Single-Cell Analysis of Experience-Dependent Transcriptomic States in Mouse Visual Cortex

Activity-dependent transcriptional responses shape cortical function. However, we lack a comprehensive understanding of the diversity of these responses across the full range of cortical cell types, and how these changes contribute to neuronal plasticity and disease. Here we applied high-throughput single-cell RNA-sequencing to investigate the breadth of transcriptional changes that occur across cell types in mouse visual cortex following exposure to light. We identified significant and divergent transcriptional responses to stimulation in each of the 30 cell types characterized, revealing 611 stimulus-responsive genes. Excitatory pyramidal neurons exhibit inter- and intra-laminar heterogeneity in the induction of stimulus responsive genes. Non-neuronal cells demonstrated clear transcriptional responses that may regulate experience-dependent changes in neurovascular coupling and myelination. Together, these results reveal the dynamic landscape of stimulus-dependent transcriptional changes that occur across cell types in visual cortex, which are likely critical for cortical function and may be sites of de-regulation in developmental brain disorders

Schizophrenia-associated somatic copy-number variants from 12,834 cases reveal recurrent NRXN1 and ABCB11 disruptions

While germline copy-number variants (CNVs) contribute to schizophrenia (SCZ) risk, the contribution of somatic CNVs (sCNVs)—present in some but not all cells—remains unknown. We identified sCNVs using blood-derived genotype arrays from 12,834 SCZ cases and 11,648 controls, filtering sCNVs at loci recurrently mutated in clonal blood disorders. Likely early-developmental sCNVs were more common in cases (0.91%) than controls (0.51%, p = 2.68e−4), with recurrent somatic deletions of exons 1–5 of the NRXN1 gene in five SCZ cases. Hi-C maps revealed ectopic, allele-specific loops forming between a potential cryptic promoter and non-coding cis-regulatory elements upon 5′ deletions in NRXN1. We also observed recurrent intragenic deletions of ABCB11, encoding a transporter implicated in anti-psychotic response, in five treatment-resistant SCZ cases and showed that ABCB11 is specifically enriched in neurons forming mesocortical and mesolimbic dopaminergic projections. Our results indicate potential roles of sCNVs in SCZ risk

Circular RNAs in the human brain are tailored to neuron identity and neuropsychiatric disease

Abstract Little is known about circular RNAs (circRNAs) in specific brain cells and human neuropsychiatric disease. Here, we systematically identify over 11,039 circRNAs expressed in vulnerable dopamine and pyramidal neurons laser-captured from 190 human brains and non-neuronal cells using ultra-deep, total RNA sequencing. 1526 and 3308 circRNAs are custom-tailored to the cell identity of dopamine and pyramidal neurons and enriched in synapse pathways. 29% of Parkinson’s and 12% of Alzheimer’s disease-associated genes produced validated circRNAs. circDNAJC6, which is transcribed from a juvenile-onset Parkinson’s gene, is already dysregulated during prodromal, onset stages of common Parkinson’s disease neuropathology. Globally, addiction-associated genes preferentially produce circRNAs in dopamine neurons, autism-associated genes in pyramidal neurons, and cancers in non-neuronal cells. This study shows that circular RNAs in the human brain are tailored to neuron identity and implicate circRNA-regulated synaptic specialization in neuropsychiatric diseases

DNA damage-induced cell death and <i>gypsy</i> ERV expression contribute hTDP-43 mediated toxicity.

<p>(A) Lifespan analysis shows that co-expression of <i>loki</i>(IR) (<i>Repo</i> > <i>loki</i>(IR) + hTDP-43) fully rescues the lifespan deficit exhibited by flies expressing glial hTDP-43 (<i>Repo</i> > hTDP-43). (B) Co-expression of <i>loki</i>(IR) (<i>ELAV</i> > <i>loki</i>(IR) + hTDP-43) likewise fully rescues the lifespan deficit exhibited by flies expressing neuronal hTDP-43 (<i>ELAV</i> > hTDP-43). (C) Central projections of whole-mount TUNEL stained brains reveal a noticeable reduction in the apoptotic activity induced by glial hTDP-43 expression (<i>Repo</i> > hTDP-43 + GFP(IR)) when <i>gypsy</i> expression is knocked down (<i>Repo</i> > hTDP-43 + <i>gypsy</i>(IR)), while knocking down <i>loki</i> completely alleviates the apoptosis induced by glial hTDP-43 expression (<i>Repo</i> > hTDP-43 + <i>loki</i>(IR)). (D) Quantification of (H), normalized to the positive control (<i>Repo</i> > hTDP-43 + GFP(IR)). <i>N</i> = 12 for <i>Repo</i> / +; <i>N</i> = 9 for <i>Repo</i> > hTDP-43 + GFP(IR); <i>N</i> = 7 for <i>Repo</i> > hTDP-43 + <i>gypsy</i>(IR); and <i>N</i> = 7 for <i>Repo</i> > hTDP-43 + <i>loki</i>(IR). <b>*</b>All of the lifespans with the exception of the NRTI feeding experiments shown in Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006635#pgen.1006635.g004" target="_blank">4</a> and 5, were performed concurrently in order to ensure comparability across groups. Therefore, appropriate controls are shared across panels.</p

