PBMC-derived extracellular vesicles in a smoking-related inflammatory disease model.

Extracellular vesicles (EVs) function as mediators of intercellular communication and as such influence the recipient cell function. EVs derived from immune cells can carry out many of the same functions as their parental cells, as they carry costimulatory molecules, antigens, and antigen-MHC complexes. As a result, there is a strong interest in understanding the composition and origin of immune cell-derived EVs in order to understand their role in the pathogenesis of diseases. This study aimed to optimize methodologies to study immune cell-derived EVs. Peripheral blood mononuclear cell-derived small EVs were isolated and observed using conventional transmission electron microscopy and sized by nanoparticle tracking analysis. They were then enumerated and profiled using imaging flow cytometry and were further characterized using a flow cytometric multiplex bead assay. These techniques were then applied to our current research, namely smoking-related inflammatory disease. We present here a comprehensive approach to analyze PBMC-derived small EVs in smoking-related inflammatory disease following the Minimal Information for Studies of Extracellular Vesicle 2018 guidelines.

The Role of Extracellular Vesicles in Allergic Sensitization: A Systematic Review.

Allergies affect approximately 10-30% of people worldwide, with an increasing number of cases each year; however, the underlying mechanisms are still poorly understood. In recent years, extracellular vesicles (EVs) have been suggested to play a role in allergic sensitization and skew to a T helper type 2 (Th2) response. The aim of this review is to highlight the existing evidence of EV involvement in allergies. A total of 22 studies were reviewed; 12 studies showed EVs can influence a Th2 response, while 10 studies found EVs promoted a Th1 or Treg response. EVs can drive allergic sensitization through up-regulation of pro-Th2 cytokines, such as IL-4 and IL-13. In addition, EVs from MRSA can induce IgE hypersensitivity in mice towards MRSA. On the other hand, EVs can induce tolerance in the immune system; for example, pre-exposing OVA-loaded EVs prevented OVA sensitization in mice. The current literature thus suggests that EVs play an essential role in allergy. Further research utilizing human in vitro models and clinical studies is needed to give a reliable account of the role of EVs in allergy.

Multi-Omic Temporal Landscape of Plasma and Synovial Fluid-Derived Extracellular Vesicles Using an Experimental Model of Equine Osteoarthritis.

Extracellular vesicles (EVs) contribute to osteoarthritis pathogenesis through their release into joint tissues and synovial fluid. Synovial fluid-derived EVs have the potential to be direct biomarkers in the causal pathway of disease but also enable understanding of their role in disease progression. Utilizing a temporal model of osteoarthritis, we defined the changes in matched synovial fluid and plasma-derived EV small non-coding RNA and protein cargo using sequencing and mass spectrometry. Data exploration included time series clustering, factor analysis and gene enrichment interrogation. Chondrocyte signalling was analysed using luciferase-based transcription factor activity assays. EV protein cargo appears to be more important during osteoarthritis progression than small non-coding RNAs. Cluster analysis revealed plasma-EVs represented a time-dependent response to osteoarthritis induction associated with supramolecular complexes. Clusters for synovial fluid-derived EVs were associated with initial osteoarthritis response and represented immune/inflammatory pathways. Factor analysis for plasma-derived EVs correlated with day post-induction and were primarily composed of proteins modulating lipid metabolism. Synovial fluid-derived EVs factors represented intermediate filament and supramolecular complexes reflecting tissue repair. There was a significant interaction between time and osteoarthritis for CRE, NFkB, SRE, SRF with a trend for osteoarthritis synovial fluid-derived EVs at later time points to have a more pronounced effect.

Extracellular vesicles and chronic obstructive pulmonary disease (COPD): a systematic review.

