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.

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.

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.

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.

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 regulates cortical intermediate progenitor cell expansion and upper layer neuron formation through repression of Cdkn1c.

Mutations in the Aristaless-related homeobox (ARX) gene are found in a spectrum of epilepsy and X-linked intellectual disability disorders. During development Arx is expressed in pallial ventricular zone (VZ) progenitor cells where the excitatory projection neurons of the cortex are born. Arx(-/Y) mice were shown to have decreased proliferation in the cortical VZ resulting in smaller brains; however, the basis for this reduced proliferation was not established. To determine the role of ARX on cell cycle dynamics in cortical progenitor cells, we generated cerebral cortex-specific Arx mouse mutants (cKO). The loss of pallial Arx resulted in the reduction of cortical progenitor cells, particularly the proliferation of intermediate progenitor cells (IPCs) was affected. Later in development and postnatally cKO brains showed a reduction of upper layer but not deeper layer neurons consistent with the IPC defect. Transcriptional profile analysis of E14.5 Arx-ablated cortices compared with control revealed that CDKN1C, an inhibitor of cell cycle progression, is overexpressed in the cortical VZ and SVZ of Arx KOs throughout corticogenesis. We also identified ARX as a direct regulator of Cdkn1c transcription. Together these data support a model where ARX regulates the expansion of cortical progenitor cells through repression of Cdkn1c.

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.

Distinct DNA binding and transcriptional repression characteristics related to different ARX mutations.

Mutations in the Aristaless-related homeobox gene (ARX) are associated with a wide variety of neurologic disorders including lissencephaly, hydrocephaly, West syndrome, Partington syndrome, and X-linked intellectual disability with or without epilepsy. A genotype-phenotype correlation exists for ARX mutations; however, the molecular basis for this association has not been investigated. To begin understanding the molecular basis for ARX mutations, we tested the DNA binding sequence preference and transcriptional repression activity for Arx, deletion mutants and mutants associated with various neurologic disorders. We found DNA binding preferences of Arx are influenced by the amino acid sequences adjacent to the homeodomain. Mutations in the homeodomain show a loss of DNA binding activity, while the T333N and P353R homeodomain mutants still possess DNA binding activities, although less than the wild type. Transcription repression activity, the primary function of ARX, is reduced in all mutants except the L343Q, which has no DNA binding activity and does not functionally repress Arx targets. These data indicate that mutations in the homeodomain result in not only a loss of DNA binding activity but also loss of transcriptional repression activity. Our results provide novel insights into the pathogenesis of ARX-related disorders and possible directions to pursue potential therapeutic interventions.

Differential effects of a polyalanine tract expansion in Arx on neural development and gene expression.

Polyalanine (poly-A) tracts exist in 494 annotated proteins; to date, expansions in these tracts have been associated with nine human diseases. The pathogenetic mechanism by which a poly-A tract results in these various human disorders remains uncertain. To understand the role of this mutation type, we investigated the change in functional properties of the transcription factor Arx when it has an expanded poly-A tract (Arx(E)), a mutation associated with infantile spasms and intellectual disabilities in humans. We found that although Arx(E) functions normally in the dorsal brain, its function in subpallial-derived populations of neurons is compromised. These contrasting functions are associated with the misregulation of Arx targets through the loss of the ability of Arx(E) to interact with the Arx cofactor Tle1. Our data demonstrate a novel mechanism for poly-A expansion diseases: the misregulation of a subset of target genes normally regulated by a transcription factor.

XLMR candidate mouse gene, Zcchc12 (Sizn1) is a novel marker of Cajal-Retzius cells.

Sizn1 (Zcchc12) is a transcriptional co-activator that positively modulates bone morphogenic protein (BMP) signaling through its interaction with Smad family members and CBP. We have demonstrated a role for Sizn1 in basal forebrain cholinergic neuron specific gene expression. Furthermore, mutations in SIZN1 have been associated with X-linked mental retardation. Given the defined role of SIZN1 in mental retardation, knowing its complete forebrain expression pattern is essential to further elucidating its role in cognition. To better define the dynamic expression pattern of Sizn1 during forebrain development, we investigated its expression in mouse brain development from embryonic day 8.0 (E8.0) to adult. We found that Sizn1 is primarily restricted to the ventral forebrain including the medial ganglionic eminence, the septum, amygdala, and striatum. In addition, Sizn1 expression is detected in the cortical hem and pallial-subpallial boundary (PSB; anti-hem); both sources of Cajal-Retzius cells. Sizn1 expression in the dorsal forebrain is restricted to a subset of cells in the marginal zone that also express Reln, indicative of Cajal-Retzius cells. These data provide novel information on brain regions and cell types that express Sizn1, facilitating further investigations into the function of Sizn1 in both development and the pathogenesis of mental retardation.

SUMO interaction motifs in Sizn1 are required for promyelocytic leukemia protein nuclear body localization and for transcriptional activation.

