ARX regulates cortical interneuron differentiation and migration.

Mutations in aristaless-related homeobox ( ARX ) are associated with neurodevelopmental disorders including developmental epilepsies, intellectual disabilities, and autism spectrum disorders, with or without brain malformations. Aspects of these disorders have been linked to abnormal cortical interneuron (cIN) development and function. To further understand ARX's role in cIN development, multiple Arx mutant mouse lines were interrogated. We found that ARX is critical for controlling cIN numbers and distribution, especially, in the developing marginal zone (MZ). Single cell transcriptomics and ChIP-seq, combined with functional studies, revealed ARX directly or indirectly regulates genes involved in proliferation and the cell cycle (e.g., Bub3 , Cspr3 ), fate specification (e.g., Nkx2.1 , Maf , Mef2c ), and migration (e.g., Nkx2.1 , Lmo1 , Cxcr4 , Nrg1 , ErbB4 ). Our data suggest that the MZ stream defects primarily result from disordered cell-cell communication. Together our findings provide new insights into the mechanisms underlying cIN development and migration and how they are disrupted in several disorders.

Transcription factors in microcephaly.

Higher cognition in humans, compared to other primates, is often attributed to an increased brain size, especially forebrain cortical surface area. Brain size is determined through highly orchestrated developmental processes, including neural stem cell proliferation, differentiation, migration, lamination, arborization, and apoptosis. Disruption in these processes often results in either a small (microcephaly) or large (megalencephaly) brain. One of the key mechanisms controlling these developmental processes is the spatial and temporal transcriptional regulation of critical genes. In humans, microcephaly is defined as a condition with a significantly smaller head circumference compared to the average head size of a given age and sex group. A growing number of genes are identified as associated with microcephaly, and among them are those involved in transcriptional regulation. In this review, a subset of genes encoding transcription factors (e.g., homeobox-, basic helix-loop-helix-, forkhead box-, high mobility group box-, and zinc finger domain-containing transcription factors), whose functions are important for cortical development and implicated in microcephaly, are discussed.

Physical Activity and Health of Koreans with Spinal Cord Injury during COVID-19.

Objectives: The primary purpose of this study was to explore the mediating roles of resilience and physical activity (PA) levels on the subjective health of people with spinal cord injury(SCI) during the COVID-19 pandemic.Methods: A total of 201 Koreans with SCI completed a survey between February and April 2021. The questionnaire measured participants' PA levels before and during the COVID-19 pandemic, resilience, and subjective health assessment. The data were analyzed using SPSS, and PROCESS macro (Model 6) was used to determine the dual mediation effects. Results: PA levels before COVID-19 positively affected resilience and PA levels during COVID-19, and resilience positively affected PA levels during COVID-19 and subjective health. PA levels during COVID-19 had a positive influence on subjective health. Moreover, resilience and PA levels during COVID-19 were independent mediators of the relationship between PA levels before COVID-19 and subjective health. PA levels before COVID-19 indirectly affected subjective health through a dual-mediated pathway of resilience and PA levels during COVID-19. Conclusion: We suggest that it is necessary to promote regular PA for people with physical disabilities to enhance resilience, and thus, maintain a healthy life even during a public health crisis.

A Comparative Analysis of E-Cigarette and Cigarette Posts on Instagram.

E-cigarette use has grown rapidly over the past decade and become a threat to public health. Marketing-especially through social media-has contributed significantly to this growth, which suggests that regulating content in social media will be critical in supporting efforts to reverse this trend. A content analysis was performed to compare 254 e-cigarette posts on Instagram with 228 cigarette posts on the same platform. The majority of e-cigarette posts were from e-cigarette companies (40.9%) and industry people (18.5%), whereas the majority of cigarette posts were from laypeople (76.8%). More e-cigarette posts than cigarette posts appeared to have a marketing intent (56.3% vs. 1.3%), and brand representation in photographs/videos was more frequent in the e-cigarette posts than in the cigarette posts (63.0% vs. 15.8%). Further, compared with the e-cigarette posts, the cigarette posts were more likely to portray daily life (73.2% vs. 41.3%) and humans (80.3% vs. 43.7%) in the photograph/video. The cigarette posts also portrayed smoking much more often than the e-cigarette posts portrayed vaping (67.1% vs. 21.3%). The study findings broaden the field's understanding of cigarette and e-cigarette content on Instagram and social media, and have implications for monitoring and regulating content for e-cigarettes and cigarettes.