Glial hTDP-43 expression results in early and dramatic de-suppression of the <i>gypsy</i> ERV.

<p>(A) Transcript levels of <i>gypsy ORF2</i> (<i>Pol</i>) as detected by qPCR in whole head tissue of flies expressing hTDP-43 in neurons (<i>ELAV</i> > hTDP-43) versus glia (<i>Repo</i> > hTDP-43) at a young (2–4 Day) or aged (8–10 Day) time point. Transcript levels normalized to <i>Actin</i> and displayed as fold change relative to flies carrying the hTDP-43 transgene with no Gal4 driver (hTDP-43 / +) at 2–4 Days (means + SEM). A two-way ANOVA reveals a significant effect of genotype (p < 0.0001) but no effect of age (p = 0.5414). <i>N</i> = 8 for all groups. (B) An equivalent analysis shows that <i>gypsy ORF3</i> (<i>Env</i>) likewise displays a significant effect of genotype (p < 0.0001) and no effect of age (p = 0.6530). <i>N</i> = 4 for the 2–4 Day cohort and <i>N</i> = 5 for the 8–10 Day cohort. (C) Central projections of whole mount brains immunostained with a monoclonal antibody directed against <i>gypsy</i> ENV protein reveals dramatic, early accumulation of ENV immunoreactive puncta in brains expressing glial hTDP-43 (5–8 Days) in comparison to both age-matched genetic controls (<i>ELAV</i> / +; <i>Repo</i> / +; hTDP-43 / +) and flies expressing neuronal hTDP-43. This effect persists out to 19–25 Days post-eclosion. <i>ELAV</i> / +, 5–8 Day (<i>N</i> = 3), 19–25 Day (<i>N</i> = 4); <i>Repo</i> / +, 5–8 Day (<i>N</i> = 3), 19–25 Day (<i>N</i> = 3); hTDP-43 / +, 5–8 Day (<i>N</i> = 5), 19–25 Day (<i>N</i> = 2); <i>ELAV</i> > hTDP-43, 5–8 Day (<i>N</i> = 2), 19–25 Day (<i>N</i> = 4); <i>Repo</i> > hTDP-43, 5–8 Day (<i>N</i> = 7), 19–25 Day (<i>N</i> = 8).</p

Neuronal and glial hTDP-43 expression results in induction of RTE expression.

<p>Differential expression of many genes and RTEs are detected in response to either neuronal or glial expression of hTDP-43 in head tissue of 8–10 day old flies (<i>N</i> = 2 biological replicates per genotype). (A) Neuronal (<i>Elav</i> > hTDP-43) expression of hTDP-43 results in both increases and decreases in expression of a broad variety of cellular transcripts (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006635#pgen.1006635.s009" target="_blank">S2A Table</a>). (B) A panel of transposons, including many RTEs, also are impacted, with most exhibiting elevated expression (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006635#pgen.1006635.s009" target="_blank">S2B Table</a>). (C) Glial expression of hTDP-43 (<i>Repo</i> > hTDP-43) also results in numerous transcriptome alterations, with many transcripts either increasing or decreasing in abundance (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006635#pgen.1006635.s010" target="_blank">S3A Table</a>). (D) Many transposons, most of which are RTEs, exhibit elevated expression levels in response to glial hTDP-43 expression (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006635#pgen.1006635.s010" target="_blank">S3B Table</a>). Several RTEs display elevated expression in response to both glial and neuronal hTDP-43 expression, however a number also exhibit specificity in response to either glial or neuronal hTDP-43 expression (compare <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006635#pgen.1006635.g001" target="_blank">Fig 1B and 1D</a>). (E) The <i>gypsy</i> ERV exhibits elevated expression only in response to glial, but not neuronal, hTDP-43 expression. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006635#sec008" target="_blank">methods</a> for details regarding analysis pipeline, including statistical analysis.</p

Glial and neuronal hTDP-43 expression erodes siRNA-mediated silencing.

