Brady J. Maher

Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure

Cerebral organoids, three-dimensional cultures that model organogenesis, provide a new platform to investigate human brain development. High cost, variability, and tissue heterogeneity limit their broad applications. Here, we developed a miniaturized spinning bioreactor (SpinΩ) to generate forebrain-specific organoids from human iPSCs. These organoids recapitulate key features of human cortical development, including progenitor zone organization, neurogenesis, gene expression, and, notably, a distinct human-specific outer radial glia cell layer. We also developed protocols for midbrain and hypothalamic organoids. Finally, we employed the forebrain organoid platform to model Zika virus (ZIKV) exposure. Quantitative analyses revealed preferential, productive infection of neural progenitors with either African or Asian ZIKV strains. ZIKV infection leads to increased cell death and reduced proliferation, resulting in decreased neuronal cell-layer volume resembling microcephaly. Together, our brain-region-specific organoids and SpinΩ provide an accessible and versatile platform for modeling human brain development and disease and for compound testing, including potential ZIKV antiviral drugs.

Direct Actions of Carbenoxolone on Synaptic Transmission and Neuronal Membrane Properties

The increased appreciation of electrical coupling between neurons has led to many studies examining the role of gap junctions in synaptic and network activity. Although the gap junctional blocker carbenoxolone (CBX) is effective in reducing electrical coupling, it may have other actions as well. To study the non-gap junctional effects of CBX on synaptic transmission, we recorded from mouse hippocampal neurons cultured on glial micro-islands. This recording configuration allowed us to stimulate and record excitatory postsynaptic currents (EPSCs) or inhibitory postsynaptic currents (IPSCs) in the same neuron or pairs of neurons. CBX irreversibly reduced evoked alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionic acid (AMPA) receptor-mediated EPSCs. Consistent with a presynaptic site of action, CBX had no effect on glutamate-evoked whole cell currents and increased the paired-pulse ratio of AMPA and N-methyl-d-aspartate (NMDA) receptor-mediated EPSCs. CBX also reversibly reduced GABA(A) receptor-mediated IPSCs, increased the action potential width, and reduced the action potential firing rate. Our results indicate CBX broadly affects several neuronal membrane conductances independent of its effects on gap junctions. Thus effects of carbenoxolone on network activity cannot be interpreted as resulting from specific block of gap junctions.

Olig2-dependent developmental fate switch of NG2 cells.

NG2-expressing cells (NG2 cells or polydendrocytes) generate oligodendrocytes throughout the CNS and a subpopulation of protoplasmic astrocytes in the gray matter of the ventral forebrain. The mechanisms that regulate their oligodendrocyte or astrocyte fate and the degree to which they exhibit lineage plasticity in vivo have remained unclear. The basic helix-loop-helix transcription factor Olig2 is required for oligodendrocyte specification and differentiation. We have found that Olig2 expression is spontaneously downregulated in NG2 cells in the normal embryonic ventral forebrain as they differentiate into astrocytes. To further examine the role of Olig2 in NG2 cell fate determination, we used genetic fate mapping of NG2 cells in constitutive and tamoxifen-inducible Olig2 conditional knockout mice in which Olig2 was deleted specifically in NG2 cells. Constitutive deletion of Olig2 in NG2 cells in the neocortex and corpus callosum but not in ventral forebrain caused them to convert their fate into astrocytes, with a concomitant severe reduction in the number of oligodendrocytes and myelin. Deletion of Olig2 in NG2 cells in perinatal mice also resulted in astrocyte generation from neocortical NG2 cells. These observations indicate that the developmental fate of NG2 cells can be switched by altering a single transcription factor Olig2.

Developmental regulation of human cortex transcription and its clinical relevance at single base resolution

Transcriptome analysis of human brain provides fundamental insight into development and disease, but it largely relies on existing annotation. We sequenced transcriptomes of 72 prefrontal cortex samples across six life stages and identified 50,650 differentially expression regions (DERs) associated with developmental and aging, agnostic of annotation. While many DERs annotated to non-exonic sequence (41.1%), most were similarly regulated in cytosolic mRNA extracted from independent samples. The DERs were developmentally conserved across 16 brain regions and in the developing mouse cortex, and were expressed in diverse cell and tissue types. The DERs were further enriched for active chromatin marks and clinical risk for neurodevelopmental disorders such as schizophrenia. Lastly, we demonstrate quantitatively that these DERs associate with a changing neuronal phenotype related to differentiation and maturation. These data show conserved molecular signatures of transcriptional dynamics across brain development, have potential clinical relevance and highlight the incomplete annotation of the human brain transcriptome.

