FTD and AD. The tag SNP association was also analyzed in selected sporadic AD patients from the same Southern Italy region. We demonstrate that CD33 and SIGLECL1 have a significantly increased level of expression in these patients.

Clinical and Molecular Characterization of a Novel Progranulin Deletion Associated with Different Phenotypes.

BACKGROUND: Mutations in the GRN gene are causative for an autosomal dominant form of frontotemporal dementia. OBJECTIVE/METHODS: The objective of the present study is to describe clinical and molecular features of three siblings harboring the GRN deletion NM_002087.3:c.295_308delTGCCCACGGGGCTT, p.(Cys99Profs*15) identified with next generation sequencing. RESULTS: Our patients demonstrated heterogeneous clinical phenotypes, such as progressive supranuclear palsy-like in the proband and the behavioral variant of frontotemporal dementia in the two affected siblings. Progranulin haploinsufficiency was revealed by both gene expression and protein analyses. CONCLUSION: The pathogenicity of the novel GRN deletion c.295_308del TGCCCACGGGGCTT is confirmed by both functional analysis and segregation in three affected siblings.

Roles of Transposable Elements in the Different Layers of Gene Expression Regulation.

The biology of transposable elements (TEs) is a fascinating and complex field of investigation. TEs represent a substantial fraction of many eukaryotic genomes and can influence many aspects of DNA function that range from the evolution of genetic information to duplication, stability, and gene expression. Their ability to move inside the genome has been largely recognized as a double-edged sword, as both useful and deleterious effects can result. A fundamental role has been played by the evolution of the molecular processes needed to properly control the expression of TEs. Today, we are far removed from the original reductive vision of TEs as "junk DNA", and are more convinced that TEs represent an essential element in the regulation of gene expression. In this review, we summarize some of the more recent findings, mainly in the animal kingdom, concerning the active roles that TEs play at every level of gene expression regulation, including chromatin modification, splicing, and protein translation.

Characterization of paralogous uncx transcription factor encoding genes in zebrafish.

The paired-type homeodomain transcription factor Uncx is involved in multiple processes of embryogenesis in vertebrates. Reasoning that zebrafish genes uncx4.1 and uncx are orthologs of mouse Uncx, we studied their genomic environment and developmental expression. Evolutionary analyses indicate the zebrafish uncx genes as being paralogs deriving from teleost-specific whole-genome duplication. Whole-mount in situ mRNA hybridization of uncx transcripts in zebrafish embryos reveals novel expression domains, confirms those previously known, and suggests sub-functionalization of paralogs. Using genetic mutants and pharmacological inhibitors, we investigate the role of signaling pathways on the expression of zebrafish uncx genes in developing somites. In identifying putative functional role(s) of zebrafish uncx genes, we hypothesized that they encode transcription factors that coordinate growth and innervation of somitic muscles.

Expression pattern dysregulation of stress- and neuronal activity-related genes in response to prenatal stress paradigm in zebrafish larvae.

Maternal stress during pregnancy adversely affects developmental fetal programming. Glucocorticoid excess is one of those conditions that underlie the prenatal stress and can lead to many pathological disorders later in life. Beyond the obvious use of mammalian model organisms to uncover the different mechanisms at the basis of prenatal stress effects, zebrafish represents a complementary fruitful model for this research field. Here we demonstrated that the application of an experimental paradigm, which simulates prenatal stress by exposing embryos to cortisol excess, produced an alteration of gene expression pattern. In particular, the transcript level of hsd11b2, a gene involved in the cortisol catabolism, was affected in prenatally stressed larvae, even after many hours from the removal of cortisol excess. Interestingly, the expression pattern of c-fos, a marker gene of neural activity, was affected in prenatally stressed larvae even in response to a swirling and osmotic stress challenge. Our data corroborate the idea of zebrafish as a useful model organism to study prenatal stress effects on vertebrate development.

Identification, Characterization, and Regulatory Mechanisms of a Novel EGR1 Splicing Isoform.

EGR1 is a transcription factor expressed in many cell types that regulates genes involved in different biological processes including growth, proliferation, and apoptosis. Dysregulation of EGR1 expression has been associated with many pathological conditions such as tumors and brain diseases. Known molecular mechanisms underlying the control of EGR1 function include regulation of transcription, mRNA and protein stability, and post-translational modifications. Here we describe the identification of a splicing isoform for the human EGR1 gene. The newly identified splicing transcript encodes a shorter protein compared to the canonical EGR1. This isoform lacks a region belonging to the N-terminal activation domain and although it is capable of entering the nucleus, it is unable to activate transcription fully relative to the canonical isoform.

Characterization of paralogous uncx transcription factor encoding genes in zebrafish.