Mutations in Sizn1 (Zcchc12), a novel transcriptional co-activator in the BMP signaling pathway, are associated with X-linked mental retardation. Previously, we demonstrated that Sizn1 positively modulates the BMP signal by interacting with Smad family members and cAMP-responsive element-binding protein-binding protein. To further define the molecular basis of Sizn1 function, we have explored its subcellular localization and generated various deletion mutants to carry out domain analyses. Here, we report that Sizn1 localizes to promyelocytic leukemia protein nuclear bodies (PML-NBs). Sizn1 deletion mutants that disrupt the MA homologous domain or the middle region fail to target to the PML-NB. We show that two SUMO interaction motifs (SIMs) in Sizn1 can bind to SUMO and govern SUMO conjugation to Sizn1 in the absence of the consensus motif for SUMO attachment. Interestingly, the SIM mutant Sizn1 localizes to nuclear bodies, but not to PML-NBs. Thus, SIMs mediate the localization of Sizn1 to PML-NB. Interestingly, mutations in SIM sequences and deletion of the MA homologous domain also affected the transcriptional co-activation function of a Sizn1. Taken together, our data indicate that the SIMs in Sizn1 are required for its PML-NB localization and for the full transcriptional co-activation function in BMP signaling.

Evidence that SIZN1 is a candidate X-linked mental retardation gene.

An estimated 1-3% of individuals within the United States are diagnosed with mental retardation (MR), yet the cause is unknown in nearly 50% of the patients. While several environmental, genetic and combined teratogenetic etiologies have been identified, many causative genes remain to be identified. Furthermore, the pathogenetic mechanisms underlying MR are known for very few of these genes. Males have a much higher incidence of MR implicating genes on the X-chromosome. We have recently identified a novel gene, SIZN1, on the X-chromosome and showed that it functions in modulating the BMP signaling pathway. Furthermore, we have shown this gene is necessary for basal forebrain cholinergic neuron (BFCN) specific gene expression. Given that cognitive function is impaired when BFCNs are lost or functionally disrupted, we undertook a screen of cognitively impaired males for SIZN1 mutations. We report on four different sequence variants in SIZN1 in 11 individuals with nonsyndromic X-linked mental retardation (XLMR). Our data implicate SIZN1 as a candidate gene for XLMR and/or as a neurocognitive functional modifier.

Identification of Arx transcriptional targets in the developing basal forebrain.

Mutations in the aristaless-related homeobox (ARX) gene are associated with multiple neurologic disorders in humans. Studies in mice indicate Arx plays a role in neuronal progenitor proliferation and development of the cerebral cortex, thalamus, hippocampus, striatum, and olfactory bulbs. Specific defects associated with Arx loss of function include abnormal interneuron migration and subtype differentiation. How disruptions in ARX result in human disease and how loss of Arx in mice results in these phenotypes remains poorly understood. To gain insight into the biological functions of Arx, we performed a genome-wide expression screen to identify transcriptional changes within the subpallium in the absence of Arx. We have identified 84 genes whose expression was dysregulated in the absence of Arx. This population was enriched in genes involved in cell migration, axonal guidance, neurogenesis, and regulation of transcription and includes genes implicated in autism, epilepsy, and mental retardation; all features recognized in patients with ARX mutations. Additionally, we found Arx directly repressed three of the identified transcription factors: Lmo1, Ebf3 and Shox2. To further understand how the identified genes are involved in neural development, we used gene set enrichment algorithms to compare the Arx gene regulatory network (GRN) to the Dlx1/2 GRN and interneuron transcriptome. These analyses identified a subset of genes in the Arx GRN that are shared with that of the Dlx1/2 GRN and that are enriched in the interneuron transcriptome. These data indicate Arx plays multiple roles in forebrain development, both dependent and independent of Dlx1/2, and thus provides further insights into the understanding of the mechanisms underlying the pathology of mental retardation and epilepsy phenotypes resulting from ARX mutations.

Sizn1 is a novel protein that functions as a transcriptional coactivator of bone morphogenic protein signaling.

Bone morphogenic proteins (BMPs) play pleotrophic roles in nervous system development, and their signaling is highly regulated at virtually every step in the pathway. We have cloned a novel gene, Sizn1 (Smad-interacting zinc finger protein), which functions as a transcriptional coactivator of BMP signaling. It positively modulates BMP signaling by interacting with Smad family members and associating with CBP in the transcription complex. Sizn1 is expressed in the ventral embryonic forebrain, where, as we will show, it contributes to BMP-dependent, cholinergic-neuron-specific gene expression. These data indicate that Sizn1 is a positive modulator of BMP signaling and provide further insight into how BMP signaling can be modulated in neuronal progenitor subsets to influence cell-type-specific gene expression and development.

Identification of a prosencephalic-specific enhancer of SALL1: comparative genomic approach using the chick embryo.

Comparative genomics is a promising approach for identifying regulatory elements governing the unique spatio-temporal expression patterns of morphogenetic genes. Conserved noncoding genomic sequences are candidate regulatory elements. Here we performed a survey for conserved noncoding elements (CNE) nested within the SALL1 gene; mutations in this gene result in the Townes-Brocks syndrome. A comparison of the genomic sequence between humans and chicken revealed five CNE. Genomic fragments corresponding to each CNE were inserted into reporter cassettes consisting of eGFP cDNA and a minimal promoter. These constructs were electroporated into chick embryos during gastrula, neurula, and pharyngula stages. Among the five CNE that were examined, one 443 bp CNE exhibited tissue-specific enhancer activity. At the neurula stage, the eGFP signal was visualized in the prosencephalon. At the pharyngula stage, the eGFP signal was confined within the anterior neural ridge, which represents one of the morphogenetic centers regulating the patterning of the anterior neural plate. This report identifies, for the first time, an enhancer element of SALL1.