Co-transmission of dopamine and GABA in periglomerular cells.

Most central neurons package and release a single transmitter. However co-transmission of fast-acting and modulatory transmitters has been observed in vertebrate and invertebrate systems. Here we describe a population of periglomerular cells in mouse brain slices (PND14-21) that co-release dopamine and GABA. We made whole cell recordings from periglomerular cells that expressed enhanced green fluorescent protein (EGFP) under the control of the tyrosine hyrdoxylase (TH) promoter. Immunolabeling confirmed that EGFP+ periglomerular cells synthesized TH as well as glutamic acid decarboxylase (GAD). Stimulation of olfactory receptor neuron (ORN) afferent input evoked excitatory postsynaptic currents (EPSCs) in EGFP+ cells that were inhibited by cocaine, which blocks dopamine transport. These effects were reversed by the D2 receptor antagonist sulpiride. Cocaine also increased the paired-pulse ratio of ORN-evoked EPSCs. These results demonstrate that TH+ periglomerular cells spontaneously release dopamine. In addition to dopamine, TH-EGFP+ cells also released GABA. Brief depolarizing voltage steps in labeled cells evoked a tail current that was completely blocked by the GABA(A) receptor antagonist gabazine and by cadmium, indicative of calcium-dependent self-inhibition in periglomerular cells. However, similar voltage steps were insufficient to cause D2-receptor mediated inhibition of ORN terminals. Our results indicate that TH+ periglomerular cells are directly activated by ORN input and release both dopamine and GABA. We suggest that concerted activation of multiple periglomerular cells may be required to detect dopamine release under normal physiological conditions.

Experience-dependent maturation of the glomerular microcircuit

Spontaneous and patterned activity, largely attributed to chemical transmission, shape the development of virtually all neural circuits. However, electrical transmission also has an important role in coordinated activity in the brain. In the olfactory bulb, gap junctions between apical dendrites of mitral cells increase excitability and synchronize firing within each glomerulus. We report here that the development of the glomerular microcircuit requires both sensory experience and connexin (Cx)36-mediated gap junctions. Coupling coefficients, which measure electrical coupling between mitral cell dendrites, were high in young mice, but decreased after postnatal day (P)10 because of a maturational increase in membrane conductance. Sensory deprivation, induced by unilateral naris occlusion at birth, slowed the morphological development of mitral cells and arrested the maturational changes in membrane conductance and coupling coefficients. As the coupling coefficients decreased in normal mice, a glutamate-mediated excitatory postsynaptic current (EPSC) between mitral cells emerged by P30. Although mitral–mitral EPSCs were generally unidirectional, they were not present in young adult Cx36−/− mice, suggesting that gap junctions are required for the development and/or function of the mature circuit. The experience-dependent transition from electrical transmission to combined chemical and electrical transmission provides a previously unappreciated mechanism that may tune the response properties of the glomerular microcircuit.

Disrupted-in-Schizophrenia (DISC1) Functions Presynaptically at Glutamatergic Synapses

The pathophysiology of schizophrenia is believed to involve defects in synaptic transmission, and the function of many schizophrenia-associated genes, including DISC1, have been linked to synaptic function at glutamatergic synapses. Here we develop a rodent model via in utero electroporation to assay the presynaptic function of DISC1 at glutamatergic synapses. We used a combination of mosaic transgene expression, RNAi knockdown and optogenetics to restrict both genetic manipulation and synaptic stimulation of glutamatergic neurons presynaptic to other layer 2/3 neocortical pyramidal neurons that were then targeted for whole-cell patch-clamp recording. We show that expression of the DISC1 c-terminal truncation variant that is associated with Schizophrenia alters the frequency of mEPSCs and the kinetics of evoked glutamate release. In addition, we show that expression level of DISC1 is correlated with the probability of glutamate release such that increased DISC1 expression results in paired-pulse depression and RNAi knockdown of DISC1 produces paired-pulse facilitation. Overall, our results support a direct presynaptic function for the schizophrenia-associated gene, DISC1.

