A direct regulatory link between microRNA-137 and SHANK2: implications for neuropsychiatric disorders

Background: Mutations in the SHANK genes, which encode postsynaptic scaffolding proteins, have been linked to a spectrum of neurodevelopmental disorders. The SHANK genes and the schizophrenia-associated microRNA-137 show convergence on several levels, as they are both expressed at the synapse, influence neuronal development, and have a strong link to neurodevelopmental and neuropsychiatric disorders like intellectual disability, autism, and schizophrenia. This compiled evidence raised the question if the SHANKs might be targets of miR-137. Methods: In silico analysis revealed a putative binding site for microRNA-137 (miR-137) in the SHANK2 3′UTR, while this was not the case for SHANK1 and SHANK3. Luciferase reporter assays were performed by overexpressing wild type and mutated SHANK2-3′UTR and miR-137 in human neuroblastoma cells and mouse primary hippocampal neurons. miR-137 was also overexpressed or inhibited in hippocampal neurons, and Shank2 expression was analyzed by quantitative real-time PCR and Western blot. Additionally, expression levels of experimentally validated miR-137 target genes were analyzed in the dorsolateral prefrontal cortex (DLPFC) of schizophrenia and control individuals using the RNA-Seq data from the CommonMind Consortium. Results: miR-137 directly targets the 3′UTR of SHANK2 in a site-specific manner. Overexpression of miR-137 in mouse primary hippocampal neurons significantly lowered endogenous Shank2 protein levels without detectable influence on mRNA levels. Conversely, miR-137 inhibition increased Shank2 protein expression, indicating that miR-137 regulates SHANK2 expression by repressing protein translation rather than inducing mRNA degradation. To find out if the miR-137 signaling network is altered in schizophrenia, we compared miR-137 precursor and miR-137 target gene expression in the DLPFC of schizophrenia and control individuals using the CommonMind Consortium RNA sequencing data. Differential expression of 23% (16/69) of known miR-137 target genes was detected in the DLPFC of schizophrenia individuals compared with controls. We propose that in further targets (e.g., SHANK2, as described in this paper) which are not regulated on RNA level, effects may only be detectable on protein level. Conclusion: Our study provides evidence that a direct regulatory link exists between miR-137 and SHANK2 and supports the finding that miR-137 signaling might be altered in schizophrenia

Substrate binding tunes the reactivity of hispidin 3-hydroxylase, a flavoprotein monooxygenase involved in fungal bioluminescence

Fungal bioluminescence was recently shown to depend on a unique oxygen-dependent system of several enzymes. However, the identities of the enzymes did not reveal the full biochemical details of this process, as the enzymes do not bear resemblance to those of other luminescence systems, and thus the properties of the enzymes involved in this fascinating process are still unknown. Here, we describe the characterization of the penultimate enzyme in the pathway, hispidin 3-hydroxylase, from the luminescent fungus Mycena chlorophos (McH3H), which catalyzes the conversion of hispidin to 3-hydroxyhispidin. 3-Hydroxyhispidin acts as a luciferin substrate in luminescent fungi. McH3H was heterologously expressed in Escherichia coli and purified by affinity chromatography with a yield of 100 mg/liter. McH3H was found to be a single component monomeric NAD(P)H-dependent FAD-containing monooxygenase having a preference for NADPH. Through site-directed mutagenesis, based on a modeled structure, mutant enzymes were created that are more efficient with NADH. Except for identifying the residues that tune cofactor specificity, these engineered variants may also help in developing new hispidin-based bioluminescence applications. We confirmed that addition of hispidin to McH3H led to the formation of 3-hydroxyhispidin as sole aromatic product. Rapid kinetic analysis revealed that reduction of the flavin cofactor by NADPH is boosted by hispidin binding by nearly 100-fold. Similar to other class A flavoprotein hydroxylases, McH3H did not form a stable hydroperoxyflavin intermediate. These data suggest a mechanism by which the hydroxylase is tuned for converting hispidin into the fungal luciferin.</p

Sex Hormones Regulate SHANK Expression

Autism spectrum disorders (ASD) have a higher prevalence in male individuals compared to females, with a ratio of affected boys compared to girls of 4:1 for ASD and 11:1 for Asperger syndrome. Mutations in the SHANK genes (comprising SHANK1, SHANK2 and SHANK3) coding for postsynaptic scaffolding proteins have been tightly associated with ASD. As early brain development is strongly influenced by sex hormones, we investigated the effect of dihydrotestosterone (DHT) and 17β-estradiol on SHANK expression in a human neuroblastoma cell model. Both sex hormones had a significant impact on the expression of all three SHANK genes, which could be effectively blocked by androgen and estrogen receptor antagonists. In neuron-specific androgen receptor knock-out mice (ArNesCre), we found a nominal significant reduction of all Shank genes at postnatal day 7.5 in the cortex. In the developing cortex of wild-type (WT) CD1 mice, a sex-differential protein expression was identified for all Shanks at embryonic day 17.5 and postnatal day 7.5 with significantly higher protein levels in male compared to female mice. Together, we could show that SHANK expression is influenced by sex hormones leading to a sex-differential expression, thus providing novel insights into the sex bias in ASD

Inherited and de novo SHANK2 variants associated with autism spectrum disorder impair neuronal morphogenesis and physiology