BACKGROUND: Chronic Obstructive Pulmonary Disease (COPD) is a common inflammatory disease of the airways characterized by irreversible airflow limitation, ranking the third highest cause of death worldwide. Extracellular vesicles (EVs) are important intercellular communication mediators released by cells into their extracellular environment with the capacity to transfer biological signals. EVs involved in COPD hold great potential to understand disease pathogenesis and identify important biomarkers. This systematic review aims to examine all available research on EVs in the pathogenesis and diagnosis of COPD to identify existing knowledge and support further research within the field. METHODS: Publications were searched using PubMed and EMBASE with the search terms (Exosomes or extracellular vesicles or microvesicles or microparticles or ectosomes) AND (chronic obstructive pulmonary disease or COPD or emphysema or bronchitis). RESULTS: Initial search yielded 512 papers of which 142 were manually selected for review and 43 were eligible for analyses. The studies were divided into groups according to the role of EVs in pathogenesis, EV origin and cargo, their role in COPD exacerbations and their diagnostic utility. EVs were found to be involved in the mechanism of pathogenesis of COPD, derived from various cell types, as well as containing modified levels of miRNAs. EVs also varied according to the pathophysiological status of disease, therefore presenting a possible method for COPD diagnosis and progress monitoring. CONCLUSION: The current findings show the limited but good quality research looking at the role of EVs in COPD, demonstrating the need for more studies to better define and provide further insight into the functional characteristics of EV in COPD pathogenesis.

Distinct small non-coding RNA landscape in the axons and released extracellular vesicles of developing primary cortical neurons and the axoplasm of adult nerves.

Neurons have highlighted the needs for decentralized gene expression and specific RNA function in somato-dendritic and axonal compartments, as well as in intercellular communication via extracellular vesicles (EVs). Despite advances in miRNA biology, the identity and regulatory capacity of other small non-coding RNAs (sncRNAs) in neuronal models and local subdomains has been largely unexplored.We identified a highly complex and differentially localized content of sncRNAs in axons and EVs during early neuronal development of cortical primary neurons and in adult axons invivo. This content goes far beyond miRNAs and includes most known sncRNAs and precisely processed fragments from tRNAs, sno/snRNAs, Y RNAs and vtRNAs. Although miRNAs are the major sncRNA biotype in whole-cell samples, their relative abundance is significantly decreased in axons and neuronal EVs, where specific tRNA fragments (tRFs and tRHs/tiRNAs) mainly derived from tRNAs Gly-GCC, Val-CAC and Val-AAC predominate. Notably, although 5'-tRHs compose the great majority of tRNA-derived fragments observed invitro, a shift to 3'-tRNAs is observed in mature axons invivo.The existence of these complex sncRNA populations that are specific to distinct neuronal subdomains and selectively incorporated into EVs, equip neurons with key molecular tools for spatiotemporal functional control and cell-to-cell communication.

The Study of Cerebrospinal Fluid microRNAs in Spinal Cord Injury and Neurodegenerative Diseases: Methodological Problems and Possible Solutions.

Despite extensive research on neurological disorders, unanswered questions remain regarding the molecular mechanisms underpinning the course of these diseases, and the search continues for effective biomarkers for early diagnosis, prognosis, or therapeutic intervention. These questions are especially acute in the study of spinal cord injury (SCI) and neurodegenerative diseases. It is believed that the changes in gene expression associated with processes triggered by neurological disorders are the result of post-transcriptional gene regulation. microRNAs (miRNAs) are key regulators of post-transcriptional gene expression and, as such, are often looked to in the search for effective biomarkers. We propose that cerebrospinal fluid (CSF) is potentially a source of biomarkers since it is in direct contact with the central nervous system and therefore may contain biomarkers indicating neurodegeneration or damage to the brain and spinal cord. However, since the abundance of miRNAs in CSF is low, their isolation and detection is technically difficult. In this review, we evaluate the findings of recent studies of CSF miRNAs as biomarkers of spinal cord injury (SCI) and neurodegenerative diseases. We also summarize the current knowledge concerning the methods of studying miRNA in CSF, including RNA isolation and normalization of the data, highlighting the caveats of these approaches and possible solutions.

Specific trophoblast transcripts transferred by extracellular vesicles affect gene expression in endometrial epithelial cells and may have a role in embryo-maternal crosstalk.