The paired-type homeodomain transcription factor Uncx is involved in multiple processes of embryogenesis in vertebrates. Reasoning that zebrafish genes uncx4.1 and uncx are orthologs of mouse Uncx, we studied their genomic environment and developmental expression. Evolutionary analyses indicate the zebrafish uncx genes as being paralogs deriving from teleost-specific whole-genome duplication. Whole-mount in situ mRNA hybridization of uncx transcripts in zebrafish embryos reveals novel expression domains, confirms those previously known, and suggests sub-functionalization of paralogs. Using genetic mutants and pharmacological inhibitors, we investigate the role of signaling pathways on the expression of zebrafish uncx genes in developing somites. In identifying putative functional role(s) of zebrafish uncx genes, we hypothesized that they encode transcription factors that coordinate growth and innervation of somitic muscles.

Temporal and spatial expression of insulin-like peptide (insl5a and insl5b) paralog genes during the embryogenesis of Danio rerio.

Relaxin (RLN) and insulin (INSL)-like peptides are member of the INSL/RLN superfamily, which are encoded by seven genes in humans and can activate the G-protein coupled receptor RXFP 1-4. These peptides evolved from a common ancestor, RLN3-like gene. Two rounds of whole genome duplication (WGD) in early vertebrate evolution, together with an additional WGD in the teleost lineage, caused an expansion of RLN genes set in the genome of Danio rerio. In particular, six RLN genes are present: a single copy of rln and insl3 genes, and two paralogs for the rln3 gene (rln3a and rln3b), and the insl5 gene (insl5a and insl5b). We have already reported the presence of rln3a and rln3b genes in the developing zebrafish brain, as well as the expression of rln gene in the developing zebrafish brain and extraneural territories, such as thyroid gland and pancreas. Here, we report for the first time the expression of the two parologs genes for insl5, insl5a, and insl5b in D. rerio embryonic development. The corresponding transcripts of both the paralogs are present in all embryonic stages analyzed by RT-qPCR. In situ hybridization analyses showed a restricted signal in intestinal cells and the pancreatic region at 72 hpf for insl5a, while at 96 hpf both genes are expressed in specific intestinal cells. Furthermore, in adult zebrafish intestine tissue, in situ hybridation experiments showed that insl5a transcript is specifically localized in the goblet cells, while insl5b transcript is in enteroendocrine cells. These data revealed a high degree of gene expression pattern conservation for such genes in vertebrate evolution.

Brain-derived neurotrophic factor modulates cholesterol homeostasis and Apolipoprotein E synthesis in human cell models of astrocytes and neurons.

In the central nervous system, cholesterol is critical to maintain membrane plasticity, cellular function, and synaptic integrity. In recent years, much attention was focused on the role of cholesterol in brain since a breakdown of cholesterol metabolism has been associated with different diseases. Brain-derived neurotrophic factor (BDNF) was previously reported to elicit cholesterol biosynthesis and promote the accumulation of presynaptic proteins in cholesterol-rich lipid rafts, but no data are available on its ability to modulate physiological mechanisms involved in cholesterol homeostasis. Major aim of this research was to investigate whether BDNF influences cholesterol homeostasis, focusing on the effect of the neurotrophin on Apolipoprotein E (ApoE) synthesis, cholesterol efflux from astrocytes and cholesterol incorporation into neurons. Our results show that BDNF significantly stimulates cholesterol efflux by astrocytes, as well as ATP binding cassette A1 (ABCA1) transporter and ApoE expression. Conversely, cholesterol uptake in neurons was downregulated by BDNF. This effect was associated with the increase of Liver X Receptor (LXR)-beta expression in neuron exposed to BDNF. The level of apoptosis markers, that is, cleaved caspase 3 and poly ADP ribose polymerase (PARP), was found increased in neurons treated with high cholesterol, but significantly lower when the cells were exposed to cholesterol in the presence of BDNF, thus suggesting a neuroprotective role of the neurotrophin, likely through its reducing effect of neuronal cholesterol uptake. Interestingly, cholesterol stimulates BDNF production by neurons. Overall, our findings evidenced a novel role of BDNF in the modulation of ApoE and cholesterol homeostasis in glial and neuronal cells.

Epigenetics and cortical spreading depression: changes of DNA methylation level at retrotransposon sequences.

Cortical spreading depression (CSD) is an evolutionarily conserved phenomenon that involves a slow and self-propagating depolarization wave associated with spontaneous depression of electrical neuronal activity. CSD plays a central role in the pathophysiology of several brain diseases and is considered to be able to promote "Preconditioning". This phenomenon consists of the brain protecting itself against future injury by adaptation. Understanding of the molecular mechanisms underlying Preconditioning has significant clinical implications. We have already proposed that the long-lasting effects of CSD could be related to silencing of retrotransposon sequences by histone methylation. We analyzed DNA methylation of two retrotransposon sequences, LINE1 and L1, and their corresponding expression pattern after CSD induction. Based on immunoprecipitation assay of the methylated DNA (meDIP), we demonstrated hypermethylation of both sequences in preconditioned rat brain cortex compared with a control 24 h after CSD induction. Using quantitative PCR, we also showed that CSD induction caused a decrease of the transcript level of both retrotransposon sequences. Our data are consistent with the hypothesis of epigenetic modifications in Preconditioning-dependent neuroprotection by increasing genome stability via the silencing of retrotransposon sequences.