TMEM161B regulates cerebral cortical gyration, Sonic Hedgehog signaling, and ciliary structure in the developing central nervous system.

Sonic hedgehog signaling regulates processes of embryonic development across multiple tissues, yet factors regulating context-specific Shh signaling remain poorly understood. Exome sequencing of families with polymicrogyria (disordered cortical folding) revealed multiple individuals with biallelic deleterious variants in TMEM161B, which encodes a multi-pass transmembrane protein of unknown function. Tmem161b null mice demonstrated holoprosencephaly, craniofacial midline defects, eye defects, and spinal cord patterning changes consistent with impaired Shh signaling, but were without limb defects, suggesting a CNS-specific role of Tmem161b. Tmem161b depletion impaired the response to Smoothened activation in vitro and disrupted cortical histogenesis in vivo in both mouse and ferret models, including leading to abnormal gyration in the ferret model. Tmem161b localizes non-exclusively to the primary cilium, and scanning electron microscopy revealed shortened, dysmorphic, and ballooned ventricular zone cilia in the Tmem161b null mouse, suggesting that the Shh-related phenotypes may reflect ciliary dysfunction. Our data identify TMEM161B as a regulator of cerebral cortical gyration, as involved in primary ciliary structure, as a regulator of Shh signaling, and further implicate Shh signaling in human gyral development.

Modified Taq DNA Polymerase for Allele-Specific Ultra-Sensitive Detection of Genetic Variants.

Allele-specific PCR (AS-PCR) has been used as a simple, cost-effective method for genotyping and gene mapping in research and clinical settings. AS-PCR permits the detection of single nucleotide variants and insertion or deletion variants owing to the selective extension of a perfectly matched primer (to the template DNA) over a mismatched primer. Thus, the mismatch discrimination power of the DNA polymerase is critical. Unfortunately, currently available polymerases often amplify some mismatched primer-template complexes as well as matched ones, obscuring AS detection. To increase mismatch discrimination, mutations were generated in the Thermus aquaticus (Taq) DNA polymerase, the most efficient variant was selected, and its performance evaluated in single nucleotide polymorphism and cancer mutation genotyping. In addition, the primer design and reaction buffer conditions were optimized for AS amplification. Our highly selective AS-PCR, which is based on an allele-discriminating priming system that leverages a Taq DNA polymerase variant with optimized primers and reaction buffer, can detect mutations with a mutant allele frequency as low as 0.01% in genomic DNA and 0.0001% in plasmid DNA. This method serves as a simple, fast, cost-effective, and ultra-sensitive way to detect single nucleotide variants and insertion or deletion mutations with low abundance.

The gendered playing field: Family socioeconomic status and national gender inequality in adolescents' out-of-school physical activity.

Research has shown that both gender and family socioeconomic status (SES) are important determinants of adolescents' physical activity (PA). This study expands our current knowledge by exploring the moderating role of family SES on gender differences in adolescents' out-of-school PA. We also examine whether this moderating effect varies across countries with different levels of gender inequality. Based on existing empirical studies and theoretical framework, it is hypothesized that 1) gender differences in out-of-school PA would be smaller among adolescents from higher SES families due to them being more likely to share egalitarian gender norms, and 2) this SES gradient in gender differences in adolescents' out-of-school PA would be more salient in countries with lower levels of gender inequality. A total of 316,657 15-year-old adolescents in 50 countries from the 2015 Programme for International Student Assessment (PISA) survey were analyzed. Our multilevel negative binomial regression models show that the gender gap in out-of-school PA is mitigated among families with higher SES, but this moderating role of family SES is less noticeable in countries with higher levels of national gender inequality. Our findings suggest the importance of egalitarian gender culture at both individual and macro-level in understanding gender disparities in adolescents' PA.