BACKGROUND: Successful establishment of pregnancy hinges on appropriate communication between the embryo and the uterus prior to implantation, but the nature of this communication remains poorly understood. Here, we tested the hypothesis that the endometrium is receptive to embryo-derived signals in the form of RNA. METHODS: We have utilized a non-contact co culture system to simulate the conditions of pre implantation environment of the uterus. We bioorthogonally tagged embryonic RNA and tracked the transferred transcripts to endometrium. Transferred transcripts were separated from endometrial transcripts and sequenced. Changes in endometrial transcripts were quantified using quantitative PCR. RESULTS: We show that three specific transcripts are transferred to endometrial cells. We subsequently demonstrate a role of extracellular vesicles (EVs) in this process, as EVs obtained from cultured trophoblast spheroids incubated with endometrial cells induced down-regulation of all the three identified transcripts in endometrial cells. Finally, we show that EVs/nanoparticles captured from conditioned culture media of viable embryos as opposed to degenerating embryos induce ZNF81 down-regulation in endometrial cells, hinting at the functional importance of this intercellular communication. CONCLUSION: Ultimately, our findings demonstrate the existence of an RNA-based communication which may be of critical importance for the establishment of pregnancy.

Women's knowledge, attitudes and practices (KAP) relating to breast and cervical cancers in rural Zimbabwe: a cross sectional study in Mudzi District, Mashonaland East Province.

BACKGROUND: Breast and cervical cancers constitute the most common cancers among women in sub-Saharan Africa. In Zimbabwe, cervical cancer accounts for more than a third of all cancers among women of African descent. Cancer knowledge levels, attitudes and practices of people in different sections of society should be assessed in order to guide current cancer interventions. This study aimed to assess breast and cervical cancer knowledge, attitudes and practices of women of reproductive age, in Mudzi District, Republic of Zimbabwe. METHODS: A cross-sectional community-based survey was conducted. A total of 409 survey household questionnaires were administered to women of reproductive age (15-49 years) in 2014. RESULTS: A total of 409 respondents were interviewed. Nearly 85% of respondents had heard of cancer. 34.2% did not know of any cervical cancer risk factors and 51% were not familiar with the signs and symptoms of cervical cancer. Fifty five percent (55%) had not discussed cancer issues with partners in the past 12 months, and only 27.4% had discussed cancer issues with partners at all. Most of the respondents (96.2%) had never undergone cervical cancer screening. The majority of the respondents (70.8%) had never discussed breast cancer issues with community members. About 70% had never discussed cervical cancer issues with community members. CONCLUSIONS: This study revealed a lack of awareness and comprehensive knowledge about breast and cervical cancer. It also revealed low self-risk perception, low uptake of cancer early detection services and low capacity of the local health institution in offering cancer services. It is recommended that the scaling-up of cancer information, dissemination, and early detection services must be prioritised, including training of local health institutions.

Effectiveness of community adolescent treatment supporters (CATS) interventions in improving linkage and retention in care, adherence to ART and psychosocial well-being: a randomised trial among adolescents living with HIV in rural Zimbabwe.

BACKGROUND: Engagement with community adolescent treatment supporters (CATS) improves adherence, psychosocial well-being, linkage and retention in care among adolescents living with HIV. However, there is an urgent need for empirical evidence of the effectiveness of this approach, in order to inform further programmatic development, national and international policy, guidelines and service delivery for adolescents living with HIV. This study set out to determine the effectiveness of CATS services on improving linkage to services and retention in care, adherence and psychosocial well-being among adolescents living with HIV in Zimbabwe. METHODS: A randomised trial was conducted in Gokwe South district, Zimbabwe over a period of 12 months. Ninety-four HIV-positive adolescents, 10-15 years old, on antiretroviral therapy were recruited to the study. 47 participants received standard of care from the Ministry of Health and Child Care and 47 received the same standard of care plus CATS services. Data collection involved a questionnaire which was administered at baseline then repeated at three, six, nine and twelve months for all participants. Survey questions on confidence, self-esteem and self-worth had a three-point Likert scale. Stigma, quality of life and the linkages to services and retention questions had a five-point Likert scale. RESULTS: Survey questionnaires were completed with response rates of 40 out of 47 (85%) for the intervention arm, and 28 out of 47 (60%) for the control arm, at end-line. The intervention group were 3.9 times more likely to adhere to treatment compared to the control group. Linkage to services and retention in care within the intervention group increased compared with a decrease in the control arm. The intervention group reported a statistically significant increase in confidence, self-esteem, self-worth (p < 0.001) and quality of life compared (p = 0.028) with a decrease in the control arm. CONCLUSIONS: This study found that adolescents receiving the CATS service had improved linkage to services and retention in care, improved adherence and improved psychosocial well-being compared to adolescents who did not have access to such services. TRIAL REGISTRATION: PACTR201711002755428 . Registered 11 November 2017. Retrospectively registered.