A human-specific AS3MT isoform and BORCS7 are molecular risk factors in the 10q24.32 schizophrenia-associated locus

Genome-wide association studies (GWASs) have reported many single nucleotide polymorphisms (SNPs) associated with psychiatric disorders, but knowledge is lacking regarding molecular mechanisms. Here we show that risk alleles spanning multiple genes across the 10q24.32 schizophrenia-related locus are associated in the human brain selectively with an increase in the expression of both BLOC-1 related complex subunit 7 (BORCS7) and a previously uncharacterized, human-specific arsenite methyltransferase (AS3MT) isoform (AS3MTd2d3), which lacks arsenite methyltransferase activity and is more abundant in individuals with schizophrenia than in controls. Conditional-expression analysis suggests that BORCS7 and AS3MTd2d3 signals are largely independent. GWAS risk SNPs across this region are linked with a variable number tandem repeat (VNTR) polymorphism in the first exon of AS3MT that is associated with the expression of AS3MTd2d3 in samples from both Caucasians and African Americans. The VNTR genotype predicts promoter activity in luciferase assays, as well as DNA methylation within the AS3MT gene. Both AS3MTd2d3 and BORCS7 are expressed in adult human neurons and astrocytes, and they are upregulated during human stem cell differentiation toward neuronal fates. Our results provide a molecular explanation for the prominent 10q24.32 locus association, including a novel and evolutionarily recent protein that is involved in early brain development and confers risk for psychiatric illness.

Whole-genome association analysis of treatment response in obsessive-compulsive disorder.

Up to 30% of patients with obsessive-compulsive disorder (OCD) exhibit an inadequate response to serotonin reuptake inhibitors (SRIs). To date, genetic predictors of OCD treatment response have not been systematically investigated using genome-wide association study (GWAS). To identify specific genetic variations potentially influencing SRI response, we conducted a GWAS study in 804 OCD patients with information on SRI response. SRI response was classified as ‘response’ (n=514) or ‘non-response’ (n=290), based on self-report. We used the more powerful Quasi-Likelihood Score Test (the MQLS test) to conduct a genome-wide association test correcting for relatedness, and then used an adjusted logistic model to evaluate the effect size of the variants in probands. The top single-nucleotide polymorphism (SNP) was rs17162912 (P=1.76 × 10−8), which is near the DISP1 gene on 1q41-q42, a microdeletion region implicated in neurological development. The other six SNPs showing suggestive evidence of association (P<10−5) were rs9303380, rs12437601, rs16988159, rs7676822, rs1911877 and rs723815. Among them, two SNPs in strong linkage disequilibrium, rs7676822 and rs1911877, located near the PCDH10 gene, gave P-values of 2.86 × 10−6 and 8.41 × 10−6, respectively. The other 35 variations with signals of potential significance (P<10−4) involve multiple genes expressed in the brain, including GRIN2B, PCDH10 and GPC6. Our enrichment analysis indicated suggestive roles of genes in the glutamatergic neurotransmission system (false discovery rate (FDR)=0.0097) and the serotonergic system (FDR=0.0213). Although the results presented may provide new insights into genetic mechanisms underlying treatment response in OCD, studies with larger sample sizes and detailed information on drug dosage and treatment duration are needed.

Crucial roles of the Arp2/3 complex during mammalian corticogenesis

The polarity and organization of radial glial cells (RGCs), which serve as both stem cells and scaffolds for neuronal migration, are crucial for cortical development. However, the cytoskeletal mechanisms that drive radial glial outgrowth and maintain RGC polarity remain poorly understood. Here, we show that the Arp2/3 complex – the unique actin nucleator that produces branched actin networks – plays essential roles in RGC polarity and morphogenesis. Disruption of the Arp2/3 complex in murine RGCs retards process outgrowth toward the basal surface and impairs apical polarity and adherens junctions. Whereas the former is correlated with an abnormal actin-based leading edge, the latter is consistent with blockage in membrane trafficking. These defects result in altered cell fate, disrupted cortical lamination and abnormal angiogenesis. In addition, we present evidence that the Arp2/3 complex is a cell-autonomous regulator of neuronal migration. Our data suggest that Arp2/3-mediated actin assembly might be particularly important for neuronal cell motility in a soft or poorly adhesive matrix environment. Summary: During mouse cortical development, the Arp2/3 actin branching complex regulates process formation and the maintenance of radial glial cell polarity, as well as affecting neuronal migration.