Removal of AMPA receptors (AMPARs) from synapses is preceded by transient endocytosis of extrasynaptic AMPARs

AMPA receptors (AMPARs) are dynamically regulated at synapses, but the time course and location of their exocytosis and endocytosis are not known. Therefore, we have used ecliptic pHluorin-tagged glutamate receptor 2 to visualize changes in AMPAR surface expression in real time. We show that synaptic and extrasynaptic AMPARs respond very differently to NMDA receptor activation; there is a rapid internalization of extrasynaptic AMPARs that precedes the delayed removal of synaptic AMPARs

The increasing diversity of functions attributed to the SAFB family of RNA/DNA binding proteins

RNA-binding proteins play a central role in cellular metabolism by orchestrating the complex interactions of coding, structural and regulatory RNA species. The SAFB (scaffold attachment factor B) proteins (SAFB1, SAFB2 and SAFB-like transcriptional modulator, SLTM), which are highly conserved evolutionarily, were first identified on the basis of their ability to bind scaffold attachment region DNA elements, but attention has subsequently shifted to their RNA-binding and protein–protein interactions. Initial studies identified the involvement of these proteins in the cellular stress response and other aspects of gene regulation. More recently, the multifunctional capabilities of SAFB proteins have shown that they play crucial roles in DNA repair, processing of mRNA and regulatory RNA, as well as in interaction with chromatin-modifying complexes. With the advent of new techniques for identifying RNA-binding sites, enumeration of individual RNA targets has now begun. This review aims to summarise what is currently known about the functions of SAFB proteins.</jats:p

Contrasting roles for DNA methyltransferases and histone deacetylases in single-item and associative recognition memory

Recognition memory enables us to judge whether we have encountered a stimulus before and to recall associated information, including where the stimulus was encountered. The perirhinal cortex (PRh) is required for judgment of stimulus familiarity, while hippocampus (HPC) and medial prefrontal cortex (mPFC) are additionally involved when spatial information associated with a stimulus needs to be remembered. While gene expression is known to be essential for the consolidation of long-term recognition memory, the underlying regulatory mechanisms are not fully understood. Here we investigated the roles of two epigenetic mechanisms, DNA methylation and histone deacetylation, in recognition memory. Infusion of DNA methyltransferase inhibitors into PRh impaired performance in novel object recognition and object-in-place tasks while infusions into HPC or mPFC impaired object-in-place performance only. In contrast, inhibition of histone deacetylases in PRh, but not mPFC, enhanced recognition memory. These results support the emerging role of epigenetic processes in learning and memory

Expression of miR-1, miR-133a, miR-133b and miR-206 increases during development of human skeletal muscle

International audienceBACKGROUND: MicroRNAs (miRNAs) are small RNA molecules that post-transcriptionally regulate gene expression and have been shown to play an important role during development. miR-1, miR-133a, miR-133b and miR-206 are expressed in muscle tissue and induced during muscle cell differentiation, a process that directs myoblasts to differentiate into mature myotubes, which are organized into myofibers. Although miR-1, miR-133a, miR-133b and miR-206 are well-studied in muscle, there is no information about their expression and function during human development. The purpose of this study was to determine the profile of these miRNAs in muscle cells isolated from different stages of human development. RESULTS: We examined the levels of miR-1, miR-133a, miR-133b and miR-206 during the development of human foetus. All four miRNA levels were found increased during late stages of human foetal muscle development. Increases in the expression levels of these miRNAs were proportional to the capacity of myoblasts to form myotubes. Changes in miRNA levels during human foetal development were accompanied by endogenous alterations in their known targets and also in their inducer, MyoD. Ectopic MyoD expression caused an induction of muscle cell differentiation in vitro, accompanied by an increase in the levels of miR-1, miR-133a, miR-133b and miR-206. CONCLUSIONS: This study provides data about the profile of four miRNAs in human muscle cells isolated during different stages of foetal development. These results may shed light on the differentiation of muscle cells and regulation of muscle formation through miRNAs, during the development of human foetus

The Application of Ribozymes and DNAzymes in Muscle and Brain

The discovery of catalytic nucleic acids (CNAs) has provided scientists with valuable tools for the identification of new therapies for several untreated diseases through down regulation or modulation of endogenous gene expression involved in these ailments. These CNAs aim either towards the elimination or repair of pathological gene expression. Ribozymes, a class of CNAs, can be mostly used to down-regulate (by RNA cleavage) or repair (by RNA trans-splicing) unwanted gene expression involved in disease. DNAzymes, derived by in vitro selection processes are also able to bind and cleave RNA targets and therefore down-regulate gene expression. The purpose of this review is to present and discuss several applications of ribozymes and DNAzymes in muscle and brain. There are several diseases which affect muscle and brain and catalytic nucleic acids have been used as tools to target specific cellular transcripts involved in these groups of diseases

Exosomes populate the cerebrospinal fluid of preterm infants with post-haemorrhagic hydrocephalus