Role of Mesenchymal Stem Cell-Derived Extracellular Vesicles in Epithelial-Mesenchymal Transition.

Epithelial-mesenchymal transition (EMT) is a process that takes place during embryonic development, wound healing, and under some pathological processes, including fibrosis and tumor progression. The molecular changes occurring within epithelial cells during transformation to a mesenchymal phenotype have been well studied. However, to date, the mechanism of EMT induction remains to be fully elucidated. Recent findings in the field of intercellular communication have shed new light on this process and indicate the need for further studies into this important mechanism. New evidence supports the hypothesis that intercellular communication between mesenchymal stroma/stem cells (MSCs) and resident epithelial cells plays an important role in EMT induction. Besides direct interactions between cells, indirect paracrine interactions by soluble factors and extracellular vesicles also occur. Extracellular vesicles (EVs) are important mediators of intercellular communication, through the transfer of biologically active molecules, genetic material (mRNA, microRNA, siRNA, DNA), and EMT inducers to the target cells, which are capable of reprogramming recipient cells. In this review, we discuss the role of intercellular communication by EVs to induce EMT and the acquisition of stemness properties by normal and tumor epithelial cells.

Autophagy receptor defects and ALS-FTLD.

Various pathophysiological mechanisms have been implicated in the ALS-FTLD clinicopathological spectrum of neurodegenerative disorders. Here we focus on the role of autophagy, an intracellular catabolic pathway, in these conditions. Growing evidence suggests that the autophagic process can be disturbed in ALS-FTLD, including by genetic mutations affecting autophagy receptor proteins (ubiquilin-2, optineurin, SQSTM1/p62) and regulators (VCP). Such mutations may impair clearance of autophagy substrates with pathological consequences. Recent studies have also uncovered a direct connection between autophagy and RNA processing, supporting an integrated model connecting several ALS-FTLD associated gene products. This article is part of a Special Issue entitled 'Neuronal Protein'.

GRIN2B mutations in West syndrome and intellectual disability with focal epilepsy.

OBJECTIVE: To identify novel epilepsy genes using a panel approach and describe the functional consequences of mutations. METHODS: Using a panel approach, we screened 357 patients comprising a vast spectrum of epileptic disorders for defects in genes known to contribute to epilepsy and/or intellectual disability (ID). After detection of mutations in a novel epilepsy gene, we investigated functional effects in Xenopus laevis oocytes and screened a follow-up cohort. RESULTS: We revealed de novo mutations in GRIN2B encoding the NR2B subunit of the N-methyl-D-aspartate (NMDA) receptor in 2 individuals with West syndrome and severe developmental delay as well as 1 individual with ID and focal epilepsy. The patient with ID and focal epilepsy had a missense mutation in the extracellular glutamate-binding domain (p.Arg540His), whereas both West syndrome patients carried missense mutations within the NR2B ion channel-forming re-entrant loop (p.Asn615Ile, p.Val618Gly). Subsequent screening of 47 patients with unexplained infantile spasms did not reveal additional de novo mutations, but detected a carrier of a novel inherited GRIN2B splice site variant in close proximity (c.2011-5_2011-4delTC). Mutations p.Asn615Ile and p.Val618Gly cause a significantly reduced Mg(2+) block and higher Ca(2+) permeability, leading to a dramatically increased Ca(2+) influx, whereas p.Arg540His caused less severe disturbance of channel function, corresponding to the milder patient phenotype. INTERPRETATION: We identified GRIN2B gain-of-function mutations as a cause of West syndrome with severe developmental delay as well as of ID with childhood onset focal epilepsy. Severely disturbed channel function corresponded to severe clinical phenotypes, underlining the important role of facilitated NMDA receptor signaling in epileptogenesis.

SMG-1 and mTORC1 act antagonistically to regulate response to injury and growth in planarians.