Mutations in the postsynaptic scaffolding gene SHANK2 have recently been identified in individuals with autism spectrum disorder (ASD) and intellectual disability. However, the cellular and physiological consequences of these mutations in neurons remain unknown. We have analyzed the functional impact caused by two inherited and one de novo SHANK2 mutations from ASD individuals (L1008_P1009dup, T1127M, R462X). Although all three variants affect spine volume and have smaller SHANK2 cluster sizes, T1127M additionally fails to rescue spine volume in Shank2 knock-down neurons. R462X is not able to rescue spine volume and dendritic branching and lacks postsynaptic clustering, indicating the most severe dysfunction. To demonstrate that R462X when expressed in mouse can be linked to physiological effects, we analyzed synaptic transmission and behavior. Principal neurons of mice expressing rAAV-transduced SHANK2-R462X present a specific, long-lasting reduction in miniature postsynaptic AMPA receptor currents. This dominant negative effect translates into dose-dependent altered cognitive behavior of SHANK2-R462X-expressing mice, with an impact on the penetrance of ASD

The adult human subventricular zone: partial ependymal coverage and proliferative capacity of cerebrospinal fluid

Neurogenesis continues throughout adulthood in specialized regions of the brain. One of these regions is the subventricular zone. During brain development, neurogenesis is regulated by a complex interplay of intrinsic and extrinsic cues that control stem-cell survival, renewal and cell lineage specification. Cerebrospinal fluid (CSF) is an integral part of the neurogenic niche in development as it is in direct contact with radial glial cells, and it is important in regulating proliferation and migration. Yet, the effect of CSF on neural stem cells in the subventricular zone of the adult human brain is unknown. We hypothesized a persistent stimulating effect of ventricular CSF on neural stem cells in adulthood, based on the literature, describing bulging accumulations of subventricular cells where CSF is in direct contact with the subventricular zone. Here, we show by immunohistochemistry on post-mortem adult human subventricular zone sections that neural stem cells are in close contact with CSF via protrusions through both intact and incomplete ependymal layers. We are the first to systematically quantify subventricular glial nodules denuded of ependyma and consisting of proliferating neural stem and progenitor cells, and showed that they are present from foetal age until adulthood. Neurosphere, cell motility and differentiation assays as well as analyses of RNA expression were used to assess the effects of CSF of adult humans on primary neural stem cells and a human immortalized neural stem cell line. We show that human ventricular CSF increases proliferation and decreases motility of neural stem cells. Our results also indicate that adult CSF pushes neural stem cells from a relative quiescent to a more active state and promotes neuronal over astrocytic lineage differentiation. Thus, CSF continues to stimulate neural stem cells throughout aging

Additional file 1: of A direct regulatory link between microRNA-137 and SHANK2: implications for neuropsychiatric disorders

Figure S1. Images of primary neuronal cultures pre‐ and posttreatment. Figure S2. High conservation of the miR‐137 binding site in the SHANK2‐3’UTR and of miR‐137 between different species. Figure S3. Relative expression levels of miR‐137 in different human tissues. Figure S4. Uncropped Western blot pictures. Table S1. ASD risk genes which are predicted or validated miR‐137 targets. Table S2. Primers used for cloning, mutagenesis, and screening. Primer sequences are all shown in 5′→3′ orientation. Table S3. Origin of total RNA samples used to measure hsa‐miR‐137 relative expression across different tissues (see Additional file 1: Figure S2 for results). Table S4. Experimentally validated miR‐137 targets. Table S5. Gene expression analysis of 69 validated miR‐137 target genes (including SHANK2) in the CommonMind RNA sequencing data. Table S6a. Gene expression analysis of validated targets from five different control microRNAs in the CommonMind RNA sequencing data. Genes labeled in gray withstand correction for multiple testing using the Benjamini-Hochberg method and a FDR of 10%. Table S6b. Comparison of the number of differentially expressed target genes of different microRNAs between SCZ and control individuals in the CommonMind RNASeq data. Table S7. Analysis of the 3′UTR of the differentially expressed miR-137 genes in the DLPFC between SCZ and control individuals for additional putative miR-124 and miR-128 binding sites. (PDF 1417 kb

Social media data for environmental sustainability : A critical review of opportunities, threats, and ethical use

Social media data are transforming sustainability science. However, challenges from restrictions in data accessibility and ethical concerns regarding potential data misuse have threatened this nascent field. Here, we review the literature on the use of social media data in environmental and sustainability research. We find that they can play a novel and irreplaceable role in achieving the UN Sustainable Development Goals by allowing a nuanced understanding of human-nature interactions at scale, observing the dynamics of so-cial-ecological change, and investigating the co-construction of nature values. We reveal threats to data ac-cess and highlight scientific responsibility to address trade-offs between research transparency and privacy protection, while promoting inclusivity. This contributes to a wider societal debate of social media data for sustainability science and for the common good.Peer reviewe

Data for the paper, "Social media data for environmental sustainability: a critical review of opportunities, threats and ethical use"

These data were collected for the paper, "Social media data for environmental sustainability: a critical review of opportunities, threats and ethical use" published in One Earth. It includes a database of studies applying social media data in environmental sustainability research, which were collected and reviewed in full by the authors. Rather than providing a comprehensive summary of all relevant literature like in a systematic review, our objective was to take stock and evaluate the previous body of work in the field in order to promote conceptual innovation from its critical examination. Building on a set of 169 studies collected in a previous systematic review of social media data applications in environmental research (Ghermandi and Sinclair 2019), the database includes additional relevant studies that were identified by snowballing previous references and adding further gray and scientific academic articles known to the authors. For studies to be included in our analysis, they had to involve the use of data from one or more social media platforms and investigate human interactions with and/or impacts on the environment. We relied on a broad definition of social media including any website or application that enables users to create and share content or to participate in social networking (e.g., blogging sites, recommendation sites, and online forums). We further strengthened the analysis by including insights from additional literature on social media that do not have a direct application to environmental sustainability (e.g., studies on biases in social media data). The final database consists of 415 studies, which were published between 2011 and 2021. Ghermandi, Andrea, and Michael Sinclair. "Passive crowdsourcing of social media in environmental research: A systematic map." Global environmental change 55 (2019): 36-47