Generation of FLAG-tagged Arx knock-in mouse model.

The Aristaless-related homeobox (ARX) is a paired-like homeodomain transcription factor playing important roles in brain development. Patients with mutations in ARX have a spectrum of neurodevelopmental disorders such as epilepsy, intellectual disability, and autism spectrum disorder, with or without structural abnormalities of the brain such as lissencephaly (smooth brain), microcephaly (small brain), and/or agenesis of the corpus callosum. Mouse models have provided important clues on the pathophysiologic roles of ARX in these disorders. However, successfully isolating specific in vivo complexes of ARX, with DNA and proteins, has remained as a challenge. To facilitate in vivo detection of ARX complexes, we generated a mouse line containing one epitope of FLAG-tag (1 × FLAG) targeted at the translational start site of the endogenous Arx gene using CRSPR/Cas9 strategy. Homozygous Flag-Arx mice are viable and fertile without gross abnormality, suggesting that the FLAG-tag does not perturb the normal function of ARX. Using a FLAG antibody, we successfully detected ARX with immunofluorescent staining and pulled down ARX in embryonic brain tissues. This Flag-Arx mouse line will be a useful tool to isolate ARX complexes from mouse tissues for many applications.

The Effects of an Online-Offline Hybrid Exercise Program on the Lives of Children with Cerebral Palsy Using Wheelchairs during the COVID-19 Pandemic in Korea.

Due to the ongoing COVID-19 pandemic, many online programs for social meetings, education, leisure, and physical activities have been developed and provided; however, children with cerebral palsy (CP) cannot enjoy online programs in the same way that those without disabilities can. The aim of this study was to investigate the differences in reintegration to normal living (RNL), social interaction, and quality of life among school-age children with CP after participation in a game-based online−offline hybrid group exercise program. The current study was conducted on 26 children with CP who participated in a hybrid exercise program. The RNL, social interaction, and quality of life were measured before and after the six-week program. The scores of RNL and quality of life were improved (p < 0.05) after program participation. Online or hybrid exercise programs incorporating interactive methods (i.e., competition and cooperating) could enhance RNL and quality of life of children with CP. Thus, well-designed online or hybrid exercise programs should be developed and provided for children with CP to enhance overall quality of life during the pandemic.

Reviews via Mobile: The Role of Mobile Cues and Typographical Errors in Online Review Adoption.

Online consumer reviews are increasingly being written on and posted from mobile devices such that some platforms have started to indicate when this is the case with cues such as "via mobile" (i.e., mobile cue). Reviews from mobile devices differ from those from non-mobile devices; for example, reviews from mobile devices are more likely to include typographical errors. For this study, a web-based experiment was conducted to investigate viewers' evaluation and adoption of online reviews in regard to a mobile cue and typographical errors. The results indicate an interaction effect between the presence of a mobile cue and typographical errors. When a review did not include typographical errors, the presence of a mobile cue negatively affected the evaluation and adoption of information (i.e., the viewer's attitude toward the reviewed restaurant). However, the effects of a mobile cue were not significant for a review with typographical errors. Further, the results suggest that the viewer's perception of the review writing effort and the review's information usefulness are sequential mediators explaining the information adoption mechanism. The findings provide interesting insights into consumers' perceptions of online reviews in the current media landscape in which the large-scale adoption of mobile devices is a well-recognized phenomenon.

When Health Organization Answers the Question: Differential Effects of Dialogic Messages in Website and Twitter through Social Presence and Psychological Distance.

Although there has been a consensus that health organizations should better utilize dialogic capabilities of online platforms, little is known about if the effectiveness of dialogic communication varies across message platforms, and if so, why. An experiment (N = 209) examined how message platforms (i.e., website vs. Twitter) moderate the effect of dialogic messages and uncovered a novel theoretical mechanism underlying such effect. Exposure to dialogic (vs. monologic) messages about a health risk yielded greater sense of direct conversation with the health organization (i.e., social presence). However, such effect was observed only when the messages were delivered via the organization's website. In the organization's Twitter, dialogic and monologic messages did not exert significantly differential influence. Enhanced social presence, in turn, reduced psychological distance to the risk, which subsequently induced stronger risk-preventive behavioral intentions.