Planarian flatworms are able to both regenerate their whole bodies and continuously adapt their size to nutrient status. Tight control of stem cell proliferation and differentiation during these processes is the key feature of planarian biology. Here we show that the planarian homolog of the phosphoinositide 3-kinase-related kinase (PIKK) family member SMG-1 and mTOR complex 1 components are required for this tight control. Loss of smg-1 results in a hyper-responsiveness to injury and growth and the formation of regenerative blastemas that remain undifferentiated and that lead to lethal ectopic outgrowths. Invasive stem cell hyper-proliferation, hyperplasia, hypertrophy, and differentiation defects are hallmarks of this uncontrolled growth. These data imply a previously unappreciated and novel physiological function for this PIKK family member. In contrast we found that planarian members of the mTOR complex 1, tor and raptor, are required for the initial response to injury and blastema formation. Double smg-1 RNAi experiments with tor or raptor show that abnormal growth requires mTOR signalling. We also found that the macrolide rapamycin, a natural compound inhibitor of mTORC1, is able to increase the survival rate of smg-1 RNAi animals by decreasing cell proliferation. Our findings support a model where Smg-1 acts as a novel regulator of both the response to injury and growth control mechanisms. Our data suggest the possibility that this may be by suppressing mTOR signalling. Characterisation of both the planarian mTORC1 signalling components and another PIKK family member as key regulators of regeneration and growth will influence future work on regeneration, growth control, and the development of anti-cancer therapies that target mTOR signalling.

SnoRNAs in cardiovascular development, function, and disease.

Small nucleolar RNAs (snoRNAs) are emerging as important regulators of cardiovascular (patho)biology. Several roles of snoRNAs have recently been identified in heart development and congenital heart diseases, as well as their dynamic regulation in hypertrophic and dilated cardiomyopathies, coronary heart disease (CHD), myocardial infarction (MI), cardiac fibrosis, and heart failure. Furthermore, reports of changes in vesicular snoRNA expression and altered levels of circulating snoRNAs in response to cardiac stress suggest that snoRNAs also function in cardiac signaling and intercellular communication. In this review, we summarize and discuss key findings and outline the clinical potential of snoRNAs considering current challenges and gaps in the field of cardiovascular diseases (CVDs).

Modulation of the pre-metastatic bone niche: molecular changes mediated by bone-homing prostate cancer extracellular vesicles.

Prostate cancer (PCa) is a leading male malignancy worldwide, often progressing to bone metastasis, with limited curative options. Extracellular vesicles (EVs) have emerged as key players in cancer communication and metastasis, promoting the formation of supportive microenvironments in distant sites. Our previous studies have highlighted the role of PCa EVs in modulating osteoblasts and facilitating tumor progression. However, the early pre-metastatic changes induced by PCa EVs within the bone microenvironment remain poorly understood. To investigate the early effects of repeated exposure to PCa EVs in vivo, mimicking EVs being shed from the primary tumor, PCa EVs isolated from cell line PC3MLuc2a were fluorescently labelled and repeatedly administered via tail vein injection to adult CD1 NuNu male mice for a period of 4 weeks. In vivo imagining, histological analysis and gene expression profiling were performed to assess the impact of PCa EVs on the bone microenvironment. We demonstrate for the first time that PCa EVs home to both bone and lymph nodes following repeated exposures. Furthermore, the accumulation of EVs within the bone leads to distinct molecular changes indicative of disrupted bone homeostasis (e.g., changes to signaling pathways such as Paxillin p = 0.0163, Estrogen Receptor p = 0.0271, RHOA p = 0.0287, Ribonucleotide reductase p = 0.0307 and ERK/MAPK p = 0.0299). Changes in key regulators of these pathways were confirmed in vitro on human osteoblasts. In addition, our data compares the known gene signature of osteocytes and demonstrates a high proportion of overlap (52.2%), suggesting a potential role for this cell type in response to PCa EV exposure. No changes in bone histology or immunohistochemistry were detected, indicating that PCa EV mediated changes were induced at the molecular level. This study provides novel insights into the alterations induced by PCa EVs on the bone microenvironment. The observed molecular changes indicate changes in key pathways and suggest a role for osteocytes in these EV mediated early changes to bone. Further research to understand these early events may aid in the development of targeted interventions to disrupt the metastatic cascade in PCa.

A novel dominant hyperekplexia mutation Y705C alters trafficking and biochemical properties of the presynaptic glycine transporter GlyT2.