<p>(A) Representative central projections show that co-expression of the hTDP-43 transgene, but not an unrelated tdTomato control transgene, interferes with the ability of a Dcr-2 processed IR (GFP(IR)) to silence a GFP transgenic reporter in glial cells using the <i>Repo-GAL4</i> driver. Quantification of GFP signal for each group is shown in the appropriate bar graph; values are represented as relative fold change over <i>Repo</i> > GFP + GFP(IR) (mean + SEM). A two-way ANOVA reveals significant effects of both genotype (p < 0.0001) and age (p < 0.0001), and a significant age x genotype interaction (p < 0.0001). <i>N</i> = 5 for <i>Repo</i> > GFP and <i>Repo</i> > GFP + GFP(IR); <i>N</i> = 10 for all other groups. (B) An equivalent analysis demonstrates that hTDP-43 has a similar effect in the neuronal cells of the <i>Drosophila</i> mushroom body using the <i>OK107-Gal4</i> driver, but with a later age of onset than hTDP-43 expression in glial cells. Quantification of GFP signal for each group is shown in the appropriate bar graph as in (A). A two-way ANOVA reveals significant effects of genotype (p = 0.0054) and age (p < 0.0001), as well as a significant age x genotype interaction (p = 0.0021). <i>N</i> = 5 for <i>OK107</i> > GFP and <i>OK107</i> > GFP + GFP(IR); <i>N</i> = 10 for all other groups. (C) Co-expression of hTDP-43, but not GFP, in the photoreceptor neurons of the fly eye under the <i>GMR-Gal4</i> driver interrupts the ability of a Dcr-2 processed IR to silence the endogenous <i>white</i>^<i>+</i> pigment gene with an age of onset similar to that observed with neuronal expression of hTDP-43 in the CNS under <i>OK107-Gal4</i>, resulting in characteristic clusters of red-pigmented ommatidia. <i>N</i> = 5 for <i>GMR</i> > <i>w</i>(IR) + Gal80^ts <i>OFF</i> and <i>GMR</i> > <i>w</i>(IR) + Gal80^ts <i>ON</i>; <i>N</i> = 20 for all other groups.</p

<i>gypsy</i> ERV expression contributes to hTDP-43 mediated toxicity.

<p>(A) Lifespan analysis shows that co-expression of <i>gypsy</i>(IR) (<i>Repo</i> > <i>gypsy</i>(IR) + hTDP-43) partially rescues the lifespan deficit exhibited by flies expressing glial hTDP-43 (<i>Repo</i> > hTDP-43). (B) Co-expression of an unrelated GFP(IR) control transgene (<i>Repo</i> > GFP(IR) + hTDP-43) does not effect the lifespan of flies expressing glial hTDP-43 (<i>Repo</i> > hTDP-43). (C) Co-expression of <i>gypsy</i>(IR) (<i>ELAV</i> > <i>gypsy</i>(IR) + hTDP-43) has no effect on lifespan in flies expressing neuronal hTDP-43 (<i>ELAV</i> > hTDP-43).</p

Neuronal and glial hTDP-43 expression induces physiological impairment and toxicity with varying severity.

<p>(A) Flies expressing glial hTDP-43 display extreme locomotor impairment at 1–5 days post-eclosion in the Benzer fast phototaxis assay, while flies expressing neuronal hTDP-43 demonstrate a slight locomotor deficit in comparison to genetic controls (one-way ANOVA, p < 0.0001). This trend continues and is exacerbated by 5–10 days post-eclosion (one-way ANOVA, p < 0.0001). Four biological replicates performed for each experiment. (B) Lifespan analysis of flies expressing neuronal versus glial hTDP-43 in comparison to genetic controls. (C) Central projections of whole-mount brains reveals a stark increase in TUNEL-positive cells in flies expressing glial hTDP-43 in comparison to genetic controls at 5 days post-eclosion. <i>N</i> = 16 for <i>Repo</i> / + and <i>N</i> = 18 for <i>Repo</i> > hTDP-43. (D) TEM likewise reveals rampant apoptosis in the neuropil of flies expressing glial hTDP-43 at 12 days post-eclosion. Arrowheads indicate pro-apoptotic nuclei, as identified by morphology.</p