Background Preterm infants are at risk of germinal matrix haemorrhage-intraventricular haemorrhage (GMH-IVH) which leads to post-haemorrhagic hydrocephalus (PHH) in 30% of infants; this is associated with moderate-severe neurodevelopmental impairment and confers significant risk of cerebral palsy. There are however no predictive indicators of the severity or long-term outcome after GMH-IVH. In recent years, endosome-derived extracellular vesicles (EVs) or exosomes have been isolated from biofluids and shown to mediate intercellular communication via selective enrichment in proteins and micro-RNAs. Methods This study aimed to isolate and characterise EVs from the cerebrospinal fluid (CSF) of 3 preterm infants with PHH using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) with immunogold protein labelling, and micro-RNA analysis. Results NTA of unaltered CSF revealed a heterogeneous and dynamic population of EVs. Exosomal-sized EVs were isolated by differential ultracentrifugation and TEM confirmed the presence of CD63+ and CD81+ exosomes. The micro-RNAs miR-9, miR-17, miR-26a, miR-124 and miR-1911 were detected within the exosome-enriched fraction and profiled over time. Conclusion This is the first reported characterisation of exosomes from the CSF of preterm infants with post-haemorrhagic hydrocephalus

Connexin 36 Expression Regulates Neuronal Differentiation from Neural Progenitor Cells

Background: Gap junction communication has been shown in glial and neuronal cells and it is thought they mediate interand intra-cellular communication. Connexin 36 (Cx36) is expressed extensively in the developing brain, with levels peaking at P14 after which its levels fall and its expression becomes entirely neuronal. These and other data have led to the hypothesis that Cx36 may direct neuronal coupling and neurogenesis during development. Methodology/Principal Findings: To investigate Cx36 function we used a neurosphere model of neuronal cell development and developed lentiviral Cx36 knockdown and overexpression strategies. Cx36 knockdown was confirmed by western blotting, immunocytochemistry and functionally by fluorescence recovery after photobleaching (FRAP). We found that knockdown of Cx36 in neurosphere neuronal precursors significantly reduced neuronal coupling and the number of differentiated neurons. Correspondingly, the lentiviral mediated overexpression of Cx36 significantly increased the number of neurons derived from the transduced neurospheres. The number of oligodendrocytes was also significantly increased following transduction with Cx36 indicating they may support neuronal differentiation. Conclusions/Significance: Our data suggests that astrocytic and neuronal differentiation during development are governed by mechanisms that include the differential expression of Cx36

The miRNA transcriptome of cerebrospinal fluid in preterm infants reveals the signaling pathways that promote reactive gliosis following cerebral hemorrhage

IntroductionGerminal Matrix-Intraventricular Haemorrhage (GM-IVH) is one of the most common neurological complications in preterm infants, which can lead to accumulation of cerebrospinal fluid (CSF) and is a major cause of severe neurodevelopmental impairment in preterm infants. However, the pathophysiological mechanisms triggered by GM-IVH are poorly understood. Analyzing the CSF that accumulates following IVH may allow the molecular signaling and intracellular communication that contributes to pathogenesis to be elucidated. Growing evidence suggests that miRs, due to their key role in gene expression, have a significant utility as new therapeutics and biomarkers.MethodsThe levels of 2,083 microRNAs (miRs) in 15 CSF samples from 10 infants with IVH were measured using miRNA whole transcriptome sequencing. Gene ontology (GO) and miR family analysis were used to uncover dysregulated signalling which were then validated in vitro in human foetal neural progenitor cells treated with IVH-CSF.ResultsFive hundred eighty-seven miRs were differentially expressed in the CSF extracted at least 2 months after injury, compared to CSF extracted within the first month of injury. GO uncovered key pathways targeted by differentially expressed miRs including the MAPK cascade and the JAK/STAT pathway. Astrogliosis is known to occur in preterm infants, and we hypothesized that this could be due to abnormal CSF-miR signaling resulting in dysregulation of the JAK/STAT pathway – a key controller of astrocyte differentiation. We then confirmed that treatment with IVH-CSF promotes astrocyte differentiation from human fetal NPCs and that this effect could be prevented by JAK/STAT inhibition. Taken together, our results provide novel insights into the CSF/NPCs crosstalk following perinatal brain injury and reveal novel targets to improve neurodevelopmental outcomes in preterm infants

Abnormal scaffold attachment factor 1 expression and localisation in spinocerebellar ataxias and huntington’s chorea

SAFB1 is a DNA and RNA binding protein that is highly expressed in the cerebellum and hippocampus and is involved in the processing of coding and non‐coding RNAs, splicing and dendritic function. We analyzed SAFB1 expression in the post‐mortem brain tissue of spinocerebellar ataxia (SCA), Huntington’s disease (HD), Multiple sclerosis (MS), Parkinson’s disease patients and controls. In SCA cases, the expression of SAFB1 in the nucleus was increased and there was abnormal and extensive expression in the cytoplasm where it co‐localized with the markers of Purkinje cell injury. Significantly, no SAFB1 expression was found in the cerebellar neurons of the dentate nucleus in control or MS patients; however, in SCA patients, SAFB1 expression was increased significantly in both the nucleus and cytoplasm of dentate neurons. In HD, we found that SAFB1 expression was increased in the nucleus and cytoplasm of striatal neurons; however, there was no SAFB1 staining in the striatal neurons of controls. In PD substantia nigra, we did not see any changes in neuronal SAFB1 expression. iCLIP analysis found that SAFB1 crosslink sites within ATXN1 RNA were adjacent to the start and within the glutamine repeat sequence. Further investigation found increased binding of SAFB1 to pathogenic ATXN1‐85Q mRNA. These novel data strongly suggest SAFB1 contributes to the etiology of SCA and Huntington’s chorea and that it may be a pathological marker of polyglutamine repeat expansion diseases