Hyperekplexia or startle disease is characterized by an exaggerated startle response, evoked by tactile or auditory stimuli, producing hypertonia and apnea episodes. Although rare, this orphan disorder can have serious consequences, including sudden infant death. Dominant and recessive mutations in the human glycine receptor (GlyR) α1 gene (GLRA1) are the major cause of this disorder. However, recessive mutations in the presynaptic Na(+)/Cl(-)-dependent glycine transporter GlyT2 gene (SLC6A5) are rapidly emerging as a second major cause of startle disease. In this study, systematic DNA sequencing of SLC6A5 revealed a new dominant GlyT2 mutation: pY705C (c.2114A→G) in transmembrane domain 11, in eight individuals from Spain and the United Kingdom. Curiously, individuals harboring this mutation show significant variation in clinical presentation. In addition to classical hyperekplexia symptoms, some individuals had abnormal respiration, facial dysmorphism, delayed motor development, or intellectual disability. We functionally characterized this mutation using molecular modeling, electrophysiology, [(3)H]glycine transport, cell surface expression, and cysteine labeling assays. We found that the introduced cysteine interacts with the cysteine pair Cys-311-Cys-320 in the second external loop of GlyT2. This interaction impairs transporter maturation through the secretory pathway, reduces surface expression, and inhibits transport function. Additionally, Y705C presents altered H(+) and Zn(2+) dependence of glycine transport that may affect the function of glycinergic neurotransmission in vivo.

Mutations in the GlyT2 gene (SLC6A5) are a second major cause of startle disease.

Hereditary hyperekplexia or startle disease is characterized by an exaggerated startle response, evoked by tactile or auditory stimuli, leading to hypertonia and apnea episodes. Missense, nonsense, frameshift, splice site mutations, and large deletions in the human glycine receptor α1 subunit gene (GLRA1) are the major known cause of this disorder. However, mutations are also found in the genes encoding the glycine receptor ß subunit (GLRB) and the presynaptic Na(+)/Cl(-)-dependent glycine transporter GlyT2 (SLC6A5). In this study, systematic DNA sequencing of SLC6A5 in 93 new unrelated human hyperekplexia patients revealed 20 sequence variants in 17 index cases presenting with homozygous or compound heterozygous recessive inheritance. Five apparently unrelated cases had the truncating mutation R439X. Genotype-phenotype analysis revealed a high rate of neonatal apneas and learning difficulties associated with SLC6A5 mutations. From the 20 SLC6A5 sequence variants, we investigated glycine uptake for 16 novel mutations, confirming that all were defective in glycine transport. Although the most common mechanism of disrupting GlyT2 function is protein truncation, new pathogenic mechanisms included splice site mutations and missense mutations affecting residues implicated in Cl(-) binding, conformational changes mediated by extracellular loop 4, and cation-π interactions. Detailed electrophysiology of mutation A275T revealed that this substitution results in a voltage-sensitive decrease in glycine transport caused by lower Na(+) affinity. This study firmly establishes the combination of missense, nonsense, frameshift, and splice site mutations in the GlyT2 gene as the second major cause of startle disease.

Distinct phenotypes in zebrafish models of human startle disease.