ß2-adrenergic receptor regulates ER-mitochondria contacts.

Interactions between the endoplasmic reticulum (ER) and mitochondria (Mito) are crucial for many cellular functions, and their interaction levels change dynamically depending on the cellular environment. Little is known about how the interactions between these organelles are regulated within the cell. Here we screened a compound library to identify chemical modulators for ER-Mito contacts in HEK293T cells. Multiple agonists of G-protein coupled receptors (GPCRs), beta-adrenergic receptors (ß-ARs) in particular, scored in this screen. Analyses in multiple orthogonal assays validated that ß2-AR activation promotes physical and functional interactions between the two organelles. Furthermore, we have elucidated potential downstream effectors mediating ß2-AR-induced ER-Mito contacts. Together our study identifies ß2-AR signaling as an important regulatory pathway for ER-Mito coupling and highlights the role of these contacts in responding to physiological demands or stresses.

PIK3R2/Pik3r2 Activating Mutations Result in Brain Overgrowth and EEG Changes.

OBJECTIVE: Mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) complex have been associated with a broad spectrum of brain and organ overgrowth syndromes. For example, mutations in phosphatidylinositol-3-kinase regulatory subunit 2 (PIK3R2) have been identified in human patients with megalencephaly polymicrogyria polydactyly hydrocephalus (MPPH) syndrome, which includes brain overgrowth. To better understand the pathogenesis of PIK3R2-related mutations, we have developed and characterized a murine model. METHODS: We generated a knock-in mouse model for the most common human PIK3R2 mutation, p.G373R (p.G367R in mice) using CRISPR/Cas9. The mouse phenotypes, including brain size, seizure activity, cortical lamination, cell proliferation/size/density, interneuron migration, and PI3K pathway activation, were analyzed using standard methodologies. For human patients with PIK3R2 mutations, clinical data (occipitofrontal circumference [OFC] and epilepsy) were retrospectively obtained from our clinical records (published / unpublished). RESULTS: The PI3K-AKT pathway was hyperactivated in these mice, confirming the p.G367R mutation is an activating mutation in vivo. Similar to human patients with PIK3R2 mutations, these mice have enlarged brains. We found cell size to be increased but not cell numbers. The embryonic brain showed mild defects in cortical lamination, although not observed in the mature brain. Furthermore, electroencephalogram (EEG) recordings from mutant mice showed background slowing and rare seizures, again similar to our observations in human patients. INTERPRETATION: We have generated a PIK3R2 mouse model that exhibits megalencephaly and EEG changes, both of which overlap with human patients. Our data provide novel insight into the pathogenesis of the human disease caused by PIK3R2 p.G373R mutation. We anticipate this model will be valuable in testing therapeutic options for human patients with MPPH. ANN NEUROL 2020;88:1077-1094.

Men and Women's Different Dreams on the Future of the Gendered Division of Paid Work and Household Work after COVID-19 in South Korea.

Men's long hours of paid work and minimal commitment to household work, combined with the comparably low-level of women's labor force participation, characterize the gendered division of work and family in South Korea. Can the changes in work and family arrangements brought on by the COVID-19 pandemic alter the persevering gendered division of paid work and household work in Korea? Along with school closures and the increased number of employees working from home during COVID-19, do Korean men and women anticipate more equal sharing of paid work and household work? We collected data from 1000 Korean adults during the period of July 3-6, 2020, and asked their predictions about various dimensions of social changes, including the gendered division, after COVID-19. Although a substantial share of both men and women anticipate a reduction in the gendered division of paid work and household work after COVID-19, Korean women are not as optimistic as their male counterparts about this potential reduction. In particular, younger women are most skeptical about the prospect that paid work and household work will be less divided by gender beyond the pandemic.

Snap29 mutant mice recapitulate neurological and ophthalmological abnormalities associated with 22q11 and CEDNIK syndrome.

Synaptosomal-associated protein 29 (SNAP29) encodes a member of the SNARE family of proteins implicated in numerous intracellular protein trafficking pathways. SNAP29 maps to the 22q11.2 region and is deleted in 90% of patients with 22q11.2 deletion syndrome (22q11.2DS). Moreover, bi-allelic SNAP29 mutations in patients are responsible for CEDNIK (cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma) syndrome. A mouse model that recapitulates abnormalities found in these syndromes is essential for uncovering the cellular basis of these disorders. In this study, we report that mice with a loss of function mutation of Snap29 on a mixed CD1;FvB genetic background recapitulate skin abnormalities associated with CEDNIK, and also phenocopy neurological and ophthalmological abnormalities found in CEDNIK and a subset of 22q11.2DS patients. Our work also reveals an unanticipated requirement for Snap29 in male fertility and supports contribution of hemizygosity for SNAP29 to the phenotypic spectrum of abnormalities found in 22q11.2DS patients.

Arx Expression Suppresses Ventralization of the Developing Dorsal Forebrain.

Early brain development requires a tight orchestration between neural tube patterning and growth. How pattern formation and brain growth are coordinated is incompletely understood. Previously we showed that aristaless-related homeobox (ARX), a paired-like transcription factor, regulates cortical progenitor pool expansion by repressing an inhibitor of cell cycle progression. Here we show that ARX participates in establishing dorsoventral identity in the mouse forebrain. In Arx mutant mice, ventral genes, including Olig2, are ectopically expressed dorsally. Furthermore, Gli1 is upregulated, suggesting an ectopic activation of SHH signaling. We show that the ectopic Olig2 expression can be repressed by blocking SHH signaling, implicating a role for SHH signaling in Olig2 induction. We further demonstrate that the ectopic Olig2 accounts for the reduced Pax6 and Tbr2 expression, both dorsal specific genes essential for cortical progenitor cell proliferation. These data suggest a link between the control of dorsoventral identity of progenitor cells and the control of their proliferation. In summary, our data demonstrate that ARX functions in a gene regulatory network integrating normal forebrain patterning and growth, providing important insight into how mutations in ARX can disrupt multiple aspects of brain development and thus generate a wide spectrum of neurodevelopmental phenotypes observed in human patients.

Ascorbate peroxidase proximity labeling coupled with biochemical fractionation identifies promoters of endoplasmic reticulum-mitochondrial contacts.

To maintain cellular homeostasis, subcellular organelles communicate with each other and form physical and functional networks through membrane contact sites coupled by protein tethers. In particular, endoplasmic reticulum (ER)-mitochondrial contacts (EMC) regulate diverse cellular activities such as metabolite exchange (Ca2+ and lipids), intracellular signaling, apoptosis, and autophagy. The significance of EMCs has been highlighted by reports indicating that EMC dysregulation is linked to neurodegenerative diseases. Therefore, obtaining a better understanding of the physical and functional components of EMCs should provide new insights into the pathogenesis of several neurodegenerative diseases. Here, we applied engineered ascorbate peroxidase (APEX) to map the proteome at EMCs in live HEK293 cells. APEX was targeted to the outer mitochondrial membrane, and proximity-labeled proteins were analyzed by stable isotope labeling with amino acids in culture (SILAC)-LC/MS-MS. We further refined the specificity of the proteins identified by combining biochemical subcellular fractionation to the protein isolation method. We identified 405 proteins with a 2.0-fold cutoff ratio (log base 2) in SILAC quantification from replicate experiments. We performed validation screening with a Split-Rluc8 complementation assay that identified reticulon 1A (RTN1A), an ER-shaping protein localized to EMCs as an EMC promoter. Proximity mapping augmented with biochemical fractionation and additional validation methods reported here could be useful to discover other components of EMCs, identify mitochondrial contacts with other organelles, and further unravel their communication.

Aristaless Related Homeobox (ARX) Interacts with ß-Catenin, BCL9, and P300 to Regulate Canonical Wnt Signaling.

Mutations in the Aristaless Related Homeobox (ARX) gene are associated with a spectrum of structural (lissencephaly) and functional (epilepsy and intellectual disabilities) neurodevelopmental disorders. How mutations in this single transcription factor can result in such a broad range of phenotypes remains poorly understood. We hypothesized that ARX functions through distinct interactions with specific transcription factors/cofactors to regulate unique target genes in different cell types. To identify ARX interacting proteins, we performed an unbiased proteomics screen and identified several components of the Wnt/ß-catenin signaling pathway, including ß-catenin (CTNNB1), B-cell CLL/lymphoma 9 (BCL9) and leucine rich repeat flightless interacting protein 2 (LRRFIP2), in cortical progenitor cells. Our data show that ARX positively regulates Wnt/ ß-catenin signaling and that the C-terminal domain of ARX interacts with the armadillo repeats in ß-catenin to promote Wnt/ß-catenin signaling. In addition, we found BCL9 and P300 also interact with ARX to modulate Wnt/ß-catenin signaling. These data provide new insights into how ARX can uniquely regulate cortical neurogenesis, and connect the function of ARX with Wnt/ß-catenin signaling.

Hereditary spastic paraplegia-linked REEP1 modulates endoplasmic reticulum/mitochondria contacts.

OBJECTIVE: Mutations in receptor expression enhancing protein 1 (REEP1) are associated with hereditary spastic paraplegias (HSPs). Although axonal degeneration is thought to be a predominant feature in HSP, the role of REEP1 mutations in degeneration is largely unknown. Previous studies have implicated a role for REEP1 in the endoplasmic reticulum (ER), whereas others localized REEP1 with mitochondria. We sought to resolve the cellular localization of REEP1 and further elucidate the pathobiology underlying REEP1 mutations in patients. METHODS: A combination of cellular imaging and biochemical approaches was used to refine the cellular localization of REEP1. Next, Reep1 mutations associated with HSP were functionally tested in neuritic growth and degeneration assays using mouse cortical culture. Finally, a novel assay was developed and used with wild-type and mutant Reep1s to measure the interactions between the ER and mitochondria. RESULTS: We found that REEP1 is present at the ER-mitochondria interface, and it contains subdomains for mitochondrial as well as ER localization. Knockdown of Reep1 and expression of pathological Reep1 mutations resulted in neuritic growth defects and degeneration. Finally, using our novel split-RLuc8 assay, we show that REEP1 facilitates ER-mitochondria interactions, a function diminished by disease-associated mutations. INTERPRETATION: Our data potentially reconcile the current conflicting reports regarding REEP1 being either an ER or a mitochondrial protein. Furthermore, our results connect, for the first time, the disrupted ER-mitochondria interactions to a failure in maintaining health of long axons in HSPs. Finally, the split-RLuc8 assay offers a new tool to identify potential drugs for multiple neurodegenerative diseases with ER-mitochondria interaction defects.

Arx together with FoxA2, regulates Shh floor plate expression.

Mutations in the Aristaless related homeodomain transcription factor (ARX) are associated with a diverse set of X-linked mental retardation and epilepsy syndromes in humans. Although most studies have been focused on its function in the forebrain, ARX is also expressed in other regions of the developing nervous system including the floor plate (FP) of the spinal cord where its function is incompletely understood. To investigate the role of Arx in the FP, we performed gain-of-function studies in the chick using in ovo electroporation, and loss-of-function studies in Arx-deficient mice. We have found that Arx, in conjunction with FoxA2, directly induces Sonic hedgehog (Shh) expression through binding to a Shh floor plate enhancer (SFPE2). We also observed that FoxA2 induces Arx through its transcriptional activation domain whereas Nkx2.2, induced by Shh, abolishes this induction. Our data support a feedback loop model for Arx function; through interactions with FoxA2, Arx positively regulates Shh expression in the FP, and Shh signaling in turn activates Nkx2.2, which suppresses Arx expression. Furthermore, our data are evidence that Arx plays a role as a context dependent transcriptional activator, rather than a primary inducer of Shh expression, potentially explaining how mutations in ARX are associated with diverse, and often subtle, defects.