Startle disease is an inherited neurological disorder that causes affected individuals to suffer noise- or touch-induced non-epileptic seizures, excessive muscle stiffness and neonatal apnea episodes. Mutations known to cause startle disease have been identified in glycine receptor subunit (GLRA1 and GLRB) and glycine transporter (SLC6A5) genes, which serve essential functions at glycinergic synapses. Despite the significant successes in identifying startle disease mutations, many idiopathic cases remain unresolved. Exome sequencing in these individuals will identify new candidate genes. To validate these candidate disease genes, zebrafish is an ideal choice due to rapid knockdown strategies, accessible embryonic stages, and stereotyped behaviors. The only existing zebrafish model of startle disease, bandoneon (beo), harbors point mutations in glrbb (one of two zebrafish orthologs of human GLRB) that cause compromised glycinergic transmission and touch-induced bilateral muscle contractions. In order to further develop zebrafish as a model for startle disease, we sought to identify common phenotypic outcomes of knocking down zebrafish orthologs of two known startle disease genes, GLRA1 and GLRB, using splice site-targeted morpholinos. Although both morphants were expected to result in phenotypes similar to the zebrafish beo mutant, our direct comparison demonstrated that while both glra1 and glrbb morphants exhibited embryonic spasticity, only glrbb morphants exhibited bilateral contractions characteristic of beo mutants. Likewise, zebrafish over-expressing a dominant startle disease mutation (GlyR α1(R271Q)) exhibited spasticity but not bilateral contractions. Since GlyR ßb can interact with GlyR α subunits 2-4 in addition to GlyR α1, loss of the GlyR ßb subunit may produce more severe phenotypes by affecting multiple GlyR subtypes. Indeed, immunohistochemistry of glra1 morphants suggests that in zebrafish, alternate GlyR α subunits can compensate for the loss of the GlyR α1 subunit. To address the potential for interplay among GlyR subunits during development, we quantified the expression time-course for genes known to be critical to glycinergic synapse function. We found that GlyR α2, α3 and α4a are expressed in the correct temporal pattern and could compensate for the loss of the GlyR α1 subunit. Based on our findings, future studies that aim to model candidate startle disease genes in zebrafish should include measures of spasticity and synaptic development.

Novel missense mutations in the glycine receptor ß subunit gene (GLRB) in startle disease.

Startle disease is a rare, potentially fatal neuromotor disorder characterized by exaggerated startle reflexes and hypertonia in response to sudden unexpected auditory, visual or tactile stimuli. Mutations in the GlyR α(1) subunit gene (GLRA1) are the major cause of this disorder, since remarkably few individuals with mutations in the GlyR ß subunit gene (GLRB) have been found to date. Systematic DNA sequencing of GLRB in individuals with hyperekplexia revealed new missense mutations in GLRB, resulting in M177R, L285R and W310C substitutions. The recessive mutation M177R results in the insertion of a positively-charged residue into a hydrophobic pocket in the extracellular domain, resulting in an increased EC(50) and decreased maximal responses of α(1)ß GlyRs. The de novo mutation L285R results in the insertion of a positively-charged side chain into the pore-lining 9' position. Mutations at this site are known to destabilize the channel closed state and produce spontaneously active channels. Consistent with this, we identified a leak conductance associated with spontaneous GlyR activity in cells expressing α(1)ß(L285R) GlyRs. Peak currents were also reduced for α(1)ß(L285R) GlyRs although glycine sensitivity was normal. W310C was predicted to interfere with hydrophobic side-chain stacking between M1, M2 and M3. We found that W310C had no effect on glycine sensitivity, but reduced maximal currents in α(1)ß GlyRs in both homozygous (α(1)ß(W310C)) and heterozygous (α(1)ßß(W310C)) stoichiometries. Since mild startle symptoms were reported in W310C carriers, this may represent an example of incomplete dominance in startle disease, providing a potential genetic explanation for the 'minor' form of hyperekplexia.

LIM-domain proteins, LIMD1, Ajuba, and WTIP are required for microRNA-mediated gene silencing.

In recent years there have been major advances with respect to the identification of the protein components and mechanisms of microRNA (miRNA) mediated silencing. However, the complete and precise repertoire of components and mechanism(s) of action remain to be fully elucidated. Herein we reveal the identification of a family of three LIM domain-containing proteins, LIMD1, Ajuba and WTIP (Ajuba LIM proteins) as novel mammalian processing body (P-body) components, which highlight a novel mechanism of miRNA-mediated gene silencing. Furthermore, we reveal that LIMD1, Ajuba, and WTIP bind to Ago1/2, RCK, Dcp2, and eIF4E in vivo, that they are required for miRNA-mediated, but not siRNA-mediated gene silencing and that all three proteins bind to the mRNA 5' m(7)GTP cap-protein complex. Mechanistically, we propose the Ajuba LIM proteins interact with the m(7)GTP cap structure via a specific interaction with eIF4E that prevents 4EBP1 and eIF4G interaction. In addition, these LIM-domain proteins facilitate miRNA-mediated gene silencing by acting as an essential molecular link between the translationally inhibited eIF4E-m(7)GTP-5(')cap and Ago1/2 within the miRISC complex attached to the 3'-UTR of mRNA, creating an inhibitory closed